Dr Marlène Gallet

Dr Pauline & Margot

Dr Subramanya Pandruvada

Dr Romain Daquin

Dr Justine Bacchetta

Dr Fabienne Coury

Kelig Pernelle

Mr Jean Wach

Mrs Lise Allard

Nathalie Demoncheaux

Ms Ms Julie Juliana Carnesechi

Dr Juliette Guttierez

Dr Sailland

Cyrielle

Dan Georgess

Marlène Mazzorana

José Terrado

Christine Delprat

Christophe Chamot ||

Rosa M Guasch

Ignacio Pérez-Roger #

Pierre Jurdic

Irma Machuca-Gayet

TRANSCRIPTOMICS REVEALS RHOE AS A NOVEL REGULATOR OF BONE RESOPTION BY OCS CHAPTER 3: DISCUSSION AND PERSPECTIVES REFERENCES ANNEX

Keywords: 3-dimensional ACPA: anti-citrullinated protein autoantibodies Ap-1: activator protein 1 BCG: bacillus Calmette-Guérin Osteoclast, bone resorption, RhoE, podosomes, migration, transcriptomics

Achievement is impossible without opportunity. First and foremost, I would like to express my gratitude to my mentor, Dr. Pierre Jurdic, for opening the gates of research when many would not and for his openness, patience and advice over the past years. Pierre, you have been a formidable boss and an exemplary role model. Thank you for all the discussions

If being apart of a superb lab wasn't in itself a wonderful experience, being apart of a community was beyond amazing. The T3-Net was more than a network of exceptional experts; it was the extended scientific family every student wishes for. I would like to particularly acknowledge Pr. Stefan Linder for his never-ending guidance and encouragement. I would also like to thank Dr. Elisabeth Genot for trusting me as a collaborator, for hosting me in Bordeaux and for her constant support.

The T3-Net experience was extremely pleasant also thanks to young peers that have become friends. Thank you Mr. Pascuale Cervero, Mr. Karima Azouzi, Mr. Franco Klingberg, Mr. Nilesh Talele, Dr. Filipa Curado, Ms. Isabel Egana, Dr. Vineetha Vijayakumar, Mr. Paolo Ciufici and Dr. Vinoth Khandelwal.

Together with Dr. Linder, I would like to specially acknowledge Dr. Isabelle Maridonneau-Parini, whom I admire for seminal work in the podosome field, for critical revision leading to significant enhancement of this manuscript.

I would like to specially acknowledge Pr. Georg Schett, Dr. Jose Terrado-Vicente and Dr. Jean-Christophe Geminard for trusting me with wonderful collaborations. These experiences have not only been pleasant and fruitful, but also giant leaps in my scientific career.

Before this thesis, I would only dream of applying for a doctoral position. These dreams became reality thanks to Dr. Andrei Popov who has taught me the essentials in experimental biology along with the proactive perseverance necessary for a professional life. I would also like to thank Pr. Stefan Nonchev for seeing potential in me and for defending it.

On a personal level, I would like to thank my parents Hossam and Penka Georgess. I owe everything I am to you. Thank you for your unconditional love and limitless belief in me. Thank you for teaching me hope and installing in me the means to realize my dreams.

Samar, thank you for hope, love, support, motivation and inspiration. You are my sunshine in the storm.

Aunt Salam, uncle Daher and Heba, thank you for all your support, encouragement and love.

The Mediterranean-Far East connection -Olimpia, Imtiaz and Esber -thank you for being my home-away-from-home.

The Welcome Committee co-founders -François and Florent -with its star artist Emilien and big sister Delphine, your friendship has brought much needed balance to my life.

L'ancien and Super G -Laurent and Audrey -thank you for continuous support, heartfelt friendship and smiles.

Résumé

Le remodelage osseux est un processus physiologique de renouvellement de l'os ancien par de l'os nouveau. Les ostéoclastes sont des cellules multinucléées géantes dont la fonction principale est de dégrader la matrice osseuse, première étape de ce remodelage. Le travail réalisé s'inscrit dans une thématique d'expertise de notre laboratoire, celle de l'organisation du cytosquelette d'actine dans les ostéoclastes résorbants. Nous avons pu élucider le rôle de la formation des podosomes et de leur organisation collective sur l'adhérence et la migration ostéoclastique. Nos résultats ont démontré que l'assemblage de podosomes sous forme de structures circulaires dites « anneaux » exerce une force centripète sur le substrat et ainsi déclenche la migration de l'ostéoclaste. L'alternance entre apparition et disparition de ces anneaux au sein de la cellule résulte en une migration saltatoire universelle pour tous les ostéoclastes. L'objectif principal de cette thèse était de trouver de nouveaux gènes impliqués dans l'organisation des podosomes. Nous avons mis en place une analyse transcriptomique comparant les ostéoclastes avec d'autres cellules multinucléées géantes qui présentent des podosomes mais sont incapables de résorber l'os. Parmi la liste de six gènes établie par cette méthode, nous avons étudié RhoE. En exploitant la culture d'ostéoclastes primaires déplétés de RhoE, nous avons démontré que ce gène est essentiel pour la migration ostéoclastique et la résorption osseuse. Nous avons ensuite établi que RhoE agit comme antagoniste de la voie de Rock pour assurer le renouvellement d'actine au sein des podosomes, ce qui entretien la fonction ostéoclastique.

ODF: osteoclast Differentiation Factor OPG: osteoprotegerin Osteoprotegerin(OPG)-Ligand. OPG OY: osteocyte PAX5: paired box protein 5 PI3K: phosphoinositide 3-kinase PLC-γ: Phospholipase C γ RA: rheumatoid arthritis RANK: receptor activator of nuclear factor κB RANKL: receptor activator of nuclear factor κB ligand RB: ruffled border RF: rheumatoid factor RGD: Arginyl-glycyl-aspartic acid motif SLP76: lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte protein of 76kDa) SZ: sealing zone SZL: sealing-zone like structure TACE : TNF-α convertase TLR: Toll-like receptor TNF-α: tumor necrosis factor α TRAF: tumor necrosis factor receptor-associated factor TRANCE: TNF-Related Activation-Induced Cytokine TRAP: tartrate-resistant acid phosphatase

List of introductory figures

I.1. The skeleton: cells and matrix

Bone is a living tissue comprised of a stiff extracellular matrix, bone cells, blood vessels and nerves. The extracellular matrix is itself mineralized and composed of Type I Collagen mostly as well as glycoproteins and proteoglycans. Besides its obvious role in mechanical support for movement, bone also provides protection of vital tissues and organs as well as regulation of the immune, endocrine and nervous systems and mineral homeostasis.

The growth of this tissue during development, its renewal during adulthood and its repair are coordinated processes ensured by the three bone cells: osteoblasts (OBs), osteoclasts (OCs) and osteocytes (OYs).

I.1.1. Osteoblasts

OBs are derived from mesenchymal stem cells. Their main function is the deposition and arrangement of the organic matrix of bone mainly composed at 90% of of Type I collagen.

OBs also secrete fibronectin, osteopontin, osteocalcin and sialoprotein which allow the mineralization of bone after crystallization of hydroxyapatite from deposited calcium and phosphate [START_REF] Khurana | Bone pathology[END_REF]. OBs are also able to regulate OC differentiation and function through a variety of cytokines such as RANKL, OPG, MCSF and interleukins IL-6 and IL-11

and growth factors such as TGF-β and BMPs. In addition, OBs can respond to two hormones that are central to mineral homeostasis and bone metabolism namely parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D. With the aging of bone, OBs are thought to transform into "bone lining cells", quiescent cells present at sites of bone where active bone formation is absent [START_REF] Bartl | Osteoporosis : diagnosis, prevention, therapy[END_REF].

I.1.2. Osteoclasts

OCs are multinucleated giant cells deriving from hematopoietic precursors. They are responsible for bone degradation, also known as bone resorption. To degrade the most compact and stiff matrix of the organism, OCs actively secrete protons and proteases thereby allowing the dissolution of its mineral phase and the breakdown of its protein scaffold, respectively (Coxon and Taylor, 2008b). This process ensures the release of minerals (Calcium, Phosphate, Magnesium,…), peptides (from degraded matrix proteins) and signaling molecules (TGF-β,…) formerly embedded in bone into the blood stream (Mulari et al., 2003a;[START_REF] Yadav | Leptin-dependent serotonin control of appetite: temporal specificity, transcriptional regulation, and therapeutic implications[END_REF]. Both osteoclastogenesis and bone resorption are regulated by OBs through RANKL and OPG expression, by OYs through RANKL and by immune cells though several cytokines such as MCSF, RANKL, TNF-α, interleukins, etc ... (Nakashima and Takayanagi, 2011a;[START_REF] O'brien | Osteocyte control of osteoclastogenesis[END_REF]. Conversely, OC are themselves able to regulate OB chemotaxis through PDGF expression [START_REF] Sanchez-Fernandez | Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling[END_REF].

I.1.3. Osteocytes

In contrast to bone surface cells, i.e. OBs and OCs, OYs are embedded in the bone matrix.

They comprise more than 90% of all bone cells and are derived from OBs that became trapped by the bone matrix that they themselves secreted and organized. OYs are mechanosensory cells able to translate loading stress into facilitation of macromolecule diffusion. OYs are not only able to communicate with each other through gap junctions but are also implicated in signaling to other bone cells [START_REF] Khurana | Bone pathology[END_REF]. For example, they can modulate OB-mediated bone formation through the expression of OB-specific factor 1 (OSF-1), dentin matrix protein 1 (DMP1) and sclerostin [START_REF] Tezuka | Isolation of mouse and human cDNA clones encoding a protein expressed specifically in osteoblasts and brain tissues[END_REF][START_REF] O'brien | Osteocyte control of osteoclastogenesis[END_REF].

OYs can control OCs as well by being the major source of RANKL, an indispensable cytokine for OC differentiation and function [START_REF] Xiong | Matrix-embedded cells control osteoclast formation[END_REF].

Bone modeling during the development and bone remodeling during adulthood occur in discrete foci or packets approximately 2-3mm long and with a diameter of 200-300µm [START_REF] Gruber | Introduction to Bone Development, Remodelling and Repair[END_REF]. These were termed "Bone remodeling units" (BRU) when first described by Frost in 1964 (Figure 1,2). Whether during bone modeling or remodeling, the first step during the lifetime of a BRU is always bone resorption by osteoclasts, followed by a reversal phase where OC-like macrophages, probably differentiating from circulating monocytes [START_REF] Kotani | Systemic circulation and bone recruitment of osteoclast precursors tracked by using fluorescent imaging techniques[END_REF], resorb the remainder of the fragmented collagen and finally a bone formation phase by OBs (Figure 1,2) [START_REF] Bartl | Osteoporosis : diagnosis, prevention, therapy[END_REF]. BRUs have a lifespan of 3-9 months and are present in an estimated number of 10 6 at any time in the adult human organism [START_REF] Bartl | Osteoporosis : diagnosis, prevention, therapy[END_REF]. These BRUs are responsible for the entire renewal of the human bone matrix every 10 years. Human BRUs outlast the lifespans of OCs (3 weeks) and OBs (3 months). Both these cell types are therefore constantly differentiating from new precursors that are thought to be delivered by blood vessels and chemically attracted or docked using matrix components [START_REF] Gruber | Introduction to Bone Development, Remodelling and Repair[END_REF]) [START_REF] Boyce | The osteoclast, bone remodelling and treatment of metabolic bone disease[END_REF].

I.2. Bone pathologies: the prevalence of osteoporosis

An imbalance in the dynamic equilibrium between bone formation and resorption can result in rare cases in an increase of bone mass and density called osteopetrosis, or, more often, in a decrease in those parameters called osteoporosis.

Osteopetrosis is due to hereditary genetic disorders that rarely occur in humans [START_REF] Balemans | A clinical and molecular overview of the human osteopetroses[END_REF]. However, it is a common bone phenotype in transgenic animals that are engineered for use in bone biology research. Examples of such animals will be mentioned in the course of this manuscript.

Osteoporosis is one of the 10 most common diseases that affect humans with a prevalence of one in every three women and one in every eight men worldwide [START_REF] Bartl | Osteoporosis : diagnosis, prevention, therapy : a practical guide for all physicians--from pediatrics to geriatrics[END_REF].

Osteoporosis is the cause of bone fractures in 70% of US individuals [START_REF] Khurana | Bone pathology[END_REF].

Besides the obvious burden of these fractures on the quality of life, they pose an increased risk of mortality [START_REF] Bartl | Osteoporosis : diagnosis, prevention, therapy : a practical guide for all physicians--from pediatrics to geriatrics[END_REF].

The cause of osteoporosis in women, and in most cases in men, is the decrease in steroid hormones with age, which always leads first to an increase in osteoclastic resorption. Steroid hormones are also important for OB formation because they promote the differentiation of HSCs into OB while blocking differentiation into adipocytes [START_REF] Gruber | Introduction to Bone Development, Remodelling and Repair[END_REF]. Most of the clinical treatments so far used to reduce pathological decrease in bone mass have aimed at blocking OC-mediated bone resorption, although emerging treatments rather stimulate OBmediated bone formation [START_REF] Marie | Osteoblasts in osteoporosis: past, emerging, and future anabolic targets[END_REF].

I.3. Osteoimmunology

Evidence of the regulation of bone cells by the immune system started emerging in the 1970s when several studies described OC-stimulating soluble factors secreted by leucocytes and myeloma cells. [START_REF] Horton | Bone resorbing activity in supernatant fluid from cultured human peripheral blood leukocytes[END_REF][START_REF] Mundy | Evidence for the secretion of an osteoclast stimulating factor in myeloma[END_REF]. The first of soluble factor to be identified was IL-1 [START_REF] Dewhirst | Purification and partial sequence of human osteoclast-activating factor: identity with interleukin 1 beta[END_REF] secreted by peripheral blood mononuclear cells.

Since then, bone and immune cells have been found to share several pathways regulating common transcription factors, cytokines, signaling factors, receptors,… In addition to these molecular findings, the immediate vicinity of bone cells with hematopoietic precursors and immune cells within the bone marrow has raised questions about the physiological crossregulation between the bone and immune systems, especially on the effect of the pathology of one system on the other. The study of functional links between these two systems has given rise to a new scientific field dubbed "Osteoimmunology" by [START_REF] Arron | Bone versus immune system[END_REF].

Advancements in this field have so far established a major axis of osteoimmune crosstalk:

T-lymphocyte and macrophage regulation of OC [START_REF] Takayanagi | Osteoimmunology and the effects of the immune system on bone[END_REF]Okamoto and Takayanagi, 2011b). It has been shown that different sub-populations of T cells, namely helper T cells, can promote OC differentiation and activity directly through RANKL, TNF expression and indirectly by IL-17-mediated stimulation of synovial fibroblasts and macrophages (Okamoto and Takayanagi, 2011a). T cells can also directly inhibit these OC processes through IL-4 and IFN-γ secretion. More recently, a second axis has emerged depicting OB regulation of hematopoietic stem cell (HSC) [START_REF] Yoon | Differential regulation of CXCL5 by FGF2 in osteoblastic and endothelial niche cells supports hematopoietic stem cell migration[END_REF].

I.4. Autoimmunity in bone diseases: the case of Rheumatoid Arthritis

A large area of osteoimmunology deals with the implication of autoimmune diseases in pathological bone remodeling. A major example of such an implication is the case of Rheumatoid arthritis (RA). RA is a systemic autoimmune disease characterized by acute inflammation of the joints with severe articular bone and cartilage erosion, often accompanied, in its late stages, by systemic inflammation-induced osteoporosis [START_REF] Schett | Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment[END_REF].

RA affects about 1% of the worldwide population but its origin is still unknown McInnes, 2011 #8386}. Current knowledge has allowed the drafting of a model of development of RA, although the exact molecular and physiological links remain elusive. At the preclinical stage, years before the actual onset of RA, the interaction of environmental factors (smoking, gut microbiome,.. ) with genetic and epigenetic regulation would result in post-translational modifications of certain self antigens [START_REF] Mcinnes | The pathogenesis of rheumatoid arthritis[END_REF]). The immune system would then loose tolerance to these "neoantigens" and lymphocytes would release autoantibodies in secondary lymphoid tissues or in the bone marrow. These autoantibodies include the "Rheumatoid factor" (RF) and anti-citrullinated protein autoantibodies (ACPA) [START_REF] Aigner | Contribution of anti-cyclic citrullinated peptide antibody and rheumatoid factor to the diagnosis of arthropathy in haemochromatosis[END_REF][START_REF] Kuna | Mutated citrullinated vimentin antibodies in rheumatoid arthritis[END_REF]. RF recognizes the Fc portion of IgGs. ACPA recognizes the posttranslationally citrullinated proteins such as α-enolase, keratin, fibrinogen, fibronectin, collagen, and vimentin [START_REF] Mcinnes | The pathogenesis of rheumatoid arthritis[END_REF]. These antibodies are currently used as early clinical markers of RA, although their suspected contributions to the onset of the disease are unknown. In a recent collaboration with Pr. Georg Schett's laboratory, we have demonstrated that ACPA, by recognizing citrullinated vimentin at the surface of OCs, markedly stimulates their differentiation by promoting an autocrine TNF-α effect in these cells (see Annex section of this manuscript: Harre*, Georgess* et al). Increased osteoclast numbers therefore result in severe bone loss in joints (Figure 3). This study provided the first proof that autoantibody response to citrullinated vimentin can induce bone loss ( [START_REF] Harre | Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin[END_REF] commented in [START_REF] Leah | Rheumatoid arthritis. Linking ACPA to bone loss in rheumatoid arthritis[END_REF]).The maturation of RF and ACPA antibody response could be triggered by infectious agents, neuroimmune factors, biomechanical stimuli and/or microvasculature anomalies. This maturation coincides with onset of synovitis, the local inflammation of the synovial membrane at the joints, thus starting the clinical/symptomatic phase of arthritis (Figure 3) [START_REF] Mcinnes | The pathogenesis of rheumatoid arthritis[END_REF]Komatsu and Takayanagi, 2012a).

Lymphocytes, macrophages, neutrophiles and mast cells are then recruited to synovial compartment and induce the formation of pannus, an abnormal layer of fibroblast-like synoviocytes (FLSs) a nd neoangiogenesis [START_REF] Pap | Osteoclast-independent bone resorption by fibroblast-like cells[END_REF]Okamoto and Takayanagi, 2011b;Komatsu and Takayanagi, 2012b). All these cell types intricately coordinate bone and cartilage erosion by secreting inflammatory cytokines, namely TNF-α. The latter is therefore the most relevant cytokine in RA because it directly promotes OC-mediated bone resorption.

In addition to this local bone and cartilage erosion, the chronic and systemic inflammation is correlated in a longer term with osteoporosis [START_REF] Schett | Synovitis--an inflammation of joints destroying the bone[END_REF]. which stimulate osteoclastogenesis. The established phase of RA is characterized by the presence of large bone erosions filled with inflamed, synovially derived pannus tissue [START_REF] Schett | Synovitis--an inflammation of joints destroying the bone[END_REF].

II. OSTEOCLAST DIFFERENTIATION

OCs are giant multinucleated cells that derive from the differentiation and fusion of mononucleated precursors. This part of the manuscript will summarize current standing on the identity of these precursors, the key signaling pathways involved in osteoclastogenesis and the link between adhesion and differentiation.

II.1. Identification of osteoclast precursors data from human and murine models

The first documented micrograph of an OC was published by Pr. A.C. Geddes in 1913 (Figure 4) [START_REF] Geddes | The Origin of the Osteoblast and of the Osteoclast[END_REF]. "In the course of the search for the origin of the osteoblast, stated Dr.

Geddes, the origin of the osteoclast was observed." The OC, found in the second metatarsal of an 8-9 week-old human embryo, was then defined as "composed of the fused bodies of one or more cartilage cells, with numerous osteoblasts living within the protoplasmic mass as cell inclusions (multiple nuclei)".

The hematopoietic origin of the OC, however, was not described before several studies in the 1970s [START_REF] Steven | Starch gel electrophoresis of hen egg white, oviduct white, yolk, ova and serum proteins[END_REF][START_REF] Kahn | Investigation of cell lineage in bone using a chimaera of chick and quial embryonic tissue[END_REF][START_REF] Walker | Bone resorption restored in osteopetrotic mice by transplants of normal bone marrow and spleen cells[END_REF][START_REF] Mundy | Direct resorption of bone by human monocytes[END_REF][START_REF] Jotereau | The development relationship between osteocytes and osteoclasts: a study using the quail-chick nuclear marker in endochondral ossification[END_REF][START_REF] Kahn | Contact-mediated bone resorption by human monocytes in vitro[END_REF][START_REF] Ash | Osteoclasts derived from haematopoietic stem cells[END_REF].

In general, hematopoietic stem cells (HSCs) can give rise to two types of multipotent lineage progenitors: common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) [START_REF] Akashi | A clonogenic common myeloid progenitor that gives rise to all myeloid lineages[END_REF]Xing et al., 2005a). CLPs can give rise to B and T lymphocytes and to natural killer cells, but not to OCs. There is one documented exception to this rule observed in Pax5 -/-mice where pro-B cells can differentiate into OCs [START_REF] Nutt | Commitment to the B-lymphoid lineage depends on the transcription factor Pax5[END_REF]. On the other hand, CMPs can give rise to megakaryocytes, erythrocytes, and granulocyte/macrophage progenitors (GMPs) which themselves can differentiate either into monocytes (Mo), dendritic cells (DCs), macrophages and osteoclasts under stimulation with M-CSF or into ganulocytes under stimulation with GF-MSF [START_REF] Geissmann | The origin of dendritic cells[END_REF]. [START_REF] Geddes | The Origin of the Osteoblast and of the Osteoclast[END_REF].

The identification of the two main osteoclastogenic cytokines, M-CSF in 1993 [START_REF] Tanaka | Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors[END_REF] and, more importantly, RANKL in 1998 [START_REF] Fuller | TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts[END_REF][START_REF] Matsuzaki | Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures[END_REF][START_REF] Yasuda | Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL[END_REF], accompanied by technological advancements, mainly Fluorescence-Activated Cell Sorting (FACS), have allowed to specifically isolate human and murine monoclonal GMP-derived populations and test their osteoclastogenic potential ex vivo. It is noteworthy to mention that mice deficient of c-Fms (the M-CSF receptor), RANKL or its receptor, RANK, do not have OCs [START_REF] Dougall | RANK is essential for osteoclast and lymph node development[END_REF][START_REF] Pettit | TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis[END_REF]. Strikingly, all the data so far obtained have described wide osteoclastogenic plasticity within the myeloid lineage where very early myeloid precursors as well as committed monocytes, macrophages and dendritic cells can differentiate into OCs.

When isolated from murine bone marrow, blood or spleen, GMPs that are expanded ex vivo under stimulation by M -CSF and GM-CSF are called Colony Forming Units-Macrophage/Ganulocyte (CFU-GMs); these can give rise to CD11b + OC precursors [START_REF] Roodman | Cell biology of the osteoclast[END_REF]. Similarly, bone marrow-derived Flt3 + cells that are expanded ex vivo in the presence of Flt3-ligand give rise to CD11b + CD11c -OC precursor colonies [START_REF] Servet-Delprat | Flt3+ macrophage precursors commit sequentially to osteoclasts, dendritic cells and microglia[END_REF]. Cells from both CD11b + expanded colonies can be directly differentiated not only into OCs, but also into macrophages and dendritic cells.

GMPs can also differentiate into osteoclast/macrophage/DC precursors (OcMDCs) characterized by CD11b + Gr-1 -/lo c-Fms -/+ expression [START_REF] Arai | Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor OE∫B (RANK) receptors[END_REF]Xing et al., 2005b).

M-CSF, through its receptor c-Fms, induces proliferation and differentiation of these C-Fms -/+ precursors. These OcDMCs can be subsequently differentiated into OCs in the presence of RANKL. Further dissection of OC precursor clonality in mice has allowed a surface marker-based distinction between bone marrow-residing OcDMCs which had the following phenotype B220 -CD3 -CD11b /low C -Fms + CD117 hi/intermediate/low and peripheral OcDMCs, residing in the blood or in the spleen, which conversely had high CD11b surface expression [START_REF] Jacome-Galarza | Identification, characterization, and isolation of a common progenitor for osteoclasts, macrophages, and dendritic cells from murine bone marrow and periphery[END_REF]. Again, similarly to the two colony-derived precursors described above, the CD117 hi subpopulation of OcDMCs can also differentiate into DCs and macrophages.

These studies have cumulatively demonstrated that OCs cannot differentiate directly neither from the original hematopoietic stem cells (HSCs), nor from myeloid-committed GMPs, but that they derive from more committed cells: OcMDCs. Within OcMDCs pool, however, several different sub-populations exist depending on their initial hematopoietic organs (CD11b-positive or -negative) or on c-fms expression (positive or negative).

The osteoclastogenic potential of terminally differentiated myeloid cells, i.e. macrophages, monocytes and DC has also been studied, adding more complexity to the scheme. In fact, human and murine Mo (CD14 + CD16 low CD3 -CD19 -CD56 -), human osteoarthritic macrophages and the murine monocytic/macrophage cell-line RAW 264.7 can all directly give rise into OCs in the presence of M-CSF and RANKL [START_REF] Sabokbar | Macrophage-osteoclast differentiation and bone resorption in osteoarthrotic subchondral acetabular cysts[END_REF][START_REF] Gallois | Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes[END_REF]. More strikingly, Mo-derived immature DCs (CD1a + CD14 -HLA a,b,ci nt , HLA DR int , and CD80l ow , CD83 low , and CD86 low ) can also directly differentiate into OCs, even more efficiently so than Mo. Indeed, a transcriptome-wide study showed that OCs are transcriptionally closer to DC than to Mo [START_REF] Rivollier | Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment[END_REF][START_REF] Speziani | Murine dendritic cell transdifferentiation into osteoclasts is differentially regulated by innate and adaptive cytokines[END_REF][START_REF] Gallois | Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes[END_REF].

In conclusion, the origin of the OCs does not seem to be unique. However diverse and complex, information about the origin of OC precursors remains physiologically relevant because it may assist in answering three other questions: (1) Where does osteoclastogenesis take place in the body? (2) Does it occur before or after recruitment on the site of resorption?

(3) Are OCs a unique population or do OC subtypes exist?

II.2. The site of osteoclast differentiation

It is unclear, in vivo, whether or when OCs are derived from circulating precursors or bonetissue-resident precursors. One hypothesis claims that precursors in bone marrow or near bone can differentiate into OCs on adjacent sites where resorption is needed. Resorption activity, unlike hematopoiesis which is widespread in the marrow, occurs focally i.e. heterogeneously across the bone matrix. This hypothesis would then suggest the presence of a mechanism that spatially directs the initiation of differentiation to where resorption activity is needed.

Although mature OCs are themselves capable of migration through cell layers [START_REF] Saltel | Transmigration: a new property of mature multinucleated osteoclasts[END_REF], data supporting the stated hypothesis per se has not yet been published. A second hypothesis claims that OC precursors leave hematopoietic tissues, invade the systemic circulation and are homed to site of bone requiring resorption [START_REF] Muto | Lineage-committed osteoclast precursors circulate in blood and settle down into bone[END_REF]. This hypothesis implies that OC precursors have a significant migration capacity. Indeed, OC precursors are capable of migrating and the genetic depletion of the Hck gene, that promotes their migration, results in impaired homing to bone sites in vivo and, consequently, to reduced differentiation [START_REF] Verollet | Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors[END_REF]. In addition to the studies mentioned in the previous paragraph depicting the high ex vivo osteoclastogenic potential of circulating myeloid cells, more recent work using in vivo two-photon microscopy in mice has showed that OC precursors originating from the spleen can transmigrate into the systemic circulation, enter the bone spaces and differentiate into OCs [START_REF] Kotani | Systemic circulation and bone recruitment of osteoclast precursors tracked by using fluorescent imaging techniques[END_REF]. Lipid mediators and some chemokines such as sphingosine-1-phosphate (S1P), SDF-1/CXCL12 and RANKL have been shown to regulate this homing process [START_REF] Ishii | Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis[END_REF]. In addition, in RA murine models, CD11b+ precursors have been shown to be mobilized by inflammatory cytokines, mainly TNF-α and RANKL, towards sites of increased OC bone erosion in the joint where the presence of mature OC was significantly increased [START_REF] Ritchlin | Mechanisms of TNF-alpha-and RANKL-mediated osteoclastogenesis and bone resorption in psoriatic arthritis[END_REF][START_REF] Li | RANK signaling is not required for TNFalpha-mediated increase in CD11(hi) osteoclast precursors but is essential for mature osteoclast formation in TNFalpha-mediated inflammatory arthritis[END_REF].

Interestingly, in RA patients, DC numbers are severely increased in the synovium. The differentiation of these DCs into OCs ex vivo has been highly induced by RA synovial fluid extracted from patients [START_REF] Rivollier | Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment[END_REF].

Given the accumulation of evidence albeit not absolutely demonstrative, the second hypothesis on the whereabouts of osteoclastogenesis seems more plausible. Finally, one common fact, regardless of the originating location of OC precursors, is that the terminal phase of differentiation of already committed OC precursors cannot occur away from the bone tissue because of the main sources of RANKL, essentially OYs and OBs, are boneresiding.

II.3. Relevant cytokines involved in osteoclast differentiation

Several cytokines and their respective receptors are essential for OC precursor differentiation and fusion. Their sources, downstream signaling pathways and spatio-temporal roles are distinct.

II.3.1. M-CSF

M-CSF is a key cytokine for the determination of precursors commitment into the macrophage/OC lineage (MacDonald et al., 1986)(Figures 5,7). Its osteoclastogenic function was identified in the early 1990s. In fact, investigators searching for the genetic factor behind the absence of OC and consequent osteopetrosis in op/op mice found that it was the alteration of the M-CSF gene expression [START_REF] Wiktor-Jedrzejczak | Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse[END_REF][START_REF] Kodama | Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colonystimulating factor[END_REF]. M-CSF is produced constitutively in the bone microenvironment by a range of cells, including stromal cells/OB and T-lymphocytes in response to high PTH levels and inflammatory molecules such as TNF-α and IL-1 [START_REF] Weir | Colony stimulating factor-1 plays a role in osteoclast formation and function in bone resorption induced by parathyroid hormone and parathyroid hormone-related protein[END_REF][START_REF] Yavropoulou | Osteoclastogenesis--current knowledge and future perspectives[END_REF]. By binding to its sole receptor, c-fms (also known as CSF1-R, at the cell membrane, M-CSF promotes the proliferation, survival, and early commitment of OcMDCs (Figures 5,7). When recognized by its ligand, the receptor dimerizes and its tyrosine kinase is activated leading to auto-phosphorylation of specific tyrosine residues. This launches a downstream signaling cascade that includes PI3K, ERK1/2, PLC-γ and the proto-oncogene c-CBL [START_REF] Ross | M-CSF, c-Fms, and signaling in osteoclasts and their precursors[END_REF]. M-CSF can induce the expression of its own receptor, c-fms, by an autocrine loop that amplifies M-CSF-mediated signals, while it has also been reported to stimulate PU.1, an essential transcription factor in osteoclastogenesis (see below section II.4.1. PU-1). M-CSF can also induce the expression of RANK, the receptor of RANKL which is another indispensible cytokine in OC differentiation (Figure 7). The mutation of the M-CSF gene in op/op mice or in tl/tl rats causes a marked decrease of tissue macrophages and a lack of OCs resulting in an osteopetrotic bone phenotype [START_REF] Wiktor-Jedrzejczak | Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse[END_REF][START_REF] Yoshida | The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene[END_REF]Dobbins et al., 2002a;Dobbins et al., 2002b)). Accordingly, the genetic knockout of c-fms also leads to osteopetrosis in mice [START_REF] Dai | Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects[END_REF].

II.3.2. RANKL/RANK/OPG triad:

A "giant leap" for OC research was the characterization of the indispensable role of RANKL in osteoclastogenesis (Figures 567). RANKL, a member of the TNF family, has been first identified on the surface of activated T-lymphocytes where it participates in the survival and activation of DCs [START_REF] Anderson | A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function[END_REF][START_REF] Wong | TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells[END_REF]. RANKL had several other names prior to the specific identification of its importance for OCs. Some of these names were related to its general functions like "TNF-Related Activation-Induced Cytokine" (TRANCE) for example. Other names, however, were more specific of its OC-targeting function as its identification became more imminent, such as Osteoclast Differentiation Factor (ODF), and more importantly, O steoprotegerin(OPG)-Ligand. OPG, a molecule also belonging to the TNF family, was identified as a secreted inhibitor of OC differentiation in vivo and in vitro (Simonet et al., 1997) (Figures 6,7). It was not before several studies in 1998 that the antagonist of OPG was found to be the immune cytokine RANKL [START_REF] Fuller | TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts[END_REF][START_REF] Lacey | Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation[END_REF][START_REF] Yasuda | Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL[END_REF]. RANKL and its receptor RANK were therefore established as important for the development of the immune system (e.g. lymph nodes) and the bone tissue [START_REF] Dougall | RANK is essential for osteoclast and lymph node development[END_REF]. In bone, secreted OPG acts as a decoy receptor for RANKL thus blocking its binding to RANK [START_REF] Simonet | Osteoprotegerin: a novel secreted protein involved in the regulation of bone density[END_REF][START_REF] Yasuda | Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL[END_REF] (Figures 6,7). RANKL is synthesized by OBs, stromal cells, hypertrophic chondrocytes, Band T-lymphocytes, osteocytes and even OCs themselves. Upon its synthesis, RANKL is relocated at the membrane of the cell and can be cleaved by proteases such as TNF-α convertase (TACE), Matrix Metalloprotease-14 (MMP-14) and A Disintegrin And Metalloprotease family member 10 (ADAM-10) [START_REF] Hikita | Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand[END_REF]. Its most functionally dominant role is when it remains at the surface of source cells and acts through cell-cell contact. Although OBs had been considered for many years as the primary physiological source of RANKL to OCs, two distinct studies using mouse genetic models recently described OYs as the major source of RANKL needed for OC differentiation (Nakashima and Takayanagi, 2011b;[START_REF] Xiong | Matrix-embedded cells control osteoclast formation[END_REF]. It is currently claimed that OYs are the major source of OPG as well [START_REF] O'brien | Osteocyte control of osteoclastogenesis[END_REF]. The ratio of RANKL/OPG is therefore crucial for bone resorption. An interesting challenge in the future of bone research would be to understand why and how the same cells, OYs, induce and inhibit OC differentiation and activity.

Because RANK lacks intrinsic enzymatic activity in its intracellular domain, after it is bound by RANKL, it recruits adaptor molecules such as TRAF family proteins, namely TRAF6 which transduces NF-κB and mitogen-activated kinases (MAPKs) pathways that include Jun p38 and N-terminal kinase (JNK), respectively [START_REF] Matsumoto | Essential role of p38 mitogen-activated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1[END_REF]Vernejoul and Marie, 2008) (Figures 5,6). RANK thereby activates several key transcription factors including AP-1 through its component c-fos and NFATc1 through NF-κB and c-fos signaling (Figure 6).

Genetic depletions is these key signaling molecules in osteoclastogenesis such as RANKL -/-, RANK -/-, TRAF6 -/-and c-fos -/-lead to osteopetrosis in mice due to the absence of differentiated OCs [START_REF] Grigoriadis | c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling[END_REF][START_REF] Dougall | RANK is essential for osteoclast and lymph node development[END_REF][START_REF] Kong | OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymphnode organogenesis[END_REF][START_REF] Lomaga | TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling[END_REF]. Conversely, OPG -/-mice are osteoporotic due to lack of OC activity [START_REF] Bucay | osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification[END_REF][START_REF] Mizuno | Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin[END_REF]. Given the central role of RANKL, a monoclonal antibody targeting it, Denosumab, is now clinically administered for the treatment of several bone pathologies such as rheumatoid arthritis, giant cell tumor of bone, … [START_REF] Dempster | Role of RANK ligand and denosumab, a targeted RANK ligand inhibitor, in bone health and osteoporosis: a review of preclinical and clinical data[END_REF]. ODF/RANKL and IL-1α through RANK and type 1 IL-1 receptor, respectively. Common signaling cascades such as NF-κB and JNK activation may be involved in the differentiation and activation of osteoclasts induced by TNF-α, ODF/RANKL, and IL-1α [START_REF] Kobayashi | Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction[END_REF].

II.3.3. TNF-α/IL-1

TNF-α is the predominant inflammatory cytokine in immuno-mediated pathological bone resorption. It can be secreted along with RANKL by activated lymphocytes, synovial fibroblasts and macrophages in the vicinity of OCs. This cytokine can induce osteoclastogenesis in two manners i.e. directly, by acting on OCs, or, indirectly by stimulating proximal cells to express RANKL, IL-1 and IL-1R [START_REF] Wei | IL-1 mediates TNF-induced osteoclastogenesis[END_REF]. Once secreted, both TNF-α and IL-1 are potent inducers of OC differentiation through their respective receptors on the surface of OC precursors. TNF-α has two cell surface receptors, TNFR-I and TNFR-II, which are expressed in various cell types including HSCs [START_REF] Kobayashi | Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction[END_REF] (Figure 5).

When TNFR-I is bound, it activates the TRADD/TRAF pathway leading to a regulation of caspase-mediated apoptosis. When TNFR II is bound, it interacts with TRAFs 2, 5 and 6 resulting in the activations of NF-κB and JNK pathways (Figure 5). Due to its ability to overlap RANKL/RANK signaling, TNF-α potently induces osteoclastogenesis [START_REF] Kobayashi | Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction[END_REF].

Like TNF-α, IL-1 is also secreted by macrophages and stromal cells and present at bone erosion sites linked with inflammation, e.g. rheumatoid arthritis. Since IL-1 and its receptor, IL-1R, expression by stromal cells and macrophages in the vicinity of OCs is promoted by TNF-α, IL-1 is considered downstream of TNF-α in this signaling interplay. When IL-1 binds to its receptor IL-1R, its leads to the activation of the TRAF6-dependent NF-κB and JNK pathways via MyD88 and IRAK recruitment [START_REF] Kwan Tat | IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology[END_REF]) (Figure 5). Due to the overlapping of their signaling with that of RANKL, TNF-α and/or IL-1R have osteoclastogenic effects that are synergetic with RANKL [START_REF] Thomson | Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption[END_REF][START_REF] Konig | Tumor necrosis factor alpha and interleukin-1 stimulate bone resorption in vivo as measured by urinary [3H]tetracycline excretion from prelabeled mice[END_REF] (Figure 5) but a minimal level of RANKL is still required for osteoclastogenesis in vivo and in vitro [START_REF] Kwan Tat | IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology[END_REF].

It is now clear that the signals induced by the pro-and anti-osteoclastogenic cytokines modulate a variety of complex intracellular processes that will determine the fate of the precursors. Once vital process that is controlled by these cytokines is the expression and activity of certain transcription factors during OC differentiation.

II.4. Transcription factors involved in OC differentiation and function

The differentiation of HSCs all the way to OCs is comprised of sequential stages that are tightly regulated by several transcription factors. For example, PU.1 and PAX5 are "early" factors responsible for steering HSCs either into CMPs or CLPs, respectively. Other factors such as MITF, NF-κB and NFATc1 control later stages of osteoclastogenesis and lymphopoiesis.

II.4.1. PU.1

PU.1 is a member of the ETS transcription factor family characterized by its DNA-binding domain ETS. PU.1 -/-mice, in addition to suffering from a lethal immune deficiency linked to the absence of B-lymphocytes and macrophages, are also osteopetrotic and devoid of OCs [START_REF] Tondravi | Osteopetrosis in mice lacking haematopoietic transcription factor PU.1[END_REF]. PUI.1 regulates therefore both the lymphoid and myeloid lineages, mainly through its expression levels (Figure 7). In other words, low levels of this transcription factor in HSCs lead to their differentiation into CLPs and high levels leads to macrophages, that can differentiate into OCs in theory, all the while inhibiting B-cell transition [START_REF] Dekoter | Regulation of B lymphocyte and macrophage development by graded expression of PU.1[END_REF].

II.4.2. PAX5

PAX5 is a member of a large family of transcription factors that are highly conserved during evolution and responsible for development and morphogenesis. PAX5 -/-mice do not have mature B-lymphocytes and therefore die a few days post-birth. Additionally, these mice exhibit a sever increase in OCs numbers and are osteopenic (i.e. with low bone mass but still higher than in osteoporosis) [START_REF] Horowitz | Pax5-deficient mice exhibit early onset osteopenia with increased osteoclast progenitors[END_REF]. Interestingly, the PAX5 gene encodes the protein B-cell-lineage-specific activator protein (BSAP) that is expressed only in Blymphopoietic lineage, downstream of PU.1; it is undetected in OBs and OCs. The ablation of PAX5 in mice results in the expansion of spleen-derived c-fms + Mac-1 + FcRγ + B220 - macrophage precursors that are highly osteoclastogenic. This population would be otherwise repressed by PAX5 expression [START_REF] Horowitz | Pax5-deficient mice exhibit early onset osteopenia with increased osteoclast progenitors[END_REF]Horowitz and Lorenzo, 2007a, b).

PAX5 has also been shown to repress other lineage-specific genes such as Flt3 (required for multipotent B-cell progenitors), c-fms (OcMDCs) and Notch 1 (T-cells) [START_REF] Nutt | Commitment to the B-lymphoid lineage depends on the transcription factor Pax5[END_REF][START_REF] Souabni | Pax5 promotes B lymphopoiesis and blocks T cell development by repressing Notch1[END_REF][START_REF] Holmes | Repression of Flt3 by Pax5 is crucial for Bcell lineage commitment[END_REF]. Given these properties, PAX5 is now considered to be an "early" repressor of osteoclastogenesis.

II.4.3. MITF

Microphtalmia-associated transcription factor (MITF) is the most characterized member of the MIT family of transcription factors. An important functional aspect of MITF activity during osteoclastogenesis lies in its cooperation with other transcription factors to promote the expression of proteinase, essential molecules for bone degradation [START_REF] Luchin | The microphthalmia transcription factor regulates expression of the tartrate-resistant acid phosphatase gene during terminal differentiation of osteoclasts[END_REF]. By interacting with PU.1, MITF can promote the expression of the TRAP gene, a protease that is critical for bone resorption [START_REF] Luchin | The microphthalmia transcription factor regulates expression of the tartrate-resistant acid phosphatase gene during terminal differentiation of osteoclasts[END_REF] (Figure 7. MITF also associates with TFE3 to drive Cathepsin K expression [START_REF] Motyckova | Linking osteopetrosis and pycnodysostosis: regulation of cathepsin K expression by the microphthalmia transcription factor family[END_REF]. The spontaneous ophthalmic mutation "mi" in the DNA-binding domain of MITF1 results in the blockade of expression of its target genes (Mansky et al., 2002a;Mansky et al., 2002b). Consequently, mi/mi mice are osteopetrotic and do not form OCs. Interestingly, RANKL is overexpressed in the bone marrow of these mice suggesting that MITF participates in the negative autoregulation of RANKL [START_REF] Tagaya | Intramedullary and extramedullary B lymphopoiesis in osteopetrotic mice[END_REF]. B-lymphopoiesis in these mice is affected in vivo but not in vitro and it has been suggested that osteopetrosis could be responsible for this in vivo phenotype by reducing the available space within the bone medulla [START_REF] Franzoso | Requirement for NF-kappaB in osteoclast and B-cell development[END_REF]) [START_REF] Tagaya | Intramedullary and extramedullary B lymphopoiesis in osteopetrotic mice[END_REF].

II.4.4. NF-κB

Transcription factors of the NF-κB/Rel family are ubiquitously expressed and include 5 members in mammals: p65 (RelA), c-Rel, Rel-B, NF-κB1 (p50/p105) and NF-κB2 (p52/p100). To be functional, these members form homo-or heterodimers translocate from the cytoplasm to the nucleus where they regulate transcription on chromatin sites. Many murine knockout models have shown the importance of NF-κB family in OC differentiation [START_REF] Franzoso | Requirement for NF-kappaB in osteoclast and B-cell development[END_REF]. For example, the expression of p50 and p52 is required in OC but not macrophages, indicating that NF-κB acts on precursors that are already specifically committed to osteoclastogenesis, i.e. downstream of PU.1 (Yamashita et al., 2007b) (Figures 567).

II.4.5. c-Fos and AP-1 complex

Along with Fra1, Fra2 and FosB, c-Fos is part of the Fos family genes. Its viral homologue, v-Fos, induces osteosarcoma in mice [START_REF] Matsuo | Osteoclasts, mononuclear phagocytes, and c-Fos: new insight into osteoimmunology[END_REF]. Fos family members each carry the highly conserved "basic leucine zipper" structure responsible for DNA-binding and for heterodimerization, typically with Jun proteins. This dimerization results in the transcription complex called Activator Protein 1 (AP-1) (Figure 6). The only component of AP-1 so far demonstrated as indispensable for OC formation is c-Fos. Mice lacking c-Fos have all suffered from impaired osteoclastogenesis coupled with osteopetrosis [START_REF] Johnson | Pleiotropic effects of a null mutation in the c-fos proto-oncogene[END_REF][START_REF] Wang | Bone and haematopoietic defects in mice lacking c-fos[END_REF][START_REF] Grigoriadis | c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling[END_REF], thus revealing the importance of this gene in the myeloid lineage. Although c-Fos depletion can be rescued in vivo and in vitro by exogenous expression of its family member Fra1, the endogenous expression of the latter is c-Fos-dependent. Furthermore, c-fos induced the expression of another OC-differentiating transcription factors, NFATc1 (see below, section II.4.6. NFATc1). RANKL and Ca 2+- signaling are both known to activate c-fos (Figure 6) while IFN-γ is known for its inhibition [START_REF] Takayanagi | T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma[END_REF]. In addition, but both c-fos and NFAT can interact at the protein level and cooperatively bind to DNA to launch transcription at promoter sites [START_REF] Matsuo | Osteoclasts, mononuclear phagocytes, and c-Fos: new insight into osteoimmunology[END_REF].

II.4.6. NFATc1

Transcription factors of the Nuclear factor of activated T cells (NFAT) family have been shown to regulate a diverse set of functions in several tissues and the immune system. This family is comprised of five members: NFAT1 (NFATc2), NFAT2 (NFATc1), NFAT3 (NFATc4), NFAT4 (NFATc3) and NFAT5. NFAT1, 2, 3 and 4 are regulated by calcineurine/calcium signaling [START_REF] Takayanagi | The role of NFAT in osteoclast formation[END_REF]. They are activated when engaged by receptor adaptors linked with calcium signaling such as FcRγ and KARAP/DAP12 of the Immunoreceptor Tyrosine-based Activation Motif (ITAM) family. The engagement of these receptors leads to the activation of phospholipase C-γ (PLC-γ) which in turn liberates calcium from the endoplasmic reticulum and thus allows its binding to calmodulin. Ca 2+ -bound calmodulin thus activates calcineurin, a serine-threonine phosphatase, which relocates NFAT to the nucleus (Figure 7) [START_REF] Vernejoul | Trait√© des maladies m√©taboliques osseuses de l'adulte[END_REF]. The importance of NFATc1 to osteoclastogenesis was first highlighted by Takayanagi and colleagues in the course of a highthroughput study of RANKL-induced genes in OCs (Takayanagi et al., 2002a). They found that this gene was the most highly induced 24 to 72 hours post-induction with RANKL.

Additionally, NFATc1 -/-myeloid progenitors could not differentiate into OCs. RANKL acts on NFATc1 in two distinct mechanisms during this process. First, RANKL induces its expression via c-fos and TRAF6 (Figures 6,7) [START_REF] Takayanagi | RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-beta[END_REF]. Indeed, in c-fos -/- and TRAF6 -/-OCs, NFATc1 is barely induced. Second, RANKL produces calcium oscillation thus resulting in calcineurin-dependent NFATc1 sequestering in the nucleus [START_REF] Takayanagi | The role of NFAT in osteoclast formation[END_REF][START_REF] Negishi-Koga | Ca2+-NFATc1 signaling is an essential axis of osteoclast differentiation[END_REF]. The chemical inhibition of calcineurin in murine bone marrow OCMDCs not only blocks this nuclear translocation but also inhibits NFATc1 mRNA transcription, indicating that NFATc1 is responsible for its auto-amplification [START_REF] Takayanagi | The role of NFAT in osteoclast formation[END_REF]. NFATc1 is translocated to the nucleus after the dephosphorylation by calcineurin that is activated by calcium (Ca 2+ ) signaling. Ca 2+ /calmodulin kinases IV is a main kinase that activates cAMP response element-binding protein (CREB), which is also important for osteoclast differentiation. Induction of c-Fos is partly mediated by CREB. In the nucleus, NFATc1 works together with other transcription factors, such as AP-1, PU.1, MITF, and CREB, to induce various osteoclast-specific genes [START_REF] Takayanagi | The role of NFAT in osteoclast formation[END_REF].

(OSCAR, TREM, …)

NFATc1 is the master regulator of osteoclastogenesis. Its central role is supported by the fact that its overexpression in c-Fos -/-mice rescues the defect in osteoclastogenesis (Matsuo K 2004 JBC). In OCs, the calcium flux needed for NFATc1 activation is mediated by the phosphorylation of the ITAM adaptors DAP12 and FcRγ (Takayanagi et al., 2002a;[START_REF] Koga | Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis[END_REF][START_REF] Despars | DAP12 Overexpression Induces Osteopenia and Impaired Early Hematopoiesis[END_REF]. This pathway is synergetic with RANKL/RANK signaling and the two converge in a signaling complex that contains phosphorylated adaptors (BLNK and SLP76) and Tec family kinases, downstream of FcRγ and RANK, respectively. This complex is responsible for the induction and activation of NFATc1 [START_REF] Ikeda | Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation[END_REF][START_REF] Koga | Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis[END_REF]Matsuo et al., 2004). However, NFATc1 expression has to be tightly tuned to avoid excessive osteoclastogenesis. Indeed, during the early stages of differentiation, strong repression is exerted on the promoter of NFATc1 by negative regulators of osteoclastogenesis such as IRF8, MafB and Bcl6. But as differentiation develops, these negative regulators are blocked by Blimp-1 thus allowing NFATc1 expression [START_REF] Nishikawa | Blimp1-mediated repression of negative regulators is required for osteoclast differentiation[END_REF]. Additionally, Leukemia Related Factor (LRF) negatively regulates NFATc1 in the early phase of differentiation but, as differentiation proceeds, its expression level increases and it becomes a positive co-factor of NFATc1 [START_REF] Tsuji-Takechi | Stage-specific functions of leukemia/lymphoma-related factor (LRF) in the transcriptional control of osteoclast development[END_REF].

Once activated, NFATc1 directly regulates a number of OC-specific genes. Using promoter analyses and chromatin immunoprecipitation techniques, it has been shown that TRAP, calcitonin receptor (Takayanagi et al., 2002a), cathepsin K [START_REF] Matsumoto | Essential role of p38 mitogen-activated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1[END_REF], β3

integrin [START_REF] Crotti | NFATc1 regulation of the human beta3 integrin promoter in osteoclast differentiation[END_REF] and OSCAR (Kim et al., 2005a) expression are NFATc1dependent in OCs (Figure 6). The AP-1 complex (previously described in the c-Fos paragraph) has been shown in some cases to be the DNA-binding partner of NFATc1. Indeed, AP-1 and NFATc1 co-binding is required for transcription of TRAP and calcitonin receptor genes as well as the autoamplification of NFATc1 (Yamashita et al., 2007a). NFATc1 can also cooperate with PU.1 and MITF in the effect on the cathepsin K and OSCAR promoters [START_REF] Matsumoto | Essential role of p38 mitogen-activated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1[END_REF]. Both PU.1 and MITF, which had been previously suspected as only relevant to the survival of early OC precursors, are therefore also important in the induction of late-stage differentiation of OCs. Together with NFATc1, these transcription factors make an OC-specific gene induction complex. This cooperation between NFATc1 and either PU.1 or MITF is however not always observed (Figure 7), such as in the case of the calcitonin receptor promoter (Kim et al., 2005a), suggesting that the component variability of this complex can contribute to gene-specific spatiotemporal regulation of transcription [START_REF] Takayanagi | The role of NFAT in osteoclast formation[END_REF]. Finally, it is worth noting that despite its unambiguous importance during osteoclastogenesis, NFATc1 is not the only member of the NFAT family to intervene during this process. NFATc2 has also been shown to be important in this context [START_REF] Ikeda | Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation[END_REF].

In conclusion of sections II.1 and II.2, it is worth noting that, although a full "molecular map" of the elements regulating OC differentiation is not yet available, serious advancements have allowed the establishment of a working model that depicts how the most essential cytokines, transcription factors and OC markers intervene during osteoclastogenesis (Figure 7). receptor; DC-STAMP, dendritic cell-specific transmembrane protein; ECM, extracellular matrix; GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; MITF, microphthalmia-associated transcription factor; OPG, osteoprotegerin; TLR, Toll-like receptor [START_REF] Zhao | Negative regulation of osteoclastogenesis and bone resorption by cytokines and transcriptional repressors[END_REF].

II.5. OC precursor fusion

As a result of stimulation by RANKL, RANK + OC monocytic precursors undergo cell-cell fusion to form giant multinucleated bone-resorbing cells. Why functional OCs need be multinucleated to efficiently degrade bone is not understood. It is widely hypothesized that mononucleated RANK + TRAP + cells are not efficient in bone resorption but there is no evidence neither supporting nor disproving this hypothesis in mammals. In teleost fish, for example, the majority of resorbing OCs is mononucleated. However, several drastic differences in the bone and immune systems exist between of mammals and fishes, including the absence of haematopoietic bone marrow and of OYs [START_REF] Witten | A comparative view on mechanisms and functions of skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function[END_REF].

Usually, bone tissue sections in mammals as well as ex vivo differentiated OCs simply show that OCs cells resorb bone in their multinucleated state. As for the precursor fusion process, extensive videomicroscopy of OCs differentiating in culture performed in our lab over the past 10 years has made it quite clear that fusion is not a random process. Prior to fusion, cells seem to select their fusing partner and to repulse other cells after a cell-cell contacts that is maintained for a certain period of time (minutes to hours) before a decision is made.

Whilst M-CSF is responsible for the proliferation, survival and early steering of myeloid precursors into the osteoclast pathway, RANKL is the main cytokine controlling cell-cell fusion and terminal osteoclastogenesis. RANKL signaling initiates a cascade of gene expression that includes the production of chemokines such as RANTES as well as MCP-1 and its two receptors CCR2 and CCR4. These are chemotactic molecules able to signal to monocytes (Kim et al., 2005b). Chemokine-mediated fusion is thought to transduce RANK - quiescent precursors to fuse with RANK + ones. In the absence of RANKL signaling, MCP-1 has been shown to stimulate TRAP and CTR by inducing NFATc1 in rheumatoid arthritis, but the resulting multinucleated cells did not resorb bone efficiently [START_REF] Kim | MCP-1-induced human osteoclast-like cells are tartrate-resistant acid phosphatase, NFATc1, and calcitonin receptor-positive but require receptor activator of NFkappaB ligand for bone resorption[END_REF]. This proves the presence of a more important RANKL-mediated mechanism of fusion: activating NFATc1 to bind to promoter region and activated the expression of DC-STAMP and ATP6V0D2 [START_REF] Lee | v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation[END_REF]. DC-STAMP-expression at the surface of OC precursors is sufficient to make them fuse with DC-STAMP -precursors. In that manner DC-STAMP expression is passed on from a single cell to a binucleated cell which in turn can recruit another DC-STAMP -mononucleated precursor, etc… (Vignery, 2005). DC-STAMP -/-and ATP6V0D2 -/-mice are osteopetrotic as a result of perturbed OC fusion leading to absence of OCs [START_REF] Lee | v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation[END_REF]. The genetic depletion of these fusion genes highlights the importance of fusion to bone resorption activity.

When differentiated ex vivo in culture-treated dishes, fully differentiated murine marrow-or spleen-derived OCs in the presence of M-CSF and RANKL can contain up to 100 nuclei per cell. However, a much lower number of nuclei is seen in murine OCs in vivo (histological section of bone tissue) or when these murine precursors are differentiated on bone (Jurdic lab, unpublished data). This puts forth the possibility of a matrix-dependent control as well as other possible physiological limitations of fusion. This discrepancy of nuclei numbers between in vivo and in vitro is not observed in human osteoclasts.

II.5. Matrix-cytokine converging pathways in osteoclastogenesis

The priming of myeloid precursors, their fusion and their homing in the bone microenvironment is accompanied by costimulatory signals from the bone matrix. Indeed, matrix proteins are recognized by activated surface receptors of differentiating OCs and contribute to (1) the differentiation process and (2) to the launching of degradation-related functions once OCs are fully differentiated. The second step is also known as OC maturation.

II.5.1. OSCAR-RANKL axis:

RANKL induces for the expression of an OC-specific member of the Leukocyte Receptor Complex (LRC) protein family: OSCAR [START_REF] Kim | A novel member of the leukocyte receptor complex regulates osteoclast differentiation[END_REF]. This immunoglobulin-like receptor is expressed in OC precursors and mature OCs in humans in mice and recognizes collagens I, II and III [START_REF] Barrow | OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice[END_REF]. Although collagen I is the predominant collagen in bone, collagen III is, along with collagen I, expressed by OBs and by RANKL + endothelial cells of the microvasculature [START_REF] Barrow | OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice[END_REF]. The matrix-independent role of OSCAR is to co-stimulate RANKL signaling through FcRγ, a member of the ITAM adaptor family (Figure 5). Another role of OSCAR has also exists. By binding to specific helical motifs in these collagens, OSCAR can potently induce osteoclastogenesis independently ITAM adaptor family members such as FcRγ and DAP12 [START_REF] Kim | A novel member of the leukocyte receptor complex regulates osteoclast differentiation[END_REF]. In fact, OSCAR overexpression was shown to recue the osteoclastogenesis defect in DAP12 -/-mice and OC differentiation ex vivo from humans suffering from DAP12 mutations [START_REF] Barrow | OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice[END_REF]. In conclusion,

given the ability of OSCAR to recognize collagen and its high osteoclastogenic potential in synergy with activate RANK, it is hypothesized that OSCAR + RANK + OC precursors can therefore be properly directed along RANKL + vessels ending in the bone microenvironment, then docked on collagen I/III + bone surface cells and naked collagen I + bone sites to reach degradation-requiring destinations [START_REF] Barrow | OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice[END_REF]. The complete molecular mechanism corresponding to matrix-dependent OSCAR signaling is not yet revealed.

II.5.2. Integrin-RANK and Integrin-MCSF axes:

The αV family of integrins recognizes the Arg-Gly-Asp (RGD) amino-acid motif that is present in several bone matrix proteins such as bone sialoproteins and osteopontin. Integrins are transmembrane receptors have a long extracellular domain and a shorter intracellular domain [START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF]. This allows them not only to bind and transmit signals from the extracellular matrix into the cell, therefore accomplishing what is called "outside-in"

signaling, but also to be modulated by signals within the cell, a process called "inside-out signaling" [START_REF] Hynes | Integrins: bidirectional, allosteric signaling machines[END_REF]. Integrins are differentially expressed by OCs along their differentiation: αVβ5 is initially expressed in M-CSF-stimulated bone marrow macrophages but its expression is lost in favor of αVβ3 after exposure to RANKL [START_REF] Lakkakorpi | Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells[END_REF][START_REF] Inoue | Granulocyte macrophage-colony stimulating factor reciprocally regulates alphav-associated integrins on murine osteoclast precursors[END_REF][START_REF] Lane | Mice lacking the integrin beta5 subunit have accelerated osteoclast maturation and increased activity in the estrogen-deficient state[END_REF][START_REF] Teitelbaum | Osteoclasts: what do they do and how do they do it?[END_REF]. OCs also express α2β1, αVβ1 and α9β1 but αVβ3 is the most abundant and therefore the principal integrin in OCs [START_REF] Rao | Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function[END_REF]. It is currently admitted that αVβ3 has 2 distinct functions during OC differentiation and maturation: (1) c-fms-dependent stimulation of osteoclastogenesis and (2) RANK-dependent cytoskeletal organization [START_REF] Teitelbaum | Osteoclasts: what do they do and how do they do it?[END_REF].

In mice lacking the β3 subunit, the resorptive capacity is abolished due to defects in OC cytoskeleton and cell spreading [START_REF] Mchugh | Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts[END_REF]. The cytoskeleton-specific interplay between αVβ3 and RANK is, in fact, mediated by another relevant molecule to actin arrangement: c-Src. In RANKL-stimulated OCs, αVβ3 and activated RANK coprecipitate in vitro uniquely in the presence of c-Src. The latter binds ligand-linked RANK via its SH2 domain and αVβ3 via SH3 domain, thus providing a possible link between these two receptors [START_REF] Teitelbaum | Osteoclasts: what do they do and how do they do it?[END_REF]. More detailed descriptions of the role of integrins and Src in OC cytoskeletal arrangement are available in section Molecular studies, however, have shown that the importance of αVβ3 during OC differentiation is actually mediated by M-CSF and its receptor c-Fms. In β3 -/-mice, OC numbers are increased in vivo although osteoclastogenesis is attenuated ex vivo.

Physiologically, mice have low circulating Ca 2+ and they suffer from osteosclerosis (i.e.

increased bone density) with age [START_REF] Mchugh | Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts[END_REF]. Having hypothesized that differential cytokine conditions between the two environments, i.e. between real bone marrow and experimental tissue culture, could be responsible for this paradox, Faccio and colleagues have demonstrated that high doses of M-CSF, but not RANKL, recues β3 -/-osteoclastogenesis in ex vivo bone marrow cultures (Faccio et al., 2003b). The authors have proposed a molecular mechanism through which c-Fms occupancy and subsequent phosphorylation on Y697 are responsible for ERK and c-fos upregulation and, ultimately, stimulation of differentiation (Faccio et al., 2003b).

II.5.3. Is adhesion required for differentiation?

Osteoclastogenesis seems to integrate signals from specific cytokines and from the matrix via integrins and other receptors. These have an unequivocal role during the bone resorption process in mature OCs, but the extent of their contribution to OC differentiation is still controversial. A study by Mochizuki and colleagues has shown that the culture of OC precursors in the presence of M-CSF and RANKL on a 2D non-adherent substrate markedly reduces RANK expression and OC differentiation efficiency [START_REF] Mochizuki | Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors[END_REF]. These precursors adopt macrophage instead of OC characteristics, thus suggesting that the adhesion of precursors to the substratum in required for osteoclastogenesis. Even more, the chemical inhibition of integrins with an RGD-containing disintegrin in OC precursors also reduces RANK expression and OC differentiation [START_REF] Mochizuki | Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors[END_REF]. The importance of adhesion, but not integrin-mediated signaling per se, in osteoclastogenesis has been confirmed by a more recent study [START_REF] Touaitahuata | Podosomes are dispensable for osteoclast differentiation and migration[END_REF]. The role of CD44, a nonintegrin OC receptor, in OC differentiation [START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF] has not yet been addressed.

Starting from the earliest stages of their differentiation, OCs precursors exhibit at their ventral surface integrin-based, actin-rich adhesive puncta called "podosomes" that are thought to maintain adhesion, cell spreading and migration in a wide range of cells (Linder et al., 2011a) [START_REF] Jurdic | Podosome and sealing zone: specificity of the osteoclast model[END_REF]. Whether podosomes are required for OC precursor fusion and differentiation in vitro is controversial. Touaitahuata and colleagues have argued that integrin signaling, assayed by podosome formation, is needed neither for fusion nor differentiation [START_REF] Touaitahuata | Podosomes are dispensable for osteoclast differentiation and migration[END_REF]. Additionally, these same authors also concluded that podosomes are dispensable for OCs migration and, ultimately, differentiation [START_REF] Touaitahuata | Podosomes are dispensable for osteoclast differentiation and migration[END_REF].

These findings are in contradiction with detailed molecular and imaging data provided by

Oikawa and colleagues depicting a central role of podosomes in OC precursor fusion [START_REF] Oikawa | Tks5dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion[END_REF]. The data showed that podosome organization at the circumference of OC precursors maintains cell spreading and thus sustains the contact of plasma membranes of apposed cells during fusion. Oikawa and colleagues provided a molecular mechanism in which the scaffold protein Tks5, known for its role in podosome formation, is activated by phosphoinositide 3-kinase and Src kinase and thus promotes podosome-mediated cell-cell contact and fusion [START_REF] Oikawa | Tks5dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion[END_REF] In order to give rise to mature multinucleated cells in vivo, OC precursors need to migrate before making cell-cell contacts and finally fusing together. There is strong evidence pointing towards mononucleated precursor recruitment to the bone and formation of mature multinucleated OCs on the bone surface. What is unknown is whether in vivo the fusion occurs within the bone marrow (i.e. in a 3D context, before reaching the bone matrix) or after reaching the bone matrix (i.e. in a 2D context). If the first case is true, it would then be possible that OC precursors can dispense of podosomes even though, macrophages, which derive from the same OC precursors and also form podosomes, are capable of exhibiting podosome-like structures in a 3D matrix [START_REF] Van Goethem | Macrophage podosomes go 3D[END_REF]. If the second case is true, podosome are likely to be important for migration and fusion because OCs precursors spontaneously make podosomes on bone and podosomes are the principal cytoskeletal structures that drive OC migration [START_REF] Jurdic | Podosome and sealing zone: specificity of the osteoclast model[END_REF][START_REF] Saltel | Actin cytoskeletal organisation in osteoclasts: a model to decipher transmigration and matrix degradation[END_REF].

Finally, matrix receptors such as integrins are, in some cases, directly involved in cell-cell interaction [START_REF] Ojakian | Integrin regulation of cell-cell adhesion during epithelial tubule formation[END_REF]. Hence, cell-cell contact is a factor that should be considered alongside adhesion and migration in order to better understand OC differentiation and fusion [START_REF] Oikawa | Tks5dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion[END_REF]. In in vitro osteoclastogenesis assays, while low seeding densities of mononucleated precursors can challenge their migration by reducing the probability of passive cell-cell contacts, a relatively high density can trigger cell aggregation and, consequently, both surface expression of αVβ3 in these precursors [START_REF] Boissy | Aggregation of mononucleated precursors triggers cell surface expression of alphavbeta3 integrin, essential to formation of osteoclast-like multinucleated cells[END_REF] as well as podosome-maintained membrane contacts [START_REF] Oikawa | Tks5dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion[END_REF]. It would therefore be interesting to investigate the balance between matrix-dependent adhesion, cell-cell adhesion and migration on different substrates: adherent and non-adherent, 2D and 3D.

As a result all the available stimuli and activated pathways that are required for their differentiation, OCs become giant multinucleated cells and acquire the proper molecular arsenal allowing them to reach and degrade bone. How they use their arsenal to fulfill their destined function will the topic of the following section III.

III. PODOSOME ORGANIZATION IN OSTEOCLASTS

At very early stages of their differentiation in vitro, OCs form integrin-based and actin-rich microscopic adhesive structures called podosomes. Although the existence of podosomes has not yet been demonstrated in vivo due to technological limitations of imaging methods, their formation in OCs seeded on their natural substrate, i.e. bone, has been documented by electronic microscopy and fluorescence microscopy [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. Hence, their formation in vivo is highly plausible. Podosomes are circular structures that are around 1µmwide and that rise from the plasma membrane into the cytoplasm reaching a height of around 0.6 µm [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF][START_REF] Labernadie | Dynamics of podosome stiffness revealed by atomic force microscopy[END_REF]Linder et al., 2011b) (Figures 9,10).

They are essential for OC adhesion, spreading, migration and bone degradation. In fact, genetic depletions of their components or regulatory proteins in mice have serious consequences on the bone phenotype through disruption of OC structures.

III.1. General principles of integrin activation

Under cytokine stimulation, specific transcription factors activated in differentiating OCs stimulate the expression of proteins required for mature OC functions. Then, signals from cytokines and substrate simultaneously orchestrate the adhesive and cytoskeletal proteins into functional structures such as podosomes.

Integrins undergo large-scale conformational changes in order to attain a high-affinity configuration during the process of integrin activation [START_REF] Paradise | Acidic extracellular pH promotes activation of integrin alpha(v)beta(3)[END_REF]. These receptors are currently understood to exist in equilibrium among three main conformational states (Figure 8). In the low-affinity state, the extracellular leg domains are bent and the headpiece is closed, with an acute angle between the I-like and hybrid domains. This conformation generally exhibits little to no binding to biological ligands but can bind to small RGD peptides in solution. In the high-affinity conformation, the leg domains are extended and separated, and the headpiece is open [START_REF] Paradise | Acidic extracellular pH promotes activation of integrin alpha(v)beta(3)[END_REF].

The third conformation, with extended legs and a closed headpiece, is expected to be of intermediate affinity [START_REF] Xiong | Crystal structure of the extracellular segment of integrin alpha Vbeta3[END_REF][START_REF] Takagi | Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling[END_REF][START_REF] Xiong | Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand[END_REF][START_REF] Paradise | Acidic extracellular pH promotes activation of integrin alpha(v)beta(3)[END_REF] (Figure 8). Although the "bent" state has a low binding affinity to RGD motifs in biological ligands, it is the physiologically dominant form, in particular in the presence of Ca 2+ and Mg 2+ [START_REF] Takagi | Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling[END_REF][START_REF] Paradise | Acidic extracellular pH promotes activation of integrin alpha(v)beta(3)[END_REF] and is sufficient for podosome formation [START_REF] Faccio | Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts[END_REF]. However, both outside-in and inside-out signaling have been demonstrated capable of activation of the heterodimer [START_REF] Takagi | Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling[END_REF] (Figure 8).

Indeed, the presence of MCSF, responsible for the inside-out activation of αVβ3 through the cytoplasmic tail of β3, can induce active αVβ3 enrichment at podosome sites (Faccio et al., 2003a). Importance of integrins in OCs has been realized through the extensive studies of αvβ3 integrin and the matrix-degradation defects observed in Glanzmann patients or mice carrying null mutations in the β3 integrin gene [START_REF] Gluckman | Pediatric bone marrow transplantation for leukemia and aplastic anemia. Report of 222 cases transplanted in a single center[END_REF][START_REF] Hodivala-Dilke | Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival[END_REF][START_REF] Mchugh | Mice lacking beta3 integrins are osteosclerotic because of dysfunctional osteoclasts[END_REF]. The reduced resorptive activity of β3-/-osteoclasts was thought to be caused by loss of αvb3-mediated signaling that regulates cell polarity and cytoskeletal reorganization (Faccio et al., 2003) [START_REF] Brunner | New insights into adhesion signaling in bone formation[END_REF]. Likely, other integrin and specifically β1 integrins are also involved in osteoclast function. Indeed, a recent report using β1-deficient osteoclast point to an important role for this integrin family in the formation of podosomes or invadopodia (Destaing et al., 2010;[START_REF] Schmidt | Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclastmediated bone resorption[END_REF]. Some studies of integrin expression on osteoclasts show that αv, α2, β1, and β3 subunits are found in osteoclasts from human bone tissue [START_REF] Nesbitt | Biochemical characterization of human osteoclast integrins. Osteoclasts express alpha v beta 3, alpha 2 beta 1, and alpha v beta 1 integrins[END_REF][START_REF] Brunner | New insights into adhesion signaling in bone formation[END_REF]. Deletion in mice of kindlin-3, an intracellular activator of integrins, leads to a severe osteoclast defect.

This mimicks osteopetrosis observed in several patients with mutations in the Kindlin-3 gene, due to a block into their resorptive capacities [START_REF] Schmidt | Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclastmediated bone resorption[END_REF]. It has shown that loss of αv, β1, and β3 subunits, through kindlin-3 inactivation, ends up in a more severe osteopetrotic phenotype than β3 mutants alone. Most of the role of integrins in osteoclasts is seen through their implication in adhesion and resorption through podosome organization [START_REF] Schmidt | Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclastmediated bone resorption[END_REF].

III.2. Recruitment of adhesion plaque molecules

Besides integrins, other structural proteins are recruited to build the podosome. These proteins are described as cytoskeletal adaptors that organize F-actin. They are called collectively "plaque proteins". One of these proteins is talin, which binds the cytodomain of β3 integtin subunit and also contributes to its inside-out activation [START_REF] Calderwood | Integrin activation[END_REF][START_REF] Paradise | Acidic extracellular pH promotes activation of integrin alpha(v)beta(3)[END_REF][START_REF] Zou | Talin1 and Rap1 are Critical for Osteoclast Function[END_REF]. Talin is an elongated antiparallel flexible dimer [START_REF] Winkler | Energy-filtered electron microscopy reveals that talin is a highly flexible protein composed of a series of globular domains[END_REF] capable of also binding F-actin and vinculin [START_REF] Papagrigoriou | Activation of a vinculin-binding site in the talin rod involves rearrangement of a five-helix bundle[END_REF]. In turn, vinculin is able to bind F-actin, α-actinin and paxillin [START_REF] Turner | Paxillin: a new vinculin-binding protein present in focal adhesions[END_REF][START_REF] Jockusch | Crosstalk between cell adhesion molecules: vinculin as a paradigm for regulation by conformation[END_REF].

The sequestration of these F-actin anchoring/docking proteins to several concentric integrin sites at the immediate vicinity of the plasma membrane, an interface usually called the "adhesion plaque", indicates that podosome assembly/growth occurs in a bottom-up direction.

Furthermore, the actin filaments anchored by these integrin-associated proteins are crosslinked by myosin II and α-actinin and organize into co-axial/radial segments linking concentric integrin sites [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. This peripheral region of the podosome is collectively called the cloud. At the centre of the podosome, the F-actin network is characterized by a higher order of density called the "podosome core" (Figures 9,10).

This high F-actin density in the podosome core is largely due to oriented and highly dynamic actin nucleation and branching machinery composed of cortactin [START_REF] Wu | Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actinbinding protein enriched in the cell cortex[END_REF]), the Arp2/3 complex, (Neuronal-)Wiskott Alrdich Syndrome Protein ((N-)WASP), WASPassociated Protein (WIP) [START_REF] Calle | Wiskott-Aldrich syndrome protein and the cytoskeletal dynamics of dendritic cells[END_REF][START_REF] Monypenny | Role of WASP in cell polarity and podosome dynamics of myeloid cells[END_REF]. The latter is necessary to the formation of the actin core for several reasons. First, WIP interacts and activates actin-organizing molecules (N)WASP, Nck and myosin [START_REF] Krzewski | Formation of a WIP-, WASp-, actin-, and myosin IIA-containing multiprotein complex in activated NK cells and its alteration by KIR inhibitory signaling[END_REF][START_REF] Anton | WASP-interacting protein (WIP): working in polymerisation and much more[END_REF][START_REF] Monypenny | Role of WASP in cell polarity and podosome dynamics of myeloid cells[END_REF]. WIP also protects WASP from proteasomal degradation (de la [START_REF] De La Fuente | WIP is a chaperone for Wiskott-Aldrich syndrome protein (WASP)[END_REF]. Second, by binding both to cortactin and actin monomers (G-actin), WIP recruits G-actin to cortactin-activated Arp2/3 nucleation sites proximal to the cytoplasmic membrane [START_REF] Kinley | Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion[END_REF] . The importance of WIP is visible through the cytoskeletal phenotype of WIP -/-murine OCs which produce podosomes that lack the core domain [START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF]. Because WIP colocalizes with the podosome core, this phenotype provides experimental proof that the podosome cloud forms independently of the podosome core.

III.3. The podosome subdomains

III.3.1. The podosome core defined by CD44

The study of WIP in OCs has underlined the importance of an unexpected podosome component: CD44. CD44 is a cell-surface transmembrane proteoglycan that binds to hyaluronic acid, collagen, osteopontin and laminin [START_REF] Goodison | CD44 cell adhesion molecules[END_REF]. The matrixdependent activation of CD44 can rescue podosome core formation in WIP -/-OCs and increases overall adhesion of these cells to their substrate. The need for outside-in activation of CD44 was additionally demonstrated by addition of activating antibodies and testing its podosome-rescuing capacity on different substrates [START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF]. Surprisingly, inside-out activation of this receptor has not been investigated so far and the detailed mechanism used by CD44 to support podosome core formation remains unclear. The nonoverlapping localizations and functions of αVβ3 and CD44 served as decisive proof for the distinction between the two podosome domains: the cloud and the core.

III.3.2. The podosome cloud defined by Src

In parallel to the core-specific function of CD44, the tyrosine kinase Src has podosome-cloud specific function. In fact, the Src -/-OCs exhibit podosomes with cores but without clouds [START_REF] Destaing | The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts[END_REF]. The SH2 or SH3 domains of Src are necessary for the docking of Src to the podosome cloud. This localization of Src can therefore allow it to exert its tyrosine kinase activity on podosomal proteins. Both the localization of Src and its enzymatic activity are essential for full podosome assembly [START_REF] Destaing | The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts[END_REF].

III.3.3. The podosome cap

Finally, a novel podosomal subdomain has been reported: the podosome "cap". The existence of this domain has been concluded from the localization of the formin FRL1 on top of the podosome core [START_REF] Mersich | The formin FRL1 (FMNL1) is an essential component of macrophage podosomes[END_REF]Linder et al., 2011a). Its potential role, however, is still to be elucidated. (Linder et al., 2011a).

III.4. Uncertainty about spatio-temporal order of podosome formation

The exact spatio-temporal order of recruitment of proteins to the site of nascent podosomes has not been fully dissected. In consequence, the chronological priority of cloud versus core formation during normal podosome assembly is still debated. A study made by Luxenburg and colleagues, however, suggested that the first step of podosome assembly is the recruitment of cloud-component paxillin followed by core-component cortactin (Luxenburg et al., 2012b). This step, which represents the formation of the adhesion plaque at the membrane-cytosol interface, is followed by the apparition of F-actin at the podosome core simultaneously with recruitment of α-actinin. β3 integrin would then be recruited to the podosome site. Although this data consolidates some aspects of the current podosome formation model, such as down-to-up assembly and inside-out activation of integrins, it raises a serious question: how are the site and the direction of actin nucleation in the core determined in the absence of integrins? A part of the answer could lie in the fact that, besides β3, other integrins subunits, such as αV, β1 and β2 and other non-integrin receptors such as CD44 are present at podosome site [START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF][START_REF] Schmidt | Kindlin-3-mediated signaling from multiple integrin classes is required for osteoclastmediated bone resorption[END_REF]. These receptors, which have not been kinetically accounted for, can therefore participate in initial docking of the adhesion plaque and F-actin. Podosome core induces tension within the actin network, which is transmitted to the ring to drive the recruitment of tension sensitive vinculin to talin/paxillin and zyxin to the actin filaments.

Glass

Alpha-actinin Unknown crosslink protein to connect the actin filaments with the core by actin polymerization at the base of the core, tension is generated within the actin network and transmitted to talin and paxillin, thereby further driving the recruitment of the tensionsensitive vinculin and zyxin to reinforce the podosome and facilitate its protrusive activity.

The connection of the actin network to the ring most likely occurs through talin and vinculin, whereas the main players crosslinking the network filaments to the core are still unknown.

Myosin IIA cross-links the actin network filaments and is essential for core shrinkage by either generating contractility or directly unbundling and depolymerizing the actin network, thereby inducing podosome core instability. The delicate balance between actin polymerization in the core and myosin IIA activity in the ring facilitates core oscillations (van den [START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF].

III.5. Podosome internal dynamics: interplay between polymerization and contractility

Once the podosome is "constructed", it has a lifespan of several minutes (2-10minutes). This seemingly long duration is however coupled with highly dynamic modulation, which is owed to intrinsic physical properties of its molecules.

First, the scaffold of the podosome, i.e. F-actin, is a polymer that constitutively undergoes fast treadmilling [START_REF] Wanger | The actin treadmill[END_REF] and is entirely renewed every 20 to 60 seconds in OC podosomes, i.e. at least 2.5 times within the podosome lifespan [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF]. This fast actin turnover requires the presence of polymerization regulating molecules such as gelsolin. This protein is in fact an actin-capping and -severing protein with cleaves fast growing filament ends and thus creates new nucleation sites [START_REF] Yin | Identification of gelsolin, a Ca2+-dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues[END_REF][START_REF] Southwick | Gelsolin and ADF/cofilin enhance the actin dynamics of motile cells[END_REF]. Gelsolin localization at podosomes has been extensively documented [START_REF] Zallone | Osteoclasts and monocytes have similar cytoskeletal structures and adhesion property in vitro[END_REF][START_REF] Marchisio | Cellsubstratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures[END_REF][START_REF] Duong | Integrins and signaling in osteoclast function[END_REF] and its indispensability to podosome formation in OCs has been confirmed (Chellaiah et al., 2000a) (Figure 11). Moreover, gelsolin depletion in mice results in mild osteopetrosis (Chellaiah et al., 2000a). The presence of cofilin, another actin-severing protein at podosome sites has been recently described [START_REF] Blangy | Cofilin activation during podosome belt formation in osteoclasts[END_REF][START_REF] Touaitahuata | Podosomes are dispensable for osteoclast differentiation and migration[END_REF]. Cofilin is characterized with a lower affinity for actin compared to gelsolin [START_REF] Southwick | Gelsolin and ADF/cofilin enhance the actin dynamics of motile cells[END_REF] but how it regulates podosomes is still unknown.

A second level of podosome dynamics is provided by acto-myosin contractility [START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF] and elastic properties of vinculin and talin [START_REF] Turner | Paxillin: a new vinculin-binding protein present in focal adhesions[END_REF] all present in the podosome cloud. These molecules convey to podosomes a property called "mechanosensitivity", which can be defined by modulation of contractility in parallel to Factin polymerization in the core and, therefore, the adaptation of size and stiffness of the entire podosome to the extracellular matrix [START_REF] Labernadie | Dynamics of podosome stiffness revealed by atomic force microscopy[END_REF][START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF]) (Figure 10). Mathematical modeling of gelsolin-mediated actin polymerization in podosomes has predicted that F-actin growth can itself be a size-limiting factor of podosome core growth (Hu et al., 2011a) (Figure 11). It is therefore conceivable that contractility and actin polymerization can interdependently modulate podosome mechanosensitivity.

III.6. OC-specific podosome properties

Podosomes are not exclusively present in OCs. Different cell types from a variety of lineages also exhibit these cytoskeletal structures. They include endothelial cells [START_REF] Moreau | Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA[END_REF], smooth muscle cells [START_REF] Kaverina | Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains[END_REF] and cells from the monocytic lineage such as DCs and macrophages [START_REF] Linder | Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages[END_REF][START_REF] Calle | The leukocyte podosome[END_REF]. The fact that podosome-presenting cells have different physiological functions shows the high adaptability of podosomes to different microenvironments. Although the pool of structural proteins that builds the podosome is conserved between different cell types, the signals that transduce podosome formation are often different. It is therefore not surprising that some experimental observations depict differences between podosomes in different cells. This section will review some of these differences within the monocytic lineage, in other words between podosomes of OCs, DCs, and macrophages.

First, given that dynamic actin polymerization and depolymerization are necessary to maintain all podosomes, the genetic ablation of gelsolin, a high-affinity actin-severing protein that promotes actin turnover, in OCs results in their inability to form podosomes and SZs (Chellaiah et al., 2000a) but has visibly no effect on podosome formation in DCs [START_REF] Hammarfjord | Gelsolin-independent podosome formation in dendritic cells[END_REF]. Second, several studies of podosomes of mature OCs seeded on culture-treated glass collectively evolve into circular patterns called rings and SZ-like structures [START_REF] Jurdic | Podosome and sealing zone: specificity of the osteoclast model[END_REF]. In human and murine macrophages seeded on 2D substrates, podosomes also collectively organize into circular structures called "rosettes" [START_REF] Cougoule | Blood leukocytes and macrophages of various phenotypes have distinct abilities to form podosomes and to migrate in 3D environments[END_REF]. In DCs however, podosomes do not exhibit such circular distributions but rather sustain their individual aspect (van den [START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF]. Third, cell migration has been

shown to be mainly exerted by podosome rings in OCs. These observations suggest that there are yet unrevealed molecular pathways that induce the collective circular patterning of podosomes in OCs and in macrophage. A subset of such pathways could be exclusive to OCs to adequately support bone resorption through SZ formation. Furthermore, the role of WIP to integral podosome formation seems to be more indispensable to DCs compared to OCs. WIP - /-DCs do not exhibit podosomes at all [START_REF] Banon-Rodriguez | The cortactin-binding domain of WIP is essential for podosome formation and extracellular matrix degradation by murine dendritic cells[END_REF] whilst Wip -/-OCs do form podosome clouds (i.e. podosomes without the core) although overall podosome formation is decreased compared to wildtype OCs [START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF].

Interplay between the actin-containing podosomes and other components of the cytoskeleton, such as microtubules, can also be differently regulated between cells. Whilst chemical microtubule depolymerization (with nocodazole) severely abolishes podosome formation in macrophages [START_REF] Linder | Microtubule-dependent formation of podosomal adhesion structures in primary human macrophages[END_REF], treatment with the same chemical at higher concentrations only partially blocks podosome formation in OCs but dramatically abrogates their collective patterning [START_REF] Destaing | A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts[END_REF].

Finally, how podosomes dynamics and patterning could be regulated in an OC-specific

manner is yet unknown. Interestingly, studies of Rho-Rock signaling in OC and OC-like cells [START_REF] Ory | Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation[END_REF][START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF] (reviewed in (Ory et al., 2008b)) have shown that cell. These patterns evolve from apparently random groups of podosomes called "clusters" to circle of podosomes "rings" and, eventually to more massive circular structures, i.e. either "sealing zones" (SZ) or "SZ-like structures" (SZL, also known as "belts") [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF][START_REF] Saltel | Apatite-mediated actin dynamics in resorbing osteoclasts[END_REF][START_REF] Jurdic | Podosome and sealing zone: specificity of the osteoclast model[END_REF][START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF] (Figure 12). The SZ-like structure is not associated with a functional activity and is at the extreme periphery of the cell. Note the increase in podosome density and interconnectivity in the SZ and the SZL compared to the cluster. Kinetic, biochemical and structural properties that accompany the patterning process are also displayed in this scheme: the pattern life span (grey), the increasing (green) and decreasing (red) factors (Adapted from [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF][START_REF] Saltel | Actin cytoskeletal organisation in osteoclasts: a model to decipher transmigration and matrix degradation[END_REF][START_REF] Novack | Osteoclast motility: putting the brakes on bone resorption[END_REF]).

The first pattern formed by a group of podosomes is the cluster. Several clusters can exist within an OC. At this point, individual podosomes have a "relaxed" conformation: the diameter of the core is ~300 nm, that of the cloud is 3µm and the average distance between the cores of two podosomes is 750nm [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. Podosomes being in this vicinity, the radial F-actin filaments of neighboring podosome clouds overlap in a common region yet without directly linking the cores to each other, suggesting the existence of some topological crosstalk between podosomes [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. The cluster pattern can last up to several hours in an OC while the lifespan of an individual podosome within the cluster is around 3 min [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF]Luxenburg et al., 2006a) (Figure 12). This means that podosome patterns are sustained by spatial commitment of de novo podosome formation through yet unknown mechanisms. Several non-exclusive hypotheses could explain this spatial confinement: (1) a local abundance of matrix-embedded or membrane-anchored molecules that directly stimulate podosome formation;

(2) a wider microenvironment-induced cell-homing mechanism that induces migration or resorption in a given direction;

(3) a cytoplasmic enrichment of podosomal proteins "left over" from disassembled podosomes thus shifting the balance from a diffusion-rate stochastic podosome formation anywhere at the cellmatrix interface in the favor of the region containing previous component.

As clusters grow, de novo podosomes are positioned towards the periphery of the clusters thus making transient circular structures that lasts for a few minutes: the rings (Figure 12).

Although individual podosomes within a given pattern are not displaced, it has been suggested that, as a consequence of actin nucleation and polymerization at the base of the podosome core, nascent podosome repel each other while growing (Hu et al., 2011a). The lifespan of individual podosomes in rings shortens down from 3 min to 1 min (Luxenburg et al., 2006c), thus conveying a more dynamic aspect to this pattern compared to clusters (Figure 12). The shorter podosome lifespan is correlated with a fast ring expansion rate at 2 µm/s [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF]. A more detailed characterization of ring properties has been technically challenging because of the transient nature of this structure. However, what is visible by microscopy of fluorescently labeled actin is that if rings stabilize, i.e. exceed their typical lifespan without changing that of individual podosomes (Luxenburg et al., 2006c), rings can give rise either to the SZ or to the SZL. When OCs are adherent to a nonmineralized substrate (glass, plastic), rings are transformed into SZL by fusing together and positioning podosomes at the very periphery of the OC [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. When OCs are on bone or on dentine, rings give rize to SZs by growing individually and making a thicker and more central and stable "super-ring" (D. Georgess, unpublished data).

The SZ and SZL are the most mature podosome patterns. The role of the SZ is to ensure osteoclastic bone resorption by confining OC-secreted degradation molecules at the vicinity of the bone matrix (see Section IV: Bone resorption processes). Its formation is substrateexclusive, meaning that the SZ forms only on mineralized substrates such as bone or hydroxyapatite-coated glass [START_REF] Saltel | Apatite-mediated actin dynamics in resorbing osteoclasts[END_REF]. The SZL is thought to be the equivalent of the SZ that forms only on non-mineralized substrates such as culture-treated plastic or glass.

Hence, the SZL is not affiliated with a specific function like the SZ, but it is used as an easier model to gather biochemical and microscopical data on collective podosome patterning in mature OCs.

The overall transition from clusters to SZs is marked not only by a collective displacement of podosomes but also by different internal actin dynamics and increased interconnectivity between podosomes (Figure 12). The amounts of actin and other structural proteins such as paxillin, vinculin and α-actinin recruited per podosome increases 3-to 4-fold in this transitions and neighboring podosomes become more tightly packed with a core-to-core distance that increases 2-fold on average, reaching 480 nm in the SZL and 210 nm in the SZ [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. With the increase of intimacy between podosomes, the density of the F-actin radial fibers that make the podosome cloud increases (Figure 12). This allows for clouds to form a continuous circular band with a thickness of 2-3 µm in the SZL and 3-6 µm in the SZ. The podosome cloud is therefore "accorded" 70 to 80 % of all the actin present in the podosome (with 20-30% of podosomal actin in the core) knowing that, at the cluster stage, the cloud only contained about 30% of actin present in the podosome (Luxenburg et al., 2012a).

Although the rate of actin turnover might change from one pattern to another, it has been shown that individual actin turnover rates in the cloud and in the ring are similar and vary simultaneously [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF]. Even if these two podosomal subdomains might have different molecular architectures, the joint actin turnover rate attests for a common regulation of actin polymerization.

Finally, although all the mentioned podosomal patterns can appear within the lifetime of a single OC, the frequency of their formation varies as a function of OC maturation. In the early stages of osteoclastogenesis, the cluster is the most frequent pattern, making around 65% of all patterns. At the final stages of OC maturation, the SZL becomes the major pattern, constituting more than 60% of all patterns in OCs [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF]. This observation reveals that OCs, in order to organize these patterns, not only respond to instantaneous extracellular cues but also commit very early along their differentiation to end-line functions such as resorption by reaching mature podosomal patterns. The fact that this differential patterning frequency occurs on the same substrate makes it tempting to speculate that the long-term regulation is directly dependent on genetic factors. Given that the frequency of SZL patterns coincides with increase OC fusion events, it would be interesting to investigate whether multinucleation is a direct genetic determinant of SZ formation.

III.7.2. Molecular mechanisms implicated in patterning

Podosome patterning is a complex molecular process that requires spatio-temporal regulation of signals and effector molecules. The molecular pathways that govern patterning were totally mysterious a decade ago but are now starting to be elucidated.

Tyrosine protein kinases: Src

During the collective patterning of scattered podosomes into the SZ/SZL, the apparent amount of certain structural proteins such as actin, vinculin, cortactin, α-actinin and paxillin increase within the individual podosome. The increase of these proteins is inversely correlated with global tyrosine phosphorylation in podosomes (Luxenburg et al., 2006b;Luxenburg et al., 2012a). This residue-specific post-translational modification has been associated with dynamic changes in adhesion structures. However, data on tyrosine kinases in OCs, albeit very revealing when available, is scarce.

In OCs, Src localizes to podosomes and is important for their assembly as well as their patterning [START_REF] Destaing | The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts[END_REF]. The dual importance of Src involves structural/docking activity and kinase activity. The first is necessary for the proper formation of the actin cloud in podosomes probably by binding the cytoplasmic tail of β3 and Pyk2 at the same time [START_REF] Zou | Syk, c-Src, the alphavbeta3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption[END_REF][START_REF] Destaing | The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts[END_REF]. Interestingly, although Src kinase activity has also been shown important for podosome initiation, it is most crucial for rearrangement of podosomes by phosphorylating several downstream targets [START_REF] Destaing | The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts[END_REF]. Src can activate another tyrosine kinase Syk, which is involved in podosomal rearrangement by facilitating Rac GTPase activation [START_REF] Faccio | Vav3 regulates osteoclast function and bone mass[END_REF]. Indeed, mice lacking Src, Syk, or vav3 expression suffer from increased bone bass due to defective OC-resorption [START_REF] Soriano | Targeted disruption of the c-src protooncogene leads to osteopetrosis in mice[END_REF][START_REF] Horne | Osteoclasts express high levels of pp60c-src in association with intracellular membranes[END_REF][START_REF] Lowell | Knockouts of Src-family kinases: stiff bones, wimpy T cells, and bad memories[END_REF][START_REF] Faccio | Vav3 regulates osteoclast function and bone mass[END_REF]. Another target of Src is cortactin, which induces slow actin turnover in podosomes in its phosphorylated form and thus contributes to SZL formation [START_REF] Martinez-Quiles | Erk/Src phosphorylation of cortactin acts as a switch on-switch off mechanism that controls its ability to activate N-WASP[END_REF]Luxenburg et al., 2006c). Finally, Src expression, has been shown to increase during osteoclastogenesis [START_REF] Verollet | Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors[END_REF] thus suggesting that it is needed for mature podosome patterns. When other tyrosine kinases that are important for podosome patterning such as Hck are deleted, OCs compensate for this deletion by overexpressing Src which in fact re-establishes mature podosome patterning, i.e. SZL formation, and above-average OC resorptive capacity.

However, Src is not expressed in OC precursors and therefore its overexpression cannot rescue defected precursor migration towards bone. As a result, Hck -/-mice, despite overexpressing Src in OCs, are ostepetrotic [START_REF] Verollet | Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors[END_REF].

Rho GTPases

This family of molecular switches mediates signal transduction and cytoskeletal remodeling related to a broad spectrum of cellular processes in all cell types [START_REF] Vega | Rho GTPases in cancer cell biology[END_REF][START_REF] Hall | Rho family GTPases[END_REF]. These molecules have been extensively studied in vitro and in vivo but the specificity of the OC cytoskeleton raises the difficulty in extrapolating their roles of into OCs.

The comprehension of the roles of these proteins has been coincidently further complicated by several technical difficulties encountered by investigators as will be briefly discussed in the following paragraphs.

• Rho

The importance of Rho to podosome organization and OC-mediated bone resorption has first been commented by Zhang and colleagues. In their study, the microinjection of C3 exoenzyme (a Clostridium botulinum toxin which inhibits RhoA/B/C) in murine OC-like cells resulted in the disassembly of the SZL structure after 20 min [START_REF] Zhang | The small GTP-binding protein, rho p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts[END_REF]. The treatment of avian OCs with C3 lead, in the first 15 min, to transient growth of podosomes but eventually resulted in their complete dissolution 2 h after the treatment (Chellaiah et al., 2000b). Also, avian macrophage polykaryons treated with TAT-C3 conjugated to the HIV TAT peptide (that ensure cell penetration) suffered from total podosomes disassembly [START_REF] Ory | Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation[END_REF]. This data shows a positive contribution of Rho to podosome stability. However, when the reversed experiments were performed, i.e. when avian OCs were transduced using a constitutively active Rho, they also suffered from podosome disassembly after 30 min (Chellaiah et al., 2000b). The converging results of Rho overactivation and inhibition suggest the necessity for a precise and time-dependent Rho activation levels during podosome formation and patterning.

Even more, a discrepancy is observed between the C3-meditated podosome disassembly in avian macrophage polykaryons and C3-mediated stabilization of podosome rings and SZ disassembly [START_REF] Ory | Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation[END_REF][START_REF] Destaing | A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts[END_REF]. Whether this experimental contradiction is species-dependent and/or due to technical differences should be investigated.

Finally, the SZL-stabilizing role of Rho is dependent on the decrease of its GTPase activity during OC maturation. Indeed, lower levels of active Rho allow for less activation of its effector, mDia. In fact, active mDia can activate HDAC6 and thus lead to deactetylation and destabilization of microtubules (MTs). Therefore, lower levels of Rho allow for the stabilization of MTs by maintaining their acetylation, resulting in enhanced OC spreading and SZL formation at the cell periphery [START_REF] Destaing | A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts[END_REF].

• Rac

Rac1 and Rac2 are both expressed in OCs. These proteins are generally involved in organization of the cytoskeleton and are also important components of the NADPH, the enzyme that generates free radicals. The NADPH-related function of Rac has not been related with podosome organization.

A study of Rac functions in OCs using a murine Cre-recombinase-based genetic depletion model has depicted distinct roles of each of the two Rac proteins during OC precursor chemotaxis and differentiation in vivo [START_REF] Wang | Identifying the relative contributions of Rac1 and Rac2 to osteoclastogenesis[END_REF]. This study has been contested by another group claiming the insufficient depletion of Rac1 and Rac2 genes in the first study [START_REF] Croke | Rac deletion in osteoclasts causes severe osteopetrosis[END_REF]. In the more recent study, it is shown that Rac1 and Rac2 are not involved in osteoclastogenesis but have overlapping roles in podosome assembly and SZL formation by localizing Arp3 at podosome sites during osteoclastogenesis. The Rac double knockout therefore results in podosome disassembly, the absence of SZs and diminished bone resorption but only if Rac deletion occurs at early OC precursor stage. The abnormalities first observed in OCs due to Rac double knockout, strangely, were not reproduced when Rac deletion was driven by a different promoter corresponding to Cathepsin-K + differentiating OCs. As the cathepsin-K promoter activity is specific to committed OCs, it is thought that the absence of effect under this promoter is due to the incorporation of much less committed, upstream mononucleated precursors where Rac1/ 2 are not deleted [START_REF] Croke | Rac deletion in osteoclasts causes severe osteopetrosis[END_REF]. The importance of Rac1/2 to the OC cytoskeleton and bone resorption is, however, confirmed by targeting them via intra-cellular blocking antibodies [START_REF] Razzouk | Rac-GTPase, osteoclast cytoskeleton and bone resorption[END_REF].

Rac activity in OCs is regulated by its Guanine nucleotide Exchange Factors (GEFs) Dock5 and vav3 [START_REF] Faccio | Vav3 regulates osteoclast function and bone mass[END_REF]Vives et al., 2011b). Dock5 is increasingly expressed along osteoclastogenesis and localizes to podosomes in SZLs. The deletion of Dock5 in mice results in an osteopetrotic phenotype explained by decreased Rac activity, absence of podosome formation and SZL patterning leading to reduced adhesion and bone resorption (Vives et al.,

IV.1. Transmigration

At the latest stages of their differentiation/maturation, OCs are multinucleated giant cells with one main task: to resorb bone. In order to accomplish this function, the OCs needs to move to adequate regions of the bone and several possible hurdles can stand in its way. Because the in vivo trajectory undertaken by the OC to reach the bone surface is still uncertain, attempts have been made to better assess the tissue invasion potential of the OC by recreating the multicomponent bone microenvironment in vitro.

The surface of bone is largely covered by osteoblasts, bone-lining cells and, in some pathological situations, fibroblast-like cells [START_REF] Pap | Osteoclast-independent bone resorption by fibroblast-like cells[END_REF] and metastatic cells (Zhao et al., 2006). These cells are able to secrete chemokines that attract OCs and locally promote bone degradation. At the same time, these cells can proliferate and/or differentiate abundantly in the vicinity of bone, thus representing a barrier between the OC and its resorption destination.

OCs, similarly to highly invasive metastatic cells, express high levels of Src and Matrix Metalloproteiases (MMPs) [START_REF] Yu | SDF-1 increases recruitment of osteoclast precursors by upregulation of matrix metalloproteinase-9 activity[END_REF] are able to, first, transmigrate through such cells layers by forming large actin rich protrusions and, second, to spread over matrix and underneath the invaded cells (Figure 13) [START_REF] Saltel | Transmigration: a new property of mature multinucleated osteoclasts[END_REF]. The actin rich protrusion observed in transmigrating OCs reaches a diameter of at least 5µm (Figure 13) and therefore seems bigger than podosome-like structures observed in 3D-migrating macrophages [START_REF] Van Goethem | Matrix architecture dictates three-dimensional migration modes of human macrophages: differential involvement of proteases and podosome-like structures[END_REF] and invadopodia observed in metastatic cells [START_REF] Linder | The matrix corroded: podosomes and invadopodia in extracellular matrix degradation[END_REF]. but could share similarities with the latter such as degradation-mediated invasion. Indeed, the chemical inhibition of either Src or MMPs hampers the transmigration process [START_REF] Saltel | Transmigration: a new property of mature multinucleated osteoclasts[END_REF].

IV.2. Bone degradation: trafficking, acidification, proteolysis

Once it reaches the bone matrix, the fully differentiated OC can start resorbing bone. In order to fulfill its task, the OC become polarized and its membrane is reorganized into four distinct and unique membrane domains: the SZ, the ruffled border (RB), the basolateral domain (BL)

and the functional secrectory domain (FSD) (Figures 14,15) [START_REF] Salo | Bone-resorbing osteoclasts reveal a dynamic division of basal plasma membrane into two different domains[END_REF]Coxon and Taylor, 2008a). The RB is the resorptive organelle of the OC. Its function is to acidify the subjacent resorption lacuna therefore dissolving the mineral phase of the bone and to secrete proteases that will degrade the organic phase of the bone (Coxon and Taylor, 2008a). The RB is a convoluted membrane with a high surface area that forms as a consequence of active and directed transport of vesicles that fuse with the plasma membrane in the basal pole of the OC, facing the bone matrix [START_REF] Abu-Amer | Substrate recognition by osteoclast precursors induces C-src/microtubule association[END_REF]Mulari et al., 2003a;[START_REF] Mulari | Osteoclast ruffled border has distinct subdomains for secretion and degraded matrix uptake[END_REF]. In fact, late endosomes/lysosomes originating from the BM are acidified by V-ATPases which pump protons into the lumen of vesicles. In parallel to this acidification, chloride channels such as ClC-7 transports chloride ions into the lumen to maintain the electroneutrality of the vesicles (Coxon and Taylor, 2008a). Along with acidification, the RD-delivered lysosomes are also responsible for the trafficking of proteolytic enzymes to the resorption lacuna. The cysteine protease Cathepsin-K, which is specific to OCs and most efficient in degrading type I collagen, is also delivered to the RB via acidic late endosomes/lysosomes [START_REF] Zaidi | Cathepsin K, osteoclastic resorption, and osteoporosis therapy[END_REF][START_REF] Zhao | Pharmacological sequestration of intracellular cholesterol in late endosomes disrupts ruffled border formation in osteoclasts[END_REF]. The matrix metalloprotease-9 (MMP-9) also contributes to the lysosome-mediated bone degradation but is activity is presumably specific to certain site of the bone tissue [START_REF] Everts | Degradation of collagen in the bone-resorbing compartment underlying the osteoclast involves both cysteine-proteinases and matrix metalloproteinases[END_REF]. This vectoral lysosome trafficking is dependent on prenylated Rab GTPases [START_REF] Feng | Rab 7: an important regulator of late endocytic membrane traffic[END_REF][START_REF] Bucci | Rab7: a key to lysosome biogenesis[END_REF] and mediated by via dynein anchorage to MTs [START_REF] Jordens | The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors[END_REF]. This dynamic transport of lysosome results in their delivery to the peripheral region of the RB, close to the SZ. The liberated acidic content upon fusion with the RB membrane allows therefore the liberation of protons that dissolve the mineral phase of the bone but also ensure acidic pH, optimal for Cathepsin-K activity [START_REF] Zaidi | Cathepsin K, osteoclastic resorption, and osteoporosis therapy[END_REF]. Conversely, in the central region of the RB, the degraded matrix components are collected by clathrin-mediated endocytosis and trafficked via the transcytosis pathway to the FSB where they are excreted to the blood stream [START_REF] Salo | Removal of osteoclast bone resorption products by transcytosis[END_REF][START_REF] Hirvonen | Novel interaction of Rab13 and Rab8 with endospanins[END_REF] (Figure 15). Hence, the central region of the RB is called the "Uptake Zone". Interestingly, another osteoclastic protease, tartrate resistant acid phosphatase (TRAP), has not been found to be transported to the RB via the already described late endocytic pathway. The delivery of TRAP is therefore suggested to be part of the biosynthetic-secretory pathway, which, like the endocytic pathway, is derived from the Golgi apparatus [START_REF] Vaaraniemi | Intracellular machinery for matrix degradation in bone-resorbing osteoclasts[END_REF]) (Figure 15).

Additionally, TRAP is also thought to be present in non-acidic biosynthetic vesicles that fuse with transcytotic [START_REF] Vaaraniemi | Intracellular machinery for matrix degradation in bone-resorbing osteoclasts[END_REF]. Because cathepsin-K can activate the phosphatase activity of TRAP by cleavage of its two subunits [START_REF] Ljusberg | Proteolytic excision of a repressive loop domain in tartrate-resistant acid phosphatase by cathepsin K in osteoclasts[END_REF], the role of TRAP might be to further process collagen fragments inside transcytotic vesicles before their liberation through the FSD (Coxon and Taylor, 2008a). Finally, a new pathway called "Reverse Transcytosis" that consists of delivering vesicles from the FSD to the peripheral zone of the RB, thus compensating for the membrane loss during the matrix uptake process [START_REF] Vaaraniemi | Intracellular machinery for matrix degradation in bone-resorbing osteoclasts[END_REF]) (Figure 14).

It is hypothesized that first event of OC polarization is SZ formation (Coxon and Taylor, 2008a) probably because the polarization does not occur on non-mineralized substrates, where the OC has a completely flat morphology (sign of the absence of polarization) and forms a SZL instead of a SZ [START_REF] Saltel | Apatite-mediated actin dynamics in resorbing osteoclasts[END_REF]. Adding further support to this hypothesis is the conceptual argument stating that RB formation and activity, i.e. secretion of degradation molecules, would be a waste of the cell's resources if the confinement of these molecules in the immediate vicinity to the bone surface were not ensured. However, the publication of few studies suggesting that protease secretion by OC is independent of polarization [START_REF] Henriksen | Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification[END_REF][START_REF] Hollberg | Osteoclast polarization is not required for degradation of bone matrix in rachitic FGF23 transgenic mice[END_REF] or that morphological differences exist between OC at different bone sites highlight that there is still much to be learned about OC polarization during bone resorption.

IV.3. The resorption-migration cycle

While adherent on the bone surface, the OC needs to search for news spots to degrade. It is thought that its migration is driven by podosomes because of their role in migration of other cells such as macrophages [START_REF] Linder | Podosomes at a glance[END_REF] but a mechanism depicting how podosomes exactly promote this 2D displacement during resorption is yet to be provided. One plausible hypothesis assumes that cyclic podosome switching between two categories of patterns, the SZ-state and a non-SZ state (i.e. cluster or ring), allows the OC to alternate between bone resorption and motility, respectively [START_REF] Saltel | Actin cytoskeletal organisation in osteoclasts: a model to decipher transmigration and matrix degradation[END_REF][START_REF] Novack | Osteoclast motility: putting the brakes on bone resorption[END_REF]. This model is derived from two in vitro observations: (1) the SZ, which is the podosomal arrangement that is maintained during bone degradation, stays in a relatively

(basaldomain)

stable position;

(2) the "resorption tracks" made by OC cultured on bone, dentine, ivory and hydroxyapatite slices consist of several individual resorption lacunae that overlap to make form of what could be described as a "chain" or also a trail of partially overlapping pits (Figure 16).

If this model were true, the OC would have to undergo fast transitioning also between two different directions of polarization. While migrating, the OC should be horizontally polarized, i.e. parallel to the substrate and with a leading edge and a trailing edge. While resorbing, the OC should be vertically polarized, in order to degrade the matrix and export its components to the systemic blood stream. overlapping circular resorption pits/lacunae (small dotted yellow circles) [START_REF] Susa | Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay[END_REF].

(C) Schematic representation of the current working model that depict OC-switching between

Basalpole

Apicalpole

an apico-basal-like polarization and an antero-posterior polarization during resorption and migration, respectively. Schema in (C) is adapted from [START_REF] Novack | Osteoclast motility: putting the brakes on bone resorption[END_REF].

I. OC MIGRATION

I.1. Introduction and rationale: How do podosomes drive OC migration?

OCs are adherent and motile cells. During their migration, they form dynamic multimolecular adhesive structures called podosomes, which are not only characterized by internal turnover, but also by collective patterning from clusters to rings. Because podosomes bridge (a) the cell to (b) its substratum, we investigated whether these structures had a particular role in mediating the mechanical interplay between the two elements (a and b). In other words, are podosomes the cause or the consequence of cell adhesion and displacement?

The cell-matrix relationship is continuous but involves several different processes: adhesion, spreading and, migration. Our broad question was therefore divided into several distinct parts to obtain experimental insight into each of these processes.

Timelapse micrographs of OCs in culture have shown that these large cells, as soon as they contact the matrix, first need to spread and, once properly spread, they start migrating in a given direction. We therefore first inspected the timing of podosome formation during the initiation of cell spreading and cell retraction using fluorescence and Nomarski contrast microscopy. By detaching and reseeding OCs, we observed that the OC first adheres then starts exhibiting podosomes concomitantly with its spreading. Inversely, the inhibition of integrin-mediated adhesion in already spread OCs resulted in podosome disassembly before subsequent cell retraction and detachment. These results indicated that podosomes are needed for maintaining adhesion during spreading but are not the exclusive mediators of these processes. Podosome exert forces to initiate and maintain the spreading of the OC.

We also sought to identify the leading podosome patterns during these sequential events. We found that podosome ring expansion is what initiates and maintains OC migration.

During the migration phase per se, we aimed at a qualitative description of the short-term forces exerted by the cell on the matrix and of the typical OC migration trajectory. To this end, we imaged short-term podosome ring movement on a fluorescently labeled substrate. We could therefore correlate the outward expansion of the podosome ring to the displacement of the substrate and we concluded that podosome rings exert on the substrate the tension needed for cell displacement. Podosomes are the structural units of the ring and the SZ but the molecular signaling that governs their collective organization into these superstructures remains elusive. In this study, our goal was to find new genes that would be relevant to this OC-exclusive functional patterning. The investigation was therefore comprised of two main parts:

We first aimed at finding a new list of candidate genes possibly involved in cytoskeletal processes during bone resorption. Such a list would be considered as grounds for selection for functional investigation of individual genes. To that end, we established an original comparative model that takes advantage of the differentiation plasticity of monocytic lineage cells. Since OCs are multinucleated cells derived from the differentiation and fusion of mononucleated monocytic precursors, we differentiated the same precursors into another type of multinucleated giant cells (MGCs) that shares several OC traits. These MGC formed podosomes and expressed osteoclastic degradation enzymes, but were unable to resorb. Based on these phenotypical properties, we used Affymetrix Genechip technology to perform a transcriptome-wide comparison between OCs and their non-resorbing counterparts with the objective of sorting out the genes that are exclusively expressed in OCs. We therefore obtained a list of genes that were highly expressed in OC. We validated this list using the Taqman Low Density Array, a method of highthroughput qPCR that provided more reliable quantification of gene expression levels. At each of these two screening steps, we observed a significant enrichment of actin-regulating genes, most of which where not studied in OCs.

This reinforced the importance of the actin cytoskeleton for OC function and suggested new possible regulators for it. We therefore chose to functionally investigate RhoE because it is a constitutively active GTP-binding protein that was previously shown to regulate actin-based structures such as focal adhesions and stress fibers.

Using primary culture of OCs derived from a RhoE gt/gt (knockout by gene trap) mouse, we observed that this gene was dispensable for OC differentiation but essential to bone resorption. Given its previously described actin-regulatory functions in other cells, we hypothesized that its role in bone resorption could be played via regulation of the OC cytoskeleton. Using confocal microscopy and Fluorescence Recovery After Photobleaching, we showed that RhoE expression maintains fast actin turnover in podosome clusters and rings and therefore regulates their patterning. Consequently, RhoE-deficient OCs suffered from impaired migration and suboptimal SZ formation, two crucial processes for bone resorption. [START_REF] Rivollier | Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment[END_REF][START_REF] Speziani | Murine dendritic cell transdifferentiation into osteoclasts is differentially regulated by innate and adaptive cytokines[END_REF][START_REF] Boyce | Advances in osteoclast biology reveal potential new drug targets and new roles for osteoclasts[END_REF]. Once differentiated, mature OCs resorb the bone matrix by secreting protons and proteases, such as tartrate-resistant acidic phosphatase (TRAP), cathepsin-K and matrix metalloprotease 9 (MMP9) onto the underlying bone matrix. A circular adhesive superstructure called "sealing zone" (SZ) allows these molecules to be within the resorption pit (lacuna) for efficient bone degradation.

Other types of MGCs can be also generated from immature DCs under various conditions.

DCs stimulated by interleukin-17 (ll-17), potentiated by IFN-γ, leads to DC-17γ-MGCs [START_REF] Coury | Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion[END_REF]. Besides resulting from cell-cell fusion of the same precursors (i.e. DCs), DC-17γ-MGCs have OC-like characteristics namely the expression of bone-degrading proteases such as TRAP, MMP9 and Cathepsin-K [START_REF] Coury | Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion[END_REF] as well as forming adhesive structures called "podosomes" [START_REF] Olsson Akefeldt | Chemoresistance of human monocyte-derived dendritic cells is regulated by IL-17A[END_REF].

Podosomes are integrin-based actin-rich dot-like structures, which are present in OCs, DCs and macrophages [START_REF] Linder | Podosomes at a glance[END_REF]. They are the structural units of OC SZ [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. A model of intra-podosomal architecture in monocytic cells that is currently widely accepted depicts two distinct domains within podosomes: a peripheral domain made of a loose F-actin meshwork with adhesion and adaptor molecules like αVβ3 integrin, paxillin, vinculin, myosin II, and a central domain comprised of a tightly connected F-actin network with adaptor molecules like cortactin and Arp2/3 [START_REF] Linder | Podosomes: adhesion hot-spots of invasive cells[END_REF][START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF]. When formed in OCs, podosomes are first assembled in clusters that self-organize into "rings" and finally into SZs on mineralized substrates or into "SZ-like" structures (also known as "podosome belts") on non-mineralized substrates. Besides driving the saltatory migration typical of OCs, podosome rings have been described as short-lived patterns, with life spans of 10-15 minutes, that mediate the transition from clusters to SZ-like patterns, more stable structures with life spans of several hours [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF][START_REF] Saltel | Apatite-mediated actin dynamics in resorbing osteoclasts[END_REF][START_REF] Jurdic | Podosome and sealing zone: specificity of the osteoclast model[END_REF]. Interestingly, the life span of individual podosomes decreases by at least two folds with the transition from clusters to rings/SZ-like structures indicating differential regulation during podosome "remodeling" [START_REF] Luxenburg | The architecture of the adhesive apparatus of cultured osteoclasts: from podosome formation to sealing zone assembly[END_REF]. The regulation of podosome assembly and disassembly as well as their inner architecture and stability are of central importance to their patterning. Therefore, fast and dynamic polymerization and depolymerization of actin, the scaffold of podosomes, has to be tightly regulated in OCs. For example, knockout of gelsolin, a high-affinity actin severing and capping protein, results in the inability of OCs to form podosomes (Chellaiah et al., 2000a).

In the same context, slowing the rate of actin turnover in OCs using cytochalasin D, an actin capping molecule, leads to overstabilized podosomes with longer life spans and seemingly larger podosome cores (Luxenburg et al., 2012b). Another important mechanism that regulates podosome size, stability and patterning is acto-myosin-II contractility around podosome cores [START_REF] Meddens | Automated podosome identification and characterization in fluorescence microscopy images[END_REF].

Some studies have provided data as to how these processes are regulated at the molecular level in OCs. Src has been shown as an indispensable regulator of phosphorylation of podosomal proteins such as cortactin and gelsolin (Luxenburg et al., 2006b;[START_REF] Destaing | The tyrosine kinase activity of c-Src regulates actin dynamics and organization of podosomes in osteoclasts[END_REF]. Control of podosome patterning by small GTPases, namely RhoA and Rac1/2 in OCs has been well documented. Rac activation following integrin-dependent signaling is essential to form a functional SZ. Indeed, mice lacking both Rac1 and Rac2 display massive osteopetrosis due to the inability of OCs to form SZs and subsequently resorb bone [START_REF] Croke | Rac deletion in osteoclasts causes severe osteopetrosis[END_REF]. Supporting this result, deletion of Rac GTPase Exchange Factors (GEFs) Vav3 and Dock5 in mice also present osteopetrotic phenotypes explained by an osteoclast functional defect [START_REF] Faccio | Vav3 regulates osteoclast function and bone mass[END_REF]Vives et al., 2011a). RhoA is critical for maintaining OC polarization and SZ formation on a mineralized matrix (Chellaiah et al., 2000b;[START_REF] Destaing | A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts[END_REF]. The complete molecular scheme of actin cytoskeleton regulation in OCs that is needed to adequately support bone resorption is still largely incomplete. Studies of Rho-Rock signaling in OC and OC-like cells have shown that this pathway can temporally regulate podosome arrangement during osteoclastogenesis and in a substrate-dependent manner but Rock-downstream signaling in OC remains unclear (Chellaiah et al., 2000b;[START_REF] Ory | Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation[END_REF]Ory et al., 2008a).

In this study, we aimed at finding new genes implicated in actin-related control of bone resorption by OCs, we have compared transcriptomic profiles of human primary Mo-derived OCs (Mo-OCs) and DC-derived OCs (DC-OCs), on one side, with those of Mo, DCs and DC-17 γ -MGC(s) with OC-like traits, on the other, to find genes that were highly upregulated in mature OCs. As a result, we obtained a short list of actin-regulating genes that were up regulated in mature OCs, considered as candidates for functional studies. We focused on the role of one candidate, RhoE (also known as Rnd3), in bone resorption using a genetic knockout model. Given its already described function in other actin structures such as focal

RESULTS

Non-resorbing human multinucleated giant cells exhibit osteoclast-like characteristics

but are unable to resorb bone.

Knowing that DC-17γ-MGCs are multinucleated giant cells expressing OC degradation markers such as TRAP, cathepsin-K and MMP9, we first asked whether they were able to resorb the mineral matrix in comparison with bone-resorbing DC-OCs. Then, we investigated their actin containing adhesion structures. Initially differentiated on tissue culture dishes, mature DC-OCs and mature DC-17γ-MGCs on day 5 and 13 of culture, respectively, were detached and seeded on apatite-collagen complexes (ACC), a bone-mimicking substrate [START_REF] Shibutani | Use of glass slides coated with apatite-collagen complexes for measurement of osteoclastic resorption activity[END_REF][START_REF] Saltel | Apatite-mediated actin dynamics in resorbing osteoclasts[END_REF], and stimulated with M-CSF and RANKL to promote resorption. Confocal microscopy of these cells fixed and stained for actin showed, as expected, that DC-OCs exhibited circular actin-rich sealing zone (SZs) and were able to resorb the mineralized matrix. DC-17γ-MGCs, however, were unable to resorb the matrix and displayed circular actin structures that were significantly less frequent and smaller in size compared to OC SZs (Figure 1A,B). We therefore named the latter actin structures "dysfunctional SZs" because of their inability to ensure proper degradation of the mineral matrix.

To gain further insight into cytoskeletal organization in DC-17γ-MGCs, we compared their actin organization with that of human Mo-OCs and DC-OCs on glass. We labeled these cells for actin, cortactin, a component of the podosome core as well as for paxillin and vinculin, components of the podosome cloud. We acquired images with a confocal microscope and visualized protein colocalization of one podosome marker at a time with actin by displaying channel merges and pixel-to-pixel multiplication of normalized and non-saturated micrographs. We found that cortactin colocalized perfectly with actin in podosome cores of Mo-OCs, DC-OCs as well as in DC-γ17-MGCs. Vinculin and paxillin also colocalized with actin in podosome clouds in all three cell types (Figure 2). These results showed that DC-17γMGCs, albeit displaying bona fide podosomes, are unable to efficiently organize SZs, which may participate to their inability to resorb bone.

Comparative transcriptomics revealed bone resorbing OC-specific genes

In order to identify new genes involved in osteoclast-mediated bone resorption, excluding ones involved in myeloid cell-cell fusion, we performed transcriptomic profiling of human bone resorbing Mo-OCs, DC-OCs as well as their precursor cells, Mo and DCs, and nonresorbing DC-derived MGCs. These latter MGCs included DC-17-MGC(s) and DC-17γ-MGCs (Figure 3A). Practically, blood monocytes from healthy human donors were differentiated in either DCs with M-CSF and IL4, or OCs with M-CSF and RANKL. DCs were then further differentiated in OCs (DC-OCs) with M-CSF and RANKL or DC-17γ-MGCs with IL17 and INF-γ. Total RNAs were extracted from these six culture conditions for two to four independent healthy donors and separately analyzed analyzed by GeneChip® Microarray technology by using 54675 distinct probes. Given the high number of probe sets used, a gene was considered as expressed in a cell type if its mean expression was superior to the overall gene expression in the same cell type (see Mat and Methods). This allowed us to establish a differential list of 115 genes that were expressed only in OCs (Figures 3,S1,S2).

The presence in this list of RNAs for NFACT1, the OC-specific transcription factor [START_REF] Takayanagi | The role of NFAT in osteoclast formation[END_REF] (reviewed in (Takayanagi et al., 2002a)) and ATP6v1c1, a proton pump needed for OC podosome ring formation [START_REF] Feng | Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts[END_REF], validated our approach. Both resorbing and non-resorbing group of cells were maintained in cell fusion culture conditions, consequently any genes known for roles in cell-cell fusion (such as DC-STAMP, ATP6v0d2, or OSCAR) were eliminated from this analysis. We therefore considered these 115 genes as new potential candidates for the investigation of OC functions. The "molecular functions" of this list compared to the human genome as determined by Gene Ontology (GO) vocabulary underlined enrichment in "Cytoskeletal protein binding" and its sub-category "Actin binding"

(Figure 3B).

We aimed at further validating candidates that would be preferentially involved in cytoskeletal regulation of bone resorption as opposed to genes rather implicated in other OC mechanisms. DC-17γ-MGCs were used as MGCs reference model. We then determined RNA expression values of the previously identified 115 genes in Mo-OCs, DC-OCs, DC-17γ-MGCs and their Mo and DC precursors by Taqman Low Density Array (TLDA), a high throughput quantitative real-time PCR. We then averaged the mean expression of a gene (X c ) from several human blood donors and determined if X c was significantly higher in both Moand DC-OCs compared to non-resorbing Mo, DCs and DC-17γ-MGCs by using the limma test. This method allowed us to spot 6 genes that were highly expressed in OCs (marked in red in Figure 3A), but were not even detected in the other cells types, namely TM4SF1, PLS3

(also known as T-fimbrin), ARHE (also known as RHOE or RND3), LNX1, AK5 and MYO1B. Three out of these six genes (PLS3, MYO1B and RHOE) have been already described as regulators of actin structures in several cell types whereas PLS3 protein has been shown as a component of podosomes in Mo-OCs (Babb 1997 Cell Motility and Cytoskeleton). Given the importance of Rho-GTPases in the regulation of podosomes in OCs, we aimed at further investigating RhoE role in OCs.

RhoE is essential for bone resorption but not for OC differentiation

RhoE has been previously shown as a regulator of focal adhesions and stress fibers in different cell types. We first investigated the implication of RhoE in osteoclastogenesis using primary murine precursors isolated from a RhoE genetrap mouse model [START_REF] Mocholi | RhoE deficiency produces postnatal lethality, profound motor deficits and neurodevelopmental delay in mice[END_REF]. Because these animals rarely surviving 15 after birth, we used either fetal liver (FL) or P14 bone marrow as a source of OC precursors. We differentiated primary OCs from fetal liver (FL) precursors from RhoE +/+ , RhoE +/gt and RhoE gt/gt E15.5 littermates in the presence of M-CSF and RANKL on culture-treated dishes and on cortical bone slices. Differentiation of FL-derived OCs (FL-OCs) on days 5, 6 and 8 post seeding was assayed by TRAP staining.

On both substrates, no difference in the number of TRAP-positive cells with 3 or more nuclei were observed between these different genotypes (Figure 4A). In the same assay, we also counted the number of nuclei per OC on days 5 and 8 of culture on plastic wells as a measure of OC precursor fusion (Figure 4B). Again, we observed no difference in the absence of RhoE expression. We therefore concluded that RhoE does not play a role in osteoclastogenesis.

To evaluate the role of RhoE in OC-mediated bone resorption, we performed resorption assays in two different conditions. First, precursors from RhoE +/+ and RhoE gt/gt littermates were differentiated directly on cortical bone slices. On day 8 of differentiation, the cultures were stopped and the resorption index was determined from surface area measurement of toluidine blue-positive resorption pits and TRAP staining of OCs on the same slices. RhoE gt/gt

OCs resorbed bone about 25 times less than wildtype OCs (Figure 4C, upper panel). In the second condition, we detached day-4 RhoE +/+ and RhoE gt/gt FL-derived OCs (FL-OCs) that were initially differentiated on culture-treated plates and seeded them for 48h on OsteoAssay substrate (OAS) plates, a bone-mimicking substrate. We counted the number of TRAPpositive OCs per well and, in replicate wells, quantified the total resorbed area per well with Silver Nitrate staining. Hence, we calculated the mean resorption index per OC. Again, RhoE gt/gt OCs resorbed the OAS matrix about 12 times less than RhoE +/+ OCs (Figure 4C, lower panel). These results accounted for an essential role of RhoE in bone resorption in vitro.

Small SZs and actin based defective structures in RhoE-lacking OCs

RhoE has been involved in the control of actin cytoskeleton in other cell types [START_REF] Guasch | RhoE regulates actin cytoskeleton organization and cell migration[END_REF]. We therefore investigated its role in SZ formation, given the critical importance of this actin structure in the bone resorption process. Day-4 FL-OCs expressing or not RhoE were lifted from culture-treated plates and seeded for 24h on bone slices. These OCs were then fixed, stained for actin and imaged using confocal microscopy. Using ImageJ software, peripheral outlines of SZs were then manually determined from acquired micrographs allowing the quantification of the surface areas of SZs. There was a slight yet significant 15% decrease in mean SZ size in RhoE gt/gt OCs compared to RhoE +/+ OCs (Figure 5). This result indicated that RhoE expression was dispensable for SZ formation but was needed to maintain normal SZ size.

Because of the observed effect of RhoE knockdown on SZ size, we investigated its importance for podosome formation and patterning into cluster, ring and SZ-like superstructures. Fixed day-4 BM-derived OCs (BM-OCs) were stained with fluorescently labeled phalloidin and imaged with confocal microscopy. Based on observed local fluorescence intensity, actin staining of OC allowed not only the identification of individual podosomes within an OC but also the discrimination between the two subdomains of podosomes: the core with high fluorescence intensity and the cloud with low intensity according to [START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF] and (van den Dries et al., 2013c) (Figure 6A). We therefore applied a method of fast and curated fluorescence intensity-based quantification of podosome core surface area. Briefly, once OC cell periphery was drawn manually, we used the "Find Peaks" plugin of ImageJ software with a set of fixed parameters (see Material and Methods) to count podosome numbers per cell and the size of individual podosome cores. We found that the mean number of podosomes per OC was decreased in RhoE gt/gt OCs (645podosomes/cell) compared to RhoE +/+ OCs (1018 podosome/cell). Podosome core size was, however, increased in the absence of RhoE expression as shown by mean values and frequency distribution of size (Figure 6B). This suggested that RhoE is not necessary for initial podosomes formation but is important for maintaining proper podosome size.

We then, quantified the number of OCs with clusters, rings and SZ-like structures per well.

The total number on average per well of OC exhibiting these podosomal structures was significantly decreased in RhoE gt/gt OCs (45 per well) compared to RhoE +/+ OCs (75 per well)

whilst the mean OC number per well was not altered as previously shown by the TRAP assay.

In more detail, the mean number of OC with clusters was decreased from 20 per well in wildtype cultures to 10 per well in RhoE gt/gt and SZ-like structures from 47 per well to 21 per well, in that same order. The number of OC with rings was however increased up to 14 per well in RhoE gt/gt OCs compared to 6 per well for wildtype OCs (Figure 6C).

Combining these data with the results from podosome size and number assays suggested that, as a result of RhoE depletion, podosomes were bigger therefore unusually stable but their number was insufficient to properly form clusters. This increase in podosome stability was also depicted by the enrichment in rings, normally transient structures required for SZ-like formation, resulting in fewer SZ-like counts per well. We concluded that RhoE is required for podosome organization and patterning.

RhoE controls actin turnover in podosome clusters and rings

The abnormalities of podosome size, number, superstructure patterning and OC-mediated bone resorption observed in RhoE gt/gt OCs raised the question of RhoE effect on actin turnover in podosomes. To address this point, we performed FRAP experiments on actin in podosome rings and clusters of day-5 FL-OCs 48h after their infection with a LifeAct-mCherry-coding lentivirus to label actin (Figure 7A). We fitted relative fluorescence intensity data with a non-exponential equation of recovery [START_REF] Negi | Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23[END_REF] to minimize constraints by mathematical functions (such as exponential growth). In wildtype OCs, mean actin turnover half times in clusters and rings were 9.8 sec ± 1.4 and 19.7sec ± 3.5, respectively. This showed that rings normally exhibit slower actin turnover. These values were significantly increased in RhoE gt/gt OC for both structures: 24 sec ± 3 in clusters and 34.4 sec ± 3.8in rings (Figure 7B). These data indicated that RhoE is involved in maintaining a fast actin turnover in podosomes.

Depletion of RhoE results in impaired OC migration

Having demonstrated a role of RhoE in actin dynamics in podosomes and in podosome stability and patterning, we hypothesized that RhoE would be implicated in OC migration. To test this hypothesis, we performed a random migration assay on OC. Differentiating FL-OCs expressing or not RhoE were detached on day 4 of culture and replated on culture-treated dishes at identical low confluence (5x10 4 cells per well) to minimize OC fusion events.

Timelapse movies were then acquired with phase-contrast microscopy and randomly migrating OCs were manually tracked. As visualized in single OC migration tracks (Figure 8A), wildtype OCs displayed typical saltatory migration consisting of displacement in a straightforward direction followed by a recognizable angular turn as we had published in a previous study (Hu et al). This pattern of migration allowed OCs to randomly cover a large surface area. RhoE gt/gt OCs displayed aberrant migration tracks where they turned in circles because of frequent consecutive angular turns to one side (Figure 8A). The frequency distribution of the instantaneous angle change (Δα i ) of several wildtype OC migration tracks nicely fitted a Gaussian distribution (R 2 = 0.91) in which the amplitude, indicating the frequency of straightforward movement (i.e. when Δα i is close to 0°), was 2.85% ± 0.07 and the absolute standard deviation (SD, i.e. the interval containing the most frequent 34% singlesided angle changes close to the mean) of 68.4° ± 2.01. Interestingly, values from RhoE gt/gt OCs deviated from the Gaussian distribution (R 2 = 0.58) with an amplitude of 1.97% ± 0.08 and an absolute standard deviation of 115° ± 6.51. The mean of Δα i Gaussian distributions did not vary between the two genotypes (-2.35° ±1,99 for RhoE +/+ OCs and -8.76° ± 5.31 for RhoE gt/gt OCs) because we pooled in our quantification several individual tracks which had negative or positive Δα i values. However, amplitudes and SDs were significantly different denoting a decrease of the frequency of straightforward displacements and the increase of the frequency of angular turns during OCs migration in the absence of RhoE expression. This aberrant distribution of angle changes therefore resulted in a significantly lower instantaneous persistence of RhoE gt/gt OCs (P i =0.335) compared to that of RhoE +/+ OCs, (P i = 0.419) (Figure 8B). Additionally, quantification of the instantaneous velocity of displacement (V i )

showed a significant decrease from 0.86 µm/sec ± 0.01 in RhoE +/+ OCs to 0.57 µm/sec ± 0.38 in RhoE gt/gt OCs (Figure 8B). These data showed that RhoE plays an essential role in the directionality as well as velocity of OC migration.

RhoE inhibits Rock-mediated cofilin phosphorylation

Having shown a role of RhoE in podosome dynamics and organization, we sought to find its subcellular localization and the molecular mechanism through which RhoE could perform this function. Day-5 BM-OCs overexpressing RhoE-GFP were fixed, stained with phalloidin and imaged with a confocal microscope. Interestingly, RhoE did not colocalize with podosomes per se but was rather ubiquitous in the cytoplasm with certain enrichments at membrane borders (Figure 9A). We therefore considered that RhoE probably acts indirectly on actin in podosomes.

It has been shown for focal adhesions and stress fibers that RhoE acts on the actin cytoskeleton through its inhibition of Rock-I kinase activity. Hence, we searched in mature OCs, phosphorylation targets downstream of Rock-I, such as Mypt1, a regulator of myosin-II (Van den Dries), and cofilin, that could mediate the effects of RhoE on podosome size and actin dynamics. We blocked Rock kinase activity in day-5 BM-OCs for 2h using an inhibitor,

DISCUSSION

Actin organization during podosomes patterning is central for osteoclast migration and bone resorption. In order to further decipher the mechanisms involved in actin remodeling in bone resorbing osteoclasts, we took advantage of human monocytic plasticity. Indeed, we are able to generate, from human blood cells, either two types of OCs derived from Mo or from DCs [START_REF] Rivollier | Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment[END_REF], or two different types of MGCs derived from DCs [START_REF] Coury | Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion[END_REF] and unpublished data). Characterization of DC-derived MGCs, revealed that, despite expressing osteoclastic proteases [START_REF] Coury | Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion[END_REF], they were unable to resorb the mineral matrix. Moreover, their actin cytoskeleton was organized into circular superstructures reminiscent of SZs that were named "dysfunctional SZs" because of their inability to ensure resorption of a mineralized matrix in comparison with OC SZs. We have showed that, in MGCs, vinculin, as previously reported [START_REF] Olsson Akefeldt | Chemoresistance of human monocyte-derived dendritic cells is regulated by IL-17A[END_REF], but also cortactin and paxillin perfectly colocalize with actin in podosomes. This indicated that the existence of bona fide podosomes in DC-derived MGCs is insufficient for SZ formation and, more importantly, it suggested that there are yet unknown SZ-specific molecular regulations which are absent in non-patterned individual podosomes.

A Comparative transcriptomics analysis between the two bone resorbing OC populations versus the two MGCs allowed us to provide a list of 115 genes that were highly expressed in OCs but not in MGCs. We hypothesized that these genes would be relevant to mature osteoclast functions. Among these, we validated six genes that were highly and exclusively expressed in OCs, namely TM4SF1, AK5, LNX1, MYO1B, RHOE/ARHE/RND3 and PLS3/T-PLASTIN/T-FIMBRIN. Previous studies have provided data linking these genes either with OCs and/or with the actin cytoskeleton. PLS3, a member of the fimbrin family, has been shown to colocalize with OC podosomes [START_REF] Babb | Fimbrin in podosomes of monocytederived osteoclasts[END_REF]. AK5 expression is stimulated by RANKL in osteosarcoma cells [START_REF] Mori | Receptor activator of nuclear factor-kappaB ligand (RANKL) directly modulates the gene expression profile of RANK-positive Saos-2 human osteosarcoma cells[END_REF]. TM4SF1, a member of the tetraspanin family described for their interaction with integrins at the cell surface, is involved in nanopodia formation and cell migration [START_REF] Zukauskas | TM4SF1: a tetraspanin-like protein necessary for nanopodia formation and endothelial cell migration[END_REF]. LNX1 is responsible for the ubiquitinilation of Src, a known OC regulator [START_REF] Weiss | c-Src is a PDZ interaction partner and substrate of the E3 ubiquitin ligase Ligand-of-Numb protein X1[END_REF]. Myo1B, a member of the myosin superfamily, is localized in filopodia in HeLa and Cos-7 cells [START_REF] Almeida | Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network[END_REF] and regulates Arp2/3 in Golgi-related actin foci. Finally, RhoE, a constitutively active RhoGTPase, inhibits stress fibers and focal adhesions by inhibiting the Rho-Rock pathway.

Considering the importance of Rho signaling pathways in OCs [START_REF] Zou | Integrins, growth factors, and the osteoclast cytoskeleton[END_REF], we focused our interest on RhoE.

RhoE depletion did not affect primary OC differentiation nor fusion but greatly decreased bone resorption. RhoE gt/gt OCs were still able to form podosomes but those were fewer in numbers per cell and with smaller cores compared to RhoE +/+ OCs. FRAP experiments targeting actin in podosomes revealed a significantly slower actin turnover in clusters and rings of RhoE deficient OCs. Such an increase in podosome stability and core size in OCs has been observed when actin turnover rate was reduced with cytochalasin-D treatment (Luxenburg et al., 2012a). Furthermore, lack of RhoE expression resulted in smaller SZs in OCs seeded on bone. Besides RhoE, RhoA is also dispensable for podosome formation but required for their patterning and SZ spreading [START_REF] Chellaiah | Rho-dependent Rho kinase activation increases CD44 surface expression and bone resorption in osteoclasts[END_REF][START_REF] Destaing | A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts[END_REF].

However, conversely to the results of RhoE knockdown, the chemical inhibition of Rho resulted in the spreading of SZ to a belt at the cell periphery of OCs seeded on ACC [START_REF] Saltel | Apatite-mediated actin dynamics in resorbing osteoclasts[END_REF]. The reversed consequences of RhoA and RhoE inhibitions suggest antagonistic functions of these proteins in OCs.

The increase in the number of OCs with rings provoked us to investigate typical ring-driven saltatory OC migration in the RhoE-depleted OCs. We have previously described OC random migration as a long phase of movement in the same direction followed by a short phase consisting of a 90°-switch in direction [START_REF] Hu | Podosome rings generate forces that drive saltatory osteoclast migration[END_REF]. In the herein study, we provide new quantifiable parameters of migration demonstrating that it is considerably altered in RhoE gt/gt OCs, namely by a decrease in displacement velocity and an increase in the frequency of 90°angle turns. This resulted in global loss of persistence of OC movement. These observations make it tempting to speculate that slow actin turnover rate in rings causes increase in rings stability and results in impaired migration and SZ organization.

To find the molecular pathway in which RhoE could take part to modulate actin kinetics in OCs, we investigated downstream phosphorylation targets of a well-described RhoE partner, Rock-I [START_REF] Riento | RhoE binds to ROCK I and inhibits downstream signaling[END_REF]. Contrary to its upstream activator Rho (Chellaiah et al., 2000b;Ory et al., 2008a), Rock-I downstream signaling towards podosomes has been poorly studied.

However, in non-myeloid cells, RhoE direct binding in 1:1 complex dimers, inhibits kinase activity of Rock-I, but not of its isoform Rock-II, [START_REF] Komander | Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure[END_REF]. This Rock-I-RhoE interaction destabilizes actin-based focal adhesions and stress fibers when RhoE is over expressed [START_REF] Klein | Rnd3 regulation of the actin cytoskeleton promotes melanoma migration and invasive outgrowth in three dimensions[END_REF]. We investigated two known Rock-I phosphorylation targets, namely Mypt1 and cofilin. Mypt1 is the main subunit of the myosin light chain phosphatase (MLCP) that activates acto-myosin-II contractility [START_REF] Ito | Myosin phosphatase: structure, regulation and function[END_REF]. We asked if Mypt1 was a Rock-I target in OCs and could explain a myosin-II-dependent regulation of podosome size and patterning as suggested in previous studies (van den Dries et al., 2013a) (Luxenburg et al., 2012b) [START_REF] Linder | Podosomes at a glance[END_REF]. The chemical inhibition of Rock activity revealed that Mypt1 phosphorylation status at its primary phosphorylation site is not Rock-dependent in OCs. Similarly, when myosin-II contractility was blocked in OCs, the size and stability of clustered podosomes were not affected (Luxenburg et al., 2012b). On the other hand, cofilin is an actin severing and capping protein that regulates F-actin assembly and disassembly thus promoting fast actin turnover [START_REF] Lappalainen | Cofilin promotes rapid actin filament turnover in vivo[END_REF][START_REF] Maekawa | Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase[END_REF]. Non-phosphorylated cofilin has been shown as the active form whilst its phosphorylation on Serine 3 blocks its activity [START_REF] Arber | Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase[END_REF] [START_REF] Abe | Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation-regulated protein essential for development[END_REF]. Furthermore, both total and phosphorylated cofilin have been already shown to localize at podosome clusters and SZ-like structures but how they regulates these structures was still unknown [START_REF] Blangy | Cofilin activation during podosome belt formation in osteoclasts[END_REF][START_REF] Touaitahuata | Podosomes are dispensable for osteoclast differentiation and migration[END_REF]. We did not investigate gelsolin, another actin-severing molecule, because gelsolin -/-OCs, contrarily to RhoE gt/gt OCs, are totally unable to form podosomes (Chellaiah et al., 2000a). This drastic effect of gelsolin depletion on the cytoskeletal phenotype of OCs is probably due to its much higher affinity to actin compared with cofilin [START_REF] Ressad | Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G-and F-actins. Comparison of plant and human ADFs and effect of phosphorylation[END_REF][START_REF] Southwick | Gelsolin and ADF/cofilin enhance the actin dynamics of motile cells[END_REF]. This suggests that gelsolin is likely to be implicated in podosome formation rather than their patterning. Inhibition of Rock severely reduced cofilin phosphorylation at serine 3. We found that p(Ser3)-cofilin levels in RhoE gt/gt OCs were much higher than in RhoE +/+ OCs. RhoE could therefore be responsible for the inhibition of Rock-mediated cofilin inactivation, thus ensuring fast actin turnover in OCs.

This Rock-RhoE-cofilin pathway highlights once again the importance of actin cytoskeleton organization in mature OC processes implicated in bone resorption such as migration and sealing zone formation. It also consolidates the emerging model that dissociates podosome dynamics from OC adhesion [START_REF] Hu | Podosome rings generate forces that drive saltatory osteoclast migration[END_REF] and differentiation [START_REF] Touaitahuata | Podosomes are dispensable for osteoclast differentiation and migration[END_REF].

were performed as shown previously [START_REF] Gallois | Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes[END_REF]. Intra-and inter-assay normalizations were performed according to manufacturer's instructions and gene expression values were averaged per cell type over all donors. When the mean expression value of a gene in a given cell type was superior to the average of all gene means in the same cell type, the gene was considered as expressed. As such, genes commonly expressed in both Mo-OCs and DC-OCs but not expressed in any other cell type used in this study were considered for further investigation.

For the mathematical analysis, after intra-and inter-chip normalization, we calculated the mean expression level of a specific RNA in a given cell type (X c ) as follows:

X c = 1 n X (i) i=1 n
" where X c is the mean expression of a RNA "X (i) " in a cell type "c" i is the number of donors for this cell type Then, we calculated the overall RNA expression per cell type ( c ) as follows:

X c = 1 n X c(i) i=1 n
" where c is the average expression of all probed genes "X c(i)" in a cell type "c" i is the number of probe sets (i = 54 675 for all cell types)

Gene Ontolgy molecular function enrichment

Molecular functions of 115 genes resulting from the transcriptomic assay were annotated according to Gene Ontology and their percentages were compared against the human genome using the online software FatiGO [START_REF] Al-Shahrour | BABELOMICS: a systems biology perspective in the functional annotation of genome-scale experiments[END_REF]. Only statistically over expressed functions in our specific gene set were represented. Statistical relevance was determined using the Fischer test.

Taqman Low Density Array (TLDA)

Mo, DCs, Mo-OCs, DC-OCs and DC-17 γ -MGCs triplicate cultures were obtained from 3 different human blood donors. RNA was extracted as mentioned in the previous paragraph, quantified with a Nanodrop (ThermoFischer Scientific, USA), quality-checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and finally reverse-transcribed using iScript™ cDNA Synthesis Kit (#170-8891, Bio-Rad, USA) following kit protocol. Amplification was performed using a made-to-order Taqman Gene Expression Assay (Agilent Technologies, USA) according to manufacturer's instructions. After verification with X X of 18S (Hs99999901_s1) amplification (standard microfluidics technical control for amplification runs), relative cDNA quantities (RQs) calculated based on cycle thresholds (Ct) were normalized with SEC61B (Hs00606455_m1) and TBP (Hs00920494_m1) housekeeping genes. Then, differential expression for each gene in resorbing cells (i.e Mo-OCs and DC-OCs) was compared with that of non resorbing cells (i.e. Mo, DC and DC-17 γ -MGCs) by using the latter as background in the limma parametric test (Limma package, Affymetrix, USA) combined with the Benjamini-Hochberg False Discovery Rate p-value adjustment.

Adjusted p-values <0.05 were considered significant.

Primary murine osteoclast cultures and transfection

OCs were differentiated from myeloid precursors extracted from either E15 to E18 fetal liver (FL) or from neonatal bone marrow (BM) or spleen (Sp) of RhoE +/+ , RhoE +/-and RhoE gt/gt mice of exactly the same age respectively described in (Schmidt JCB 2011, Destaing MBoC 2003[START_REF] Chabadel | CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts[END_REF]. For each experiment, all cultures were started from the same source tissue (i.e. FL, BM or Sp) from littermates sacrificed at the same time. Once extracted from their respective tissues by flushing (in the case of BM) or grinding against a 100µm-pore nylon cell strainer (in the case of FL and Sp), these precursor cells were purified with a gradient of Leukocyte Separation Medium at 1250g for 20min and 20°C by aspiration of the gradient interface. Cells were then washed by centrifugation and put into culture with αMEM with 10% fetal bovine serum, 100 units/ml Penicillin, 100µg/ml Streptomycin, 2mM Lglutamine, 20ng/ml murine recombinant M-CSF (produced by the "Production d'analyse des protéines" platform, UMS3444, Lyon-Gerland, France) and murine RANKL. The source of precursors for each experiment is mentioned in the Results section. For transfection, day-4 OCs were detached with 0.25mM EDTA in warm PBS and transfected using the Neon® MP-100 electroporator (Life Technologies, USA). Briefly, 0.5x10 6 cells were resuspended with 2µg of pEGFP-C-RhoE (#23229, Addgene) plasmid DNA in 10µl of Buffer "R" (see kit protocol) then jolted with a 1720V current in 2x10ms pulses. Electroporated cells were then seeded on coverslips with pre-warmed medium and kept overnight. Medium was renewed the following day.

Differentiation assays

To quantify differentiation and fusion of FL-derived OCs (FL-OCs) from RhoE +/+ , RhoE +/gt and RhoE gt/gt mice [START_REF] Mocholi | RhoE deficiency produces postnatal lethality, profound motor deficits and neurodevelopmental delay in mice[END_REF], the same number of precursors (2x10 4 cells/well) from E15.5 littermates was seeded in 96-well culture-treated plates and on bones slices of the same size. They were then fixed at several time points (day 5, day 6 and day 8 post-seeding) with 4% (m/v) parafolmaldehyde in PBS for 15 min at room temperature then stained at 37°C using a TRAP assay kit (Sigma-Aldrich, USA) according to manufacturer's instructions. Cells with 3 or more nuclei that turned violet were counted as OCs.

Resorption assays

To quantify the resorbed surface per murine OC (i.e. resorption index), two methods were used. For resorption on bovine cortical bone slices, day-8 FL-OCs were assayed for differentiation then resorption was measured as mentioned in [START_REF] Harre | Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin[END_REF] [START_REF] Destaing | Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein[END_REF]. For resorption on bone-mimicking OsteoAssay Surfaces® (OAS) (#3988, Corning, USA), day-4 FL-OCs were detached from plastic wells by flushing after incubation at 37°C for 5min in PBS + 0.25mM EDTA, counted then seeded at the same number (2x10 4 cells/well in 96-well plates) in replicate plates and cultured for 48h. To measure the total surface of the resorbed matrix, OCs were washed off with distilled water then the matrix was stained with a 5% (m/v) Silver Nitrate solution. In parallel wells, the number of OCs was determined by TRAP staining as mentioned previously. Finally, the resorption index was obtained by diving the total resorbed area per well by the total number of OCs per well.

To determine the resorption index of human DC-OCs and DC-17ϒ-MGCs on coverslips coated with apatite-collagen complexes (ACC) [START_REF] Shibutani | Use of glass slides coated with apatite-collagen complexes for measurement of osteoclastic resorption activity[END_REF] [START_REF] Saltel | Apatite-mediated actin dynamics in resorbing osteoclasts[END_REF], day-4 DC-OCs and day-14 DC-17ϒ-MGCs (i.e. stages of differentiation where both cell types a terminally differentiated [START_REF] Coury | Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion[END_REF] [START_REF] Rivollier | Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment[END_REF] were detached using 1X Accutase® (Sigma Aldrich, USA), replated on ACC coverslips with RANKL and M-CSF stimulation to promote resorption. The resorbed area per cell was determined by the absence of matrix crystal complexes subjacent to the cell.

Images of all substrates were captured with an AxioImager microscope (Carl Zeiss, Germany) topped with a CCD Coolsnap color camera (Photometrics, USA) and manually quantified with ImageJ software.

Random migration assay

Day-5 Sp-OCs were detached and replated at low confluence (5x10 4 cells/well in a 24-well plate) to solicit random (non-directed) OC migration. Cells were given 2h to adhere then were imaged in phase contrast on an inverted microscope Axiovert 100M (Zeiss) for 14 hours at 37°C with saturated humidity. Images were acquired every 5min. Consecutive cell positions were determined manually using the MTrackJ plugin in ImageJ at 20min intervals so that displacements were significantly greater than imprecisions due to manual tracking. Small front-to-back polarized OCs that< were not in contact with other cells were tracked. In other words, OCs that were fusing or without visible leading and trailing edges were not considered for this assay. Instantaneous velocity (V i ) was defined as the distance travelled between two consecutive points divided by the corresponding time interval (20min). Instantaneous angular change (Δα i ) was defined as the result of the subtraction between the angles of velocity vectors of two consecutive displacements. Persistence of movement "P i " was defined as the ratio of "d i " divided by "D i ", with "d i " defined as the direct distance between the position "i"

and the position at the start of the track and D i defined as over the length "D" of the track at a given position "i".

Fluorescent labeling and confocal microscopy of fixed cells

OCs in culture on coverslips or glass-bottom plates (Mattek Corp., USA) were fixed with 4% parafolmaldehyde in phosphate buffered saline (PBS) for 10min at 37°C and permeabilized using PBS + 0.2% Triton-X-100 Triton-X-100 in PBS. All antibody dilutions and washes were thereafter performed with PBS + 0.1% Triton-X-100. Used primary antibodies were:

anti-vinculin (5µg/ml final, clone hVIN-1, #V9264, Sigma-Aldrich, USA), anti-cortactin (10µg/ml final, #05-180, Clone 4F11, Millipore, USA), anti-paxillin (10µg/ml final, #610051, BD Biosciences, USA). Secondary antibodies Alexa Fluor® 488 anti-Mouse IgG (A11029, Life Technologies) and Alexa Fluor® 488 anti-Rabbit IgG (A-21245, Life Technologies, USA) were used at 2µg/ml. Then Alexa Fluor® 647 Phalloidin (A22287, Life Technologies, USA) was diluted 1:100. Image acquisition was performed with either a DMI4000 microscope (Leica Microsystems, Germany) equipped with a spinning disk unit CUS22 (Yokogawa, Tokyo, Japan) or an SP7 spectral confocal microscope (Leica Microsystems, Germany). For podosome colocalization studies, all images were acquired with the same excitation and detection parameters without reaching signal saturation. To perform pixel-topixel multiplication of 16-bit images using ImageJ, non-specific signal obtained from negative control micrographs (cells with only secondary antibodies) was subtracted from the "green micrographs" on coverslips treated with primary and secondary antibodies.

Background noise in "red micrographs" (where Alexa Fluor® 647 Phalloidin was used) was measured outside the cells and subtracted from each image. For each cell, the "green channel image" was multiplied with the red channel using the "multiplication" option of the Image Calculator plugin in ImageJ and the result was represented as a "32-bit float" image with a "Ratio" lookup table. In the resulting image, the intensity value of a given pixel is therefore the results of the multiplication of the intensity values of the pixels with the same coordinates from the "red channel" and the "green channel". For the measurement of SZ sizes on bone slices, contrast minima per field were raised so that bone autofluorescence was no more observed. For podosome core size measurement, background noise was measured outside of the cells and subtracted from entire images. The podosome core was delimited based on local intensity thresholds using the Find Peaks plugin in ImageJ with the following parameters:

Gaussian blur "0.72", Background method: "Std.Dev above mean", Background parameter "0", Statistics mode "Both", Background level "24", Search method "Half peak value", Remove edge maxima "On".

Fluorescence recovery after photobleaching (FRAP) acquisition and analysis

Day-4 FL-OCs were infected with 2 nd generation packaging viral particles (provided by the "Anira-Vectorologie" platform, UMS3444, Lyon-Gerland, France) containing a pLVX-LifeAct-mCherry lentiviral construct (obtained by inserting LifeAct coding sequence between EcoR1 and BamH1 site in pLVX-mCherry-N1 vector, #632562, Clonetech, USA) at a multiplicity of infection of 10:1 in 1ml per well of a 6-well plate. OCs were incubated overnight and medium was changed in the morning. OCs with clusters and rings were fluorescent on day 6 (48h post infection) and imaged using a DMI4000 microscope (Leica Microsystems, Germany) equipped with a spinning disk unit and a FRAP module piloted by iLas2 software (Roper Scientific, USA). mCherry was excited with a 561nm laser and bleached with a 473nm laser at 100% in 3 iterations. Acquisition was optimized to obtain maximal signal without any saturated pixels and defined in 3 temporal phases: Prebleach phase (5 acquisitions, 5sec time interval between consecutive acquisitions), first postbleach phase (20 acquisitions, 3sec interval) and second postbleach phase (11 acquisitions, 30sec interval). The mean relative fluorescence intensity (RFI) of a given region of interest was measured and normalized as mentioned in [START_REF] Negi | Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23[END_REF]. The obtained RFIs were curve fitted and plotted using the formula in [START_REF] Negi | Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23[END_REF] with consideration of the guidelines in [START_REF] Hardy | Fluorescence recovery after photobleaching (FRAP) with a focus on F-actin[END_REF]. Statistical analysis of recovery half-times was calculated from data fitting using the "Do the best fit values of selected parameters differ between datasets differ" function in Prism 6 (Graphpad software, USA) [START_REF] Weisswange | The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility[END_REF].

Immunoblotting

Day-5 BM-derived OCs (BM-OCs) were lysed in 2X Lameli sampling buffer, ran on SDS-PAGE and blotted on PVDF membranes. Primary antibodies were: anti-cofilin (1:1000, clone we found that increased differentiation but not resorption capacity of OCs was the reason behind this bone loss. RA-induced osteoclastogenesis mainly consisted of three phases. First, autoantibodies that rise years before the onset of RA recognize a genetically mutated and post-translationally modified protein, mutated citrullinated vimentin (MCV), in OCs. This recognition stimulates OC secretion of an immune cytokine TNF-α, which known for its acute osteoclastogenic potential. Finally, by an amplified autocrine loop, TNF-α not only induces OC precursor proliferation and differentiation, but also promotes its own expression in these cells [START_REF] Harre | Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin[END_REF]. Therefore, this study constituted the first link between the three main elements in RA: the major autoantibody (anti-MCV), the prevalent clinical aspect (bone loss) and the dominant cytokine (TNF-α) (Figure 17).

Although it is now clear that MCV can be exported to the surface of OC, future efforts should be made to elucidate the molecular mechanisms induced by these autoantibodies after they bind MCV at the surface of the cell. Autoantibodies against citrullinated proteins (ACPAs), with high specificity against citrullinated vimentin are produced by plasma cells. These ACPAs bind to osteoclast precursor cells and induce the release of TNF, which in turn enhances the differentiation of these cells into mature osteoclasts [START_REF] Schett | Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment[END_REF].

ORGANIZATION OF THE OSTEOCLAST CYTOSKELETON DURING BONE RESORPTION

Differentiated OCs dispose of a unique cytoskeletal machinery that allows them to degrade bone. Although podosome formation is not exclusive to OCs, these cells are the only ones that arrange their podosomes to form sealing zones (SZs). As its name suggests, the role of the SZ is to ensure proper confinement ("sealing") of degradation molecules secreted via the ruffled border into the subjacent resorption lacuna. In fact, the formation of the SZ is concomitant with OC apico-basal polarization and subcellular compartmentalization. Not only does the OC form a ruffled border that is characterized by vesicular secretion and proton pump activity and surrounded by a SZ, the cell also commits to other resorption-related functions such as microtubule stabilization and transcytosis through the apical pole. Furthermore, the OC also transitions into an active podosome-driven migration phase between two resorption phases, thus adding a second type of polarization, i.e. antero-lateral, to the dynamic process of bone resorption. Given its involvement in all the processes of resorption, the OC cytoskeleton was the main subject of research in this thesis. Discussed below are the significant findings from the two main studies, Hu et al and Georgess et al: how they elucidate some aspects of OC function but also why they make grounds for new perspectives.

Novel actin-organizing genes indentified in osteoclasts

With the perspective of finding new pathways involved in OC function, we have performed a differential transcriptomic screen comparing, on one hand, bone resorbing human OCs and, on the other hand, non-resorbing multinucleated cells from the same OC precursors. We were then able to establish a list of 115 genes that were highly and exclusively expressed in OCs.

Gene Ontology annotation of these genes has revealed that this list contained an enrichment of certain cellular functions in OCs compared to the percentage of the functions in the Human genome. One of the enriched functions was "Cytoskeleton binding" including is sub-category "Actin binding", thus highlighting the importance of the cytoskeleton in general and actin filaments in particular to OCs. Additionally, other enriched functions never before addressed in OC have been identified, e.g. "Active transporter activity".

In a second step of this comparative study, we verified with higher quantitative precision the expression levels of the 115 genes in the same set of cells used for the transcriptome analysis.

This allowed the validation of 6 candidate genes (out of the original 115) as OC-exclusive genes in our model: Tm4sf1, Pls3, Myo1b, AK5, LNX1 and RhoE. Previous studies have provided data linking these genes either with OCs and/or with the actin cytoskeleton in other cells. This highlights once more the necessity of tight cytoskeletal regulation in OCs. PLS3, a member of the fimbrin family known for organizing actin filaments, has been shown to colocalize with OC podosomes but how it might actually regulate podosomes is unknown.

AK5 expression is RANKL-dependent in osteosarcoma cells but its function in OCs has not been addressed. TM4SF1, a member of the tetraspanin family described for their interaction with integrins at the cell surface, organizes adhesion structures and cell migration.

The role of podosomes in adhesion and spreading

Before the OC migrates and resorbs bone, it needs to adhere the extracellular matrix. The relationship between cell adhesion and podosome organization has been long admitted by scientists, but its details were poorly understood. We have shown that, as soon as the OC contacts it substrate but before it is properly spread, intracellular waves of unorganized actin are pooled in membrane protrusions at the edge of the cells. After 10-20 minutes, podosome assembly begins and the cell membrane is laterally stretched to make even bigger protrusions than ensure the complete spreading of the cell. Now given the fact that actin recruitment in podosomes occurs at the level of the adhesion plaque at the immediate vicinity of the cell membrane (see Chapter I -sections III.2 and III.3), it would mean that podosome assembly (and namely podosome core growth) in a bottom-up manner exerts a force on the membrane itself, pushing it away from the center of the cell and thus induces spreading. Our findings converge with other data describing matrix-dependent podosome dynamics [START_REF] Labernadie | Dynamics of podosome stiffness revealed by atomic force microscopy[END_REF][START_REF] Van Den Dries | Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations[END_REF].

Conversely, the inhibition of integrins in an already-spread OC results in two sequential phases of detachment: (1) within the first seconds, podosomes disassemble but the cell remains spread, then (2) the cell retracts and detaches from the substrate. These observations allow the conclusion that podosomes are not the only mediators of adhesion in OCs, at least in a short period of time, but that they greatly enhance and maintain it.

Osteoclast saltatory migration

An adherent and spread OC forms podosomes that grow in a confined space during their assembly phase. The growth of podosomes in close vicinity within a cluster leads to them "pushing" each other, thus resulting in a ring pattern. While rings expand by rapid podosome assembly and disassembly in an outward radial direction, they apply traction forces on the matrix thus steering a part of the cell that becomes a leading edge. Our observations have

shown that, within one OC, at least two podosome rings grow simultaneously, hence creating two leading edges at opposite directions. At this point, the migratory OC adopts a triangular form with two tips occupied by podosome rings and the third tip constituting the trailing edge of the cell. Due to the transient nature of rings, one of the two migratory rings eventually collapses before the other. The most stable ring then determines the migratory direction of the entire cell, after a characteristic delay period, in following that of its proper growth. These events translate into a cyclic biphasic migration scheme: (1) Straightforward movement:

while the two rings grow at the front of the cell (in positions similar to two tips of a triangle) and the trailing edge of the OC passively follows behind (positioned at the third tip of the triangle), the OC moves forward in a straight direction, then (2) Angular switch: the rings reach a maximal distance between them, they stretch and flatten the triangle, one rings collapses and the other determines the sudden "jump" of the entire cell in its direction. This jump has a characteristic deviation of 90° from the previous straightforward phase direction and marks the start of a new straightforward phase which will eventually perpendicularly deviate from itself. The cycling of angular switching in the OC trajectory allows for a large mean squared displacement, which is probably needed by the OC to cover a maximal surface area in the search for spots to degrade.

Although the saltatory migration of OC is intrinsically heterogeneous, its characteristic parameters can be experimentally quantified. In the normal situation, the OC migrates at an average instantaneous velocity of 0.8µm/sec and with a track persistence value of 0.45 ("0" being a constant rotation of the cell around itself and "1" being a perfectly straight line). From now on, these parameters can be used by OC biologists for quantitative assessment of OC migration in different experimental conditions. We have used them to assay for the role of RhoE in OC migration. For example, while investigating the function of RhoE in OCs, we saw that RhoE -/-OCs migrated with reduced instantaneous velocity compared to wildtype

OCs, but also that their persistence was reduced due to increased angular turns and decreased straightforward motion. Therefore RhoE deletion in OCs changed their migration from saltatory to simply "turning in circles" around themselves.

Regulation of podosome internal dynamics and collective patterning

Podosome dynamics need to be tightly regulated by the OC in order to properly organize into a SZ. The presence of classical structural markers such as actin, vinculin, paxillin and cortactin within podosomes of OCs or of other multinucleated giant cells from the same lineage can lead to circular patterning of podosomes but is insufficient to form a functional SZ capable of ensuring bone resorption. This finding supports the existence of OC-specific molecular mechanisms to podosomes that distally support SZ formation by upstream regulation of podosomal proteins. Indeed, although their upstream distal regulators are not always known, some podosomal proteins like cortactin and cofilin have been previously

shown to undergo posttranslational modification, such as tyrosine phosphorylation, which have consequences on actin dynamics in podosome. The idea of "distal upstream regulation" is further confirmed by data derived from a single bone marrow-derived OC population. In absence of the expression of RhoE, which is not a structural component of the podosome, podosomes can still assemble but SZ formation is abnormal. In fact, RhoE regulates podosomes by modulating cofilin-mediated actin turnover.

The resorption-migration cycle

In order for OCs to efficiently degrade bone and ensure its renewal, this cell needs to fulfill two functions: resorption and migration to find new resorption target sites. This would mean that the podosomes in OCs needed to maintain dynamic patterning transition between SZs rings. The data obtained on the role of RhoE in OCs has demonstrated how one molecular pathway links internal podosome dynamics to both ring-driven migration and SZ-mediated resorption. This provides proof, additionally to that already found in the literature, of the necessity of podosomes to these cellular processes. However, the gathered data did not bring new insight as to the spatio-temporal regulation of the hypothetical resorption-migration cycle. To address this aspect, one would need to distinguish podosome migratory rings from SZs, which is not possible in a static, fixed setting because they are both circular podosomal patterns that contain the same proteins. However, the discrimination between rings and SZ could be addressed by timelapse microscopy of OCs with fluorescently labeled actin and seeded on bone (Figure 18). The quantification of morphological and kinetic parameters such as pattern lifespan and size, circular expansion rate, pattern displacement, cell displacement, etc…, followed by principal component analysis should allow the identification of two subsets (rings and SZ) within these circular podosome patterns. This approach might provide, once and for all, a clear demonstration of how the OC coordinates displacement with resorption. Between the 2 nd to the 3 rd position (magenta), at 19 min and 39 min 40 sec, respectively, podosome assembly site have been partially conserved and the circular pattern has expanded.

This suggests ring stabilization and transition to SZ.

Signaling in osteoclasts

Given the dynamic nature of the "OC cytoskeleton in action", its study using biophysical approaches is both intriguing and insightful. However, the increasingly acquired data demands biochemical explanations: What are the signals transmitted to the OC cytoskeleton?

What are the genes and proteins that transmit them? How does the OC synchronize its cytoskeleton with its entire polarization?

Although these questions are very broad, partial answers might be provided by identifying new molecular pathways in OCs. The list of candidate genes that we have provided could serve as starting points for the identification of such pathways.

Another source of insight into OC signaling might be the characterization of the interplay between members of the Rho GTPase family in OCs. These small proteins are involved in a variety of cellular mechanisms and act as differential molecular switches in different parts of the cell in response to different intra-and extracellular stimuli.

We Another GTPase that has been shown important in OCs is Rac. Rac has been shown to be essential for OCs and to be activated by Dock5 in OCs.

These three GTPases, Rho, RhoE and Rac, are present in OCs. The functions of Rho and Rac is conveyed by their ability to catalyze GTP to GDP and can be therefore be induced or repressed in different parts of the cell. These 2 GTPases are also known for their antagonistic functions vis-à-vis one another. RhoE, on the other hand, is an atypical Rho family member, that is constitutively active. Its lack of GTPase activity allows is be continuously linked to GTP and therefore remains active, although post-translational modifications have been shown

to modulate its activity. RhoE and Rho are also known to have antagonistic functions pertaining to the actin cytoskeleton, mainly through modulating Rock activity.

A more detailed molecular scheme is needed for a full understanding of the GTPases in OCs:

how they are spatially and temporally expressed, activated and localized along the OC lifespan.

Figure 1 .

 1 Figure 1. Chronological order of phases in a bone remodeling cycle. ...................................................17

Figure 2 .

 2 Figure 2. Histological sections of human bone remodeling units. .........................................................17

Figure 3 .

 3 Figure 3. Evolution of periarticular bone erosion in the course of rheumatoid arthritis. .......................20 Figure 4. The first documented micrograph of an osteoclast in a histological section by Pr. Geddes..23 Figure 5. Schematic representation of major ligand-receptor systems in OC differentiation and function. .........................................................................................................................................28 Figure 6. Schematic of signaling cascades in osteoclast differentiation. ..............................................33 Figure 7. Regulation of OC differentiation. ...........................................................................................35 Figure 8. Integrins exhibit three distinct conformations correlated with binding affinity. ....................43 Figure 9. General schema of podosome architecture. ............................................................................46 Figure 10. Model depicting the interplay of actin network and myosin IIA at podosomes. .................47 Figure 11. Schema of a mathematical model depicting steady-state actin turnover in the podosome...49 Figure 12. Current model of dynamic podosome patterning in OCs. ....................................................52 Figure 13. Model of osteoclast transmigration through cell layers.. ......................................................60 Figure 14. Transmission electron micrograph of a bone resorbing human osteoclast. ..........................62 Figure 15. Vesicular trafficking pathways in osteoclasts. ......................................................................63 Figure 16. The osteoclast resorption-migration model. .........................................................................64 Figure 17. The current model of autoantibody-induced osteoclastogenesis in RA..............................124 Figure 18. Merged micrographs of the actin cytoskeleton in a primary osteoclast in three time points. .....................................................................................................................................................129

Figure 1 .Figure 2 .

 12 Figure 1. Chronological order of phases in a bone remodeling cycle. The first step is resorption of old bone by OCs. Secondly, OC-like macrophages thought to be differentiated from circulating monocytes (Kotani et al., 2013) remove matrix debris during the reversal phase. Third, osteoblasts secrete proteins that form the osteoid layer (unmineralized bone), which is later mineralized by the same cells to become the new bone. Osteoblasts either remain on the surface of bone and become lining cells or are embeded within the bone matrix that they secreted to become osteocytes. Osteocytes contact both osteoclasts and osteoblasts and can regulate their activities. (http://imueos.wordpress.com/2010/09/29/bone-physiology/)

Figure 3 .

 3 Figure 3. Evolution of periarticular bone erosion in the course of rheumatoid arthritis (RA). During the preclinical phase of RA, autoantibodies that target citrullinated proteins, namely vimentin, are produced early on by plasma cells. Citrullination is posttranslational modification consisting of the replacement of an Arginine by a Citrulline. The anticitrullinated protein autoantibodies (ACPA) can stimulate the differentiation of osteoclasts and therefore lead to initial bone loss. These early changes may initiate in the bone marrow adjacent to the joint. Synovitis at the onset of clinical disease leads to production of cytokines

Figure 4 .

 4 Figure 4. The first documented micrograph of an osteoclast in a histological section by Pr. Geddes. The figure is explained by its legend from the original publication in 1913

Figure 5 .

 5 Figure 5. Schematic representation of major ligand-receptor systems in OC differentiation and function regulated by TNF-α, ODF/RANKL, and IL-1α. TNF-α and RANKL (also known as Osteoclast Differentiation Factor, ODF) independently stimulate osteoclast differentiation. OC differentiation induced by TNF-α occurs via TNFR1 and TNFR2 expressed by osteoclast precursors (here abbreviated as M-BMMϕ for Monocytes-

Figure 6 .

 6 Figure 6. Schematic of signaling cascades in osteoclast differentiation. Osteoclastogenesis is supported by osteoblasts or bone marrow stromal cells, which provide RANKL, M-CSF, and poorly characterized ligands for costimulatory receptors. RANKL binding to RANK results in the recruitment of TRAF6, which activates NF-κB and MAPKs. The induction of NFATc1, a key transcription factor for osteoclastogenesis, is dependent on the transcription factors AP-1 (containing c-Fos) and NF-κB. Costimulatory signals for RANK: immunoreceptors associated with ITAM-harboring adaptors stimulate Ca 2+ signaling.

Figure 7 .

 7 Figure 7. Regulation of OC differentiation. OCs are derived from myeloid precursors. Macrophage colony-stimulating factor (M-CSF) induces myeloid precursors to differentiate to osteoclast precursors that express RANK (Receptor activator of NF-κB) and TREM2 (Triggering receptor expressed by myeloid cells-2) receptors. Upon RANK ligand (RANKL) stimulation and ITAM (Immunoreceptor tyrosine-based activation motif) activation, osteoclast precursors undergo further differentiation to mononuclear osteoclasts with NFATc1 (Nuclear factor of activated T cells, cytoplasmic 1) induction and express osteoclast-related genes such as those encoding Tartrate-resitant acidic phosphatase (TRAP), cathepsin K (CtsK) and αvβ3. Mononuclear osteoclasts then fuse to multinuclear osteoclasts and function as polarized bone resorbing cells. This process of osteoclast differentiation is regulated by various transcription factors and exogenous factors at different stages. Inflammatory factors that promote osteoclastogenesis are shown in red. Inhibitors of osteoclastogenesis are shown in blue. Calc, calcitonin; Calc R, calcitonin receptor; CSF-1R, colony stimulating factor 1

Figure 8 .

 8 Figure 8. Integrins exhibit three distinct conformations correlated with binding affinity.Headpiece domains are depicted in black. In the low affinity conformation, the integrin leg domains are bent and the headpiece is closed. In the intermediate affinity conformation, the leg domains are extended and the headpiece is closed. In the high affinity conformation the leg domains are extended, and the headpiece is open[START_REF] Paradise | Acidic extracellular pH promotes activation of integrin alpha(v)beta(3)[END_REF].

Figure 9 .

 9 Figure 9. General schema of podosome architecture. The cloud subdomain of the podosome links the cell to the extracellular matrix (ECM) by integrins, and is contains F-actin (yellow) that is organized by adaptor (or plaque-type) proteins (like paxillin and talin) and crosslinked by myosin II. The core subdomain of the podosome is linked to the ECM via CD44 and contains dense network of branched actin (red). The cap subdomain rests on top of the F-actin dome of the core. Its function is not yet characterized(Linder et al., 2011a).

Figure 10 .

 10 Figure 10. Model depicting the interplay of actin network and myosin IIA at podosomes. The amount of core and ring components in existing podosomes fluctuates, reflecting their variable protruding activity over time. Paxillin and talin are already present at their maximum levels in small podosome cores. As a reaction to matrix rigidity, podosomes grow vertically

Figure 11 .

 11 Figure 11. Schema of a mathematical model depicting steady-state actin turnover in the podosome. For simplification purposes, the model is considered as consisting of 2 dynamics elements: actin and gelsolin. Actin polymerization takes place at the base of the podosome core (with a diameter = σ) by recruitment of actin monomers at a characteristic speed v. After reaching an inherent limit of height, these polymers are severed by gelsolin thus releasing smaller polymers into the podosome cloud. In the cloud, gelsolin (diffusion rate = D g ) cleaves again the small polymers (diffusion rate = D n ) at a defined severing rate β (Hu et al., 2011a).

  this pathway can temporally regulate podosome patterning during osteoclastogenesis and in a substrate-dependent manner. Signaling upstream of Rho and downstream of Rock though remain unclear.III.7. Podosome patterning in osteoclastsIII.7.1. Structural and kinetic propertiesRegardless of the debated importance of podosomes and adhesion in general to the OC differentiation process itself, these structures are undoubtedly crucial for the support of the mature function of OCs: bone resorption. As early as individual podosomes form within an OC, they are collectively and sequentially organized into patterns along the life of the same

Figure 12 .

 12 Figure 12. Current model of dynamic podosome patterning in OCs. Podosomes (surrounded by red dotted line) are collectively arranged into clusters the rings then finally either sealing zones (SZ) or SZ-like structures (also called "belts"). In the cluster, podosomes are grouped in close vicity as can be seen by immunofluorescence (IF) in a region of the cell and have a "relaxed" architecture as shown in the scanning electron micrograph (SEM). As podosomes transition from clusters to rings, transient cicrular patterns. Finally, rings can transform into SZs, if the osteoclast is seeded on bone or into SZ-like structures if the osteoclast is seeded on a non-mineralized substrate. The SZ ensures proper bone resorption by confining the degradation molecules secreted by the OC ruffled border into the resorption lacuna. The SZ-like structure is not associated with a functional activity and is at the extreme

Figure 13 .

 13 Figure 13. Model of osteoclast transmigration through cell layers. Multinucleated mature osteoclasts seeded on top of osteoblast layers (A) rapidly extend actin-rich protrusions through the stromal layer (B) until they contact the underlying substrate (C) on which they spread (D). Micrographs in insets: (*) show actin (red) and integrin β3 (green) in X/Y sections at the level indicated by black dotted lines. Left inset in (B) is a 3D reconstruction of the protrusion, bars: 5µm[START_REF] Saltel | Transmigration: a new property of mature multinucleated osteoclasts[END_REF].

Figure 14 .

 14 Figure 14. Transmission electron micrograph of a bone resorbing human osteoclast. The ruffled border (localised between the white arrows) penetrates deep into the bone matrix. An area of translucent vacuolar structures is visible directly adjacent to the ruffled border inside of the cell (indicated by the long black arrows). Numerous vacuolar (v) structures are also visible close to the basolateral plasma membrane; secretory-type vacuoles are also abundant (filled arrowhead). N: nucleus.[START_REF] Stenbeck | Formation and function of the ruffled border in osteoclasts[END_REF].

Figure 15 .

 15 Figure 15. Vesicular trafficking pathways in osteoclasts. The ruffled border is formed as a consequence of trafficking of vesicles in the endosomal pathway, and therefore has characteristics of a late endosomal membrane. A distinct biosynthetic pathway may also contribute to the formation of the ruffled border. Degradation products from the resorption process are removed from the resorption lacuna by a transcytotic path-way and released at the FSD, which exhibits characteristics of an apical membrane. Recently, a reverse pathway from the FSD to the ruffled border has been identified. FSD, functional secretory domain; BL, basolateral domain; SZ, sealing zone(Coxon and Taylor, 2008a).

Figure 16 .

 16 Figure 16. The osteoclast resorption-migration model. (A) Scanning electron micrograph of a resorbing osteoclast on dentine showing consecutive and partially overlapping circular resorption pits/lacunae (dotted yellow circles). (B) Resorption tracks of osteoclasts seeded on dentine, then removed for the staining of resorption tracks with Toluidine blue, again showing

Finally, we described

  the typical long-term OC migration trajectory and quantified displacement parameters in the perspective of establishing a physical correlation between the cell and its actin cytoskeleton. The displacement of the cellular actin mass always preceded the global cell displacement by a characteristic lag time, demonstrating once again that the actin-based structures are at the basis of OC movement. Moreover, the ring-dependent trajectory was characterized by periodic lateral "jumps" resulting from asymmetrical stability of rings in the migrating OC.Finally, the mechanical data obtained from this study, combined with previously demonstrated data on podosome dynamics, allowed us to form a working model that governs OC motility. The model elaborates that podosomes grow in close vicinity during assembly on a confined area, such as the area of a cluster or a ring. Hence, because of a steric frustration event that accompanies their growth, podosomes repel each other and create the tension needed for migration. migration and SZ formation are essential to OC-mediated bone resorption.

Finally, we

  ABBREVIATIONSBM: bone marrow OC(s): osteoclast(s) DC(s): immature dendritic cell(s) DC-MGC(s): multinucleated giant cell(s) derived from dendritic cells, not including OCs DC-17-MGC(s): multinucleated giant cell(s) derived from dendritic cells by stimulation with IL-17 DC-17 γ -MGC(s): multinucleated giant cell(s) derived from dendritic cells by stimulation with IL-17 and IFN-γ DC-OC: dendritic-cell derived osteoclast FL: Foetal liver FRAP: Fluorescence recovery after photobleaching IL-17: interleukin-17 IFN-γ: interferon-γ M-CSF: macrophage colony stimulating factor MGC(s): multinucleated giant cell(s) Mo: monocytes Mo-OC: monocyte-derived osteoclast RANKL: receptor activator of nuclear factor κB SZ: sealing zone TRAP: tartrate-resistant acid phosphatase gt: gene trap

Figure 1 .

 1 Figure 1. DC-17 γ -MGC, like OCs, are able to make podosomes but not functional SZs for resorption. (A) Cells seeded on a resorbable apatite-collagen coated matrix. As expected, DC-OCs form SZs (actin is in white in the left panel, in red in the middle panel) and are able to resorb bone (resorbed area in outlined with a white dotted line, right panel). DC-17 γ -MGCs make dysfunctional SZ structures and are unable to resorb the matrix. Yellow line outlines the cell periphery. Scale bar is 20µm. (B) Quantification of actin-containing structures and resorption areas per cell. Left: Percentage of fixed DC-OCs exhibiting SZs compared with DC-17 γ -MGCs exhibiting dysfunctional SZ structures. Middle: box-andwhiskers plot showing surface area of SZ and dysfunctional SZs. Represented on the plot are minimum and maximum values (whiskers), mean (+), median (middle line in grey box), and 25% and 75% quartiles (limits of grey box). Right: histogram of the resorption index (area of resorbed matrix per cell) of the two cell-types. Error bars are SEM.

Figure 2 .

 2 Figure 2. Cytoskeleton organization in DC-17 γ -MGC compared to Mo and DC-Ocs. Cells seeded on glass showing colocalization of paxillin or vinculin (green) with actin (red) at the podosome cloud (upper panel and middle panel, respectively) and cortactin (green) with actin (red) at the podosome core. Multiplication images where calculated pixel-to-pixel from actin combined with one podosomal protein at a time and are represented in 32-bit float images with "Ratio" lookup table. The calibration bar indicates the correspondence of false color to 32-bit pixel fluorescence intensities. Scale bar is 10µm in first 4 columns and 2µm in magnified square insets.

Figure 3 .

 3 Figure 3. Workflow and results of the comparative transcriptomic study used to probe for OC-specific genes. (A) In vitro treatments used for differentiation and fusion of human Mo into resorbing OCs, of immature DCs into resorbing OCs and of DCs into non resorbing MGCs by addition of IL-17 and/or IFN-γ. Genechip® Microarray technique was applied to total RNAs obtained from each of cell type followed by mathematical comparison to sort out 115 mRNAs highly and exclusively expressed in Mo-OCs and DC-OCs. Validation of the upregulation of the 115 genes in OCs by Taqman Low Density Array performed on cDNAs from Mo, DCs, Mo-OCs, DC-OCs, and DC-17ϒ-MGCs. Due to the high cut-off value for detection, only 56 genes out of 115 were found to be expressed in OCs. Among the these, 6 genes (grey columns, names in red) were undetected neither in Mo, DCs nor in MGCs. (B) Enriched Gene Ontology Molecular functions in the 115 OC-speficic genes compared to the

Figure 4 .

 4 Figure 4. RhoE is dispensable for primary murine OC differentiation and fusion but is required for bone resorption. (A) Micrographs showing TRAP staining of FL-OCs differentiated with M-CSF and RANKL on tissue culture-treated plates after 5, 6 and 8 days of culture. Scale bars are 200µm. Histogram shows a time-scale quantification of the number of OCs (TRAP-positive cells with 3 or more nuclei) per well on tissue culture plastic and bone slices. (B) Plot of the number of nuclei per OC as indicator of OC fusion on tissue culture-treated plastic. (C) Upper panel: micrographs and plot showing resorption index of OCs seeded for 8 days on bone. Resorbed bone is stained with toluidine blue (violet surface outline with white dotted lines). Scale bar is 200µm. Graphs shown mean ± SEM. Lower panel: micrographs and plot showing resorption index of OCs seeded for 48 hours on a bonemimicking substrate (OsteoAssay plates). Unresorbed matrix is stained with silver nitrate (grey). Resorbed surface is not stained (white). Scale bar is 500µm.

Figure 5 .

 5 Figure 5. RhoE gt/gt OCs exhibit smaller SZs. Micrographs of actin-stained OC seeded on cortical bone slices showing smaller SZs sizes. Yellow lines show cell periphery inferred from overcontrasted images. SZs were determined by their circular shape and strong fluorescence. SZ outlines were manually drawn and inner surface areas where quantified and calculated mean ± SEM was plotted in the graph. Scale bar is 50µm.

Figure 6 .

 6 Figure 6. Podosome size, number and superstructure patterning are regulated by RhoE in OCs. (A) Upper lane: micrographs of FL-OC podosomes stained with actin showing a reduced number of podosomes in RhoE gt/gt OCs Yellow line in the zoomed images (lower lane) shows cell periphery (Upper lane). Lower lane: zoom of white insets in upper lane using "Ratio" lookup table colors. White circles depict peripheries of podosome cores as identified by local intensity thresholds using ImageJ (see Materials and Methods) followed by manual curating. The calibration bar indicates the correspondence of false color to 16-bit pixel fluorescence intensities (B) Left: Whisker-box plot depicts minimum and maximum number of podosomes per cell (whiskers), 25% and 75% quartiles (lower and upper limits of boxes) and median (midline of boxes). Middle: Histogram showing mean podosome core size with SEM bars. Left: Frequency distribution histogram of podosome core sizes showing a shift toward bigger sizes in RhoE gt/gt OCs. Horizontal axis shows the centers of intervals. (C)

Figure 7 .

 7 Figure 7. FRAP of LifeAct-mCherry reveals slower turnover in podosome clusters and rings in RhoE gt/gt FL-OCs. (A) Micrographs of timeframes pre-and post-photobleaching of LifeAct-mCherry-expressing FL-OCs on day 5. Yellow insets show bleached areas. Scale bar is 10µm. (B) Mean fluorescence recovery of photobleached areas normalized to mean fluorescence of the same area before photobleaching and to the non-bleached remainder of the cell during recovery. Data was gathered from n>12 samples per genotype and per structure from 2 independent cultures. Each culture was differentiated using a pool of precursors from at least 3 animals of the same genotype. Dots are means of mean relative fluorescence. Lines are fitted curves. Bars are SD. (C) Actin recovery half-times calculated from recovery curves.Recovery half-times are increased in clusters from 9.8 sec ± 1.4 in RhoE +/+ to 19.7sec ± 3.5 in RhoE gt/gt OCs and in rings from 24 sec ± 3 in RhoE +/+ to 34.4 sec ± 3.8 in RhoE gt/gt .

Figure 8 .Figure 9 .

 89 Figure 8. RhoE gt/gt OCs show impaired migration. (A) Upper panel: micrographs of timelapse imaging showing day-5 Sp-OC migration tracks (red). Scale bar is 40µm. Lower panel: Representative rose plots of 5 RhoE +/+ and 5 RhoE gt/gt OC migration tracks for a total duration of 8h per track. (B) Plots of migration parameters of migrating OCs (RhoE +/+ : n-=84 tracks, 1870 time points; RhoE gt/gt : n=39 tracks, 1214 time points). Left: instantaneous velocity of displacement (i.e. speed between 2 consecutive positions). Middle: instantaneous persistence of OC migration (P i =D i /d i ). Right: Frequency distribution of angle change between 2 consecutive movements. Raw values are represented with dots and fitted with a Gaussian function. Error bars are SEM

Figure 17 .

 17 Figure 17. The current model of autoantibody-induced osteoclastogenesis in RA.

  It would be interesting to find if it is implicated in integrin signaling during OC migration. LNX1 is responsible for the ubiquitinilation of Src, a major podosome regulator. Myo1B, a member of the myosin superfamily, is localized in filopodia, protrusive adhesion-type structures, and regulates Arp2/3, a known podosome component, in Golgi-related actin foci. Finally, RhoE, a constitutively active GTP-binding protein of the Rho family, inhibits stress fibers and focal adhesions by inhibiting the Rho-Rock pathway. In summary, our comparative model has provided new putative regulators of OC pathways.Because of their high expression in OC and the cytoskeletal functions that these gene play in other cell types, their study should bring new information on the regulation of OC cytoskeleton.Considering the importance of RhoGTPase signaling pathways in OCs, we focused our interest on RhoE. We confirmed the importance of RhoE for podosome organization during bone resorption by its ability to inhibit Rock-activity in vitro. Details of the cellular and molecular mechanisms controlled by RhoE and how they have provided new insight into the OC mode of function are given in the subsequent sections of this chapter. In the future, investigation of the role of RhoE will evolve around its function as a physiological regulator of bone remodeling. Two distinct in vivo models should be used to investigate the physiological role of RhoE. First, RhoE OC-specific deletion in mice could be achieved by crossing RhoE-floxed mice with mice expressing Cre recombinase under the Cathepsin-K promoter, which is specific to OCs. Both mouse lines are available. The finding of an osteopetrotic bone phenotype in OC-specific RhoE-deleted mice would then be the ultimate proof of the dependency of bone resorption on this gene. The second model, which already exists and should be used for the in vivo investigation, is the RhoE ubiquitous knockout by genetrap (RhoE gt/gt ). These mice present severe developmental defects which lead to their premature postnatal death. Among the existing abnormalities, RhoE gt/gt mice are strikingly smaller at birth and display growth retardation compared to wildtype littermates. The underlying causes of this smaller size could be arrested chondrogenesis, arrested osteogenesis, and/or the absence of bone resorption, which is the first step of the cartilage-to-bone transition. Thus, to uncover the global role of RhoE in bone, its tissue-specific functions should be assessed. Histological analysis of bone sections should provide indications of RhoE-depleted OB differentiation and bone formation. Furthermore, micro-computed tomography (µCT) studies of bone tissues in each of the proposed murine models would finalize the analyses by allowing accurate quantification of bone histomorphometric parameters.

Figure 18 .

 18 Figure 18. Merged micrographs of the actin cytoskeleton in a primary osteoclast in three time points. A murine bone marrow-derived OC expressing fluorescent LifeAct (an F-actin probe) displays evolving circular podosome patterns (either ring or SZ) during resorption of bovine bone. Between the 1 st position at 0 min (red) to the 2 nd position at 19 min (green), de novo podosome assembly sites have moved suggesting a ring-driven migration phase.

  have provided a new molecular mechanism depicting the role of RhoE in the regulation of podosome dynamics and, subsequently, bone resorption. RhoE acts as an inhibitor of Rho kinase (Rock) and thus prevents Rock-mediated cofilin phosphorylation. Finally, nonphosphorylated cofilin ensures fast actin turnover in podosomes. Although cofilin has been confirmed as a Rock target and therefore an indirect target of RhoE, Rock has other downstream targets in OCs and differentially modulated by binding to Rho or to RhoE. In fact, while RhoE inhibits Rock, Rho promotes its. Besides Rock, Rho (A/B/C) can also act via other effectors in the OC such as mDia to regulate microtubules and podosome patterning.

  The cell biology of the OC is an exciting field of research. It allows the understanding of fundamental processes such as cell migration, fusion, matrix degradation, and cytoskeletal remodeling in a physiologically relevant context, that of bone remodeling and homeostasis.OCs differentiate and fuse from mononuclear monocytic precursors to become large multinucleated giant cells. After reaching maturity, these "big" cells roam on the bone surface and degrade it. Meticulous regulation of OC differentiation and mature function is necessary for a healthy bone tissue and, therefore, a healthy organism. Conversely, deregulations of OC processes can lead to bone pathologies. The general purpose of my thesis was to indentify new molecular mechanisms that regulate OC-mediated bone resorption with particular interest to OC differentiation and OC cytoskeletal organization.

OSTEOCLAST DIFFERENTIATION IN PATHOLOGY: THE LINK BETWEEN AUTOIMMUNITY AND BONE LOSS

Osteoclastogenesis is the first rate-determining step in bone resorption. While studying the mechanisms promoting massive bone loss in Rheumatoid Arthritis (RA) in vivo and in vitro,

Acknowledgments

ACKNOWLEDGMENTS The authors would like to acknowledge the contribution of the following members of SFR Biosciences Gerland-Lyon Sud (UMS344/US8) facilities: C. Lionnet at PLATIM platform for valuable input on microscopy, AniRA-PBES for animal care, AniRA-Vectorologie for lentiviral particle production, PAP platform for murine RANKL production. D.G. is an Early Stage Researcher at Tissue Transmigration Training Network (T3-Net) funded by the European Commission under Framework Program 7. D.G is also funded by Fondation-ARC ("Aides individuelles jeunes chercheurs", ref. DOC20120605264). This project has received research grants from Fondation-ARC ("Subvention fixes", ref. SFI20101201877), Fondation Arthritis Courtin and PRCEU-UCH22/12.

Abbreviations CHAPTER 2: RESULTS

Title:

Comparative transcriptomics reveals RhoE as a novel regulator of actin dynamics in bone resorbing osteoclasts.

Abstract

Osteoclasts (OCs) are multinucleated giant cells (MGCs) of the monocytic lineage, responsible for bone resorption. Podosomes are actin-rich adhesive structures important for OC functions because they pattern into "rings", that drive osteoclast migration, and into "sealing-zones" (SZs) that ensure optimal enzyme and proton confinement in the resorption lacuna. Although changes in actin dynamics during podosome patterning have been documented, the mechanisms that regulate these changes are largely unknown. From human monocytic precursors, we differentiated MGCs that express OC degradation enzymes, but are unable to resorb the mineral matrix. We demonstrated that, despite exhibiting bona fide podosomes, these cells presented dysfunctional SZs. We then performed two-step differential transcriptomic profiling of bone resorbing OCs versus non resorbing MGCs to generate a list of genes implicated in bone resorption. Among the generated list of candidate genes, we investigated the role of RhoE/Rnd3. Using primary RhoE-deficient OCs, we demonstrated that RhoE is indispensable for OC migration and bone resorption. Using FRAP analysis, we found that RhoE is required for maintaining fast actin turnover in podosomes probably by inhibiting Rock-dependent cofilin phosphorylation. Consequently, the role of RhoE in podosome dynamics is central for OC migration and SZ formation and therefore to bone resorption.

adhesions and stress fibers, we investigated its implication in OC podosome-related aspects such as size, actin dynamics, patterning and cell migration. Finally, given that RhoE interaction with Rock-I has been widely shown to be a central pathway of RhoE function [START_REF] Riento | RhoE binds to ROCK I and inhibits downstream signaling[END_REF][START_REF] Riento | Function and regulation of RhoE[END_REF], we checked whether RhoE function in OCs was Rock-dependent.

Y-27632, and probed by immunoblotting of total lysates the canonical phosphorylation sites of each of the two Rock targets: cofilin phosphorylation on serine 3 and Mypt1 phosphorylation on threonine 696 (Figure S3). We observed a decrease in p(Ser3)-Cofilin but not p(Th696)-Mypt1 ratios normalized to their respective total protein controls. Thus we validated cofilin as a downstream target of Rock in mature OCs.

Then, to investigate whether cofilin phosphorylation was altered in the absence of RhoE expression in a Rock-dependent manner, we probed p(Ser3)-cofilin levels in RhoE +/+ and RhoE gt/gt BM-OCs with or without inhibition of Rock activity by Y-27632. In the absence of Y-27632 treatment, p(Ser3)-cofilin was 2.3 times higher in OCs lacking RhoE expression than in wild type OCs. The addition of Y-27632 reduced cofilin in both situations whilst Rock-I levels did not vary in any of these conditions (Figure 9B) suggesting that the observed changes in cofilin phosphorylation were independent of Rock-I expression levels but dependent on its kinase activity. Taken together, these experiments revealed that, when expressed in OCs, RhoE partially blocks Rock-mediated cofilin phosphorylation and therefore the presence of active i.e. non-phosphorylated cofilin.

Ethics Statements

All animal procedures were approved by local ethics comitee for animal welfare of the Universidad CEU Cardenal Herrera, ID#CEBA03/2007,met the local guidelines, European regulations (EUdirective86/609) and standards for Use of Laboratory Animals nu 5388-01 (NIH).

Primary human cell cultures

All the human cell types used in this study were differentiated directly or indirectly from peripheral monocytes from healthy donor blood (Etablissement Français du Sang, Lyon Gerland, France). All the human recombinant cytokines used for human cultures were obtained from Peprotech (USA). Briefly, CD14 + Mo were isolated from blood after two successive gradient centrifugations in Leukocyte Separation Medium (CMSMSL01-01, Eurobio, France) and 50% Percoll [START_REF] Gallois | Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes[END_REF], respectively, followed by negative antibody selection. CD14 + CD16 + Mo were then differentiated into OCs in α-minimal essential medium (α-MEM) (22561, Life Technologies, USA) with 10% fetal bovine serum (Bio West, France), 100units/ml Penicillin, 100µg/ml Streptomycin (#15070-063, Life Technologies, USA), 2mM L-glutamine (#25030081, Life Technologies, USA) 50ng/ml human recombinant M-CSF and 30ng/ml human recombinant RANKL. After the first 3 days of culture, medium was changed every 48h with 25ng/ml M-CSF and 100ng/ml RANKL until the end of the culture (5 to 6 days). Mo were also differentiated in suspension into immature (i.e. CD1a + , CD14 -, CD16 -) DCs in RPMI with 10% fetal bovine serum, 10mM HEPES, 2mM L-glutamine, 100ng/ml GM-CSF and 10ng/ml IL-4 for 6 days. Experimental details of Mo isolation and differentiation and DC differentiation can be found in [START_REF] Gallois | Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes[END_REF]. DCs were then differentiated into either OCs using the same conditions as for Mo-OCs [START_REF] Rivollier | Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment[END_REF] or into different MGCs such as DC-γ17-MGC [START_REF] Coury | Langerhans cell histiocytosis reveals a new IL-17A-dependent pathway of dendritic cell fusion[END_REF] with or without IFN-γ.

Affymetrix GeneChip® assays and comparative analysis

Primary human cultures of the following cell types were performed from several donors: Mo