Anja Heinemann

Yinghong He

Elena Zimina

Melanie Boerries

Hauke Busch

Nadja Chmel

Thorsten Kurz

Dr Leena Bruckner-Tuderman
email: bruckner-tuderman@uniklinik-freiburg.de

John Wiley

Sons

Cristina Has

Induction of Phenotype Modifying Cytokines by FERMT1 Mutations

Keywords: Kindler syndrome, epidermolysis bullosa, fibrosis, TGF, cytokine, fermitin, kindlin Kindler syndrome, kindlin, fermitin, epidermolysis bullosa, fibrosis, TGFβ, cytokine

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INTRODUCTION

Kindlins are evolutionarily conserved FERM (Four-point-one, ezrin, radixin, moesin) domain-containing proteins, which have recently emerged as key regulators of integrin activation [START_REF] Moser | The tail of integrins, talin, and kindlins[END_REF]. Among them, kindlin-1 is expressed in epithelial cells, predominantly in the skin, the intestine and the kidney, and loss-of-function mutations in its gene FERMT1 (KIND1; MIM#607900) cause the Kindler syndrome (KS; MIM 173650). KS manifests with skin blistering in the childhood, but later photosensitivity and progressive generalized poikiloderma dominate the phenotype [START_REF] Fine | The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB[END_REF][START_REF] Lai-Cheong | Kindler syndrome: a focal adhesion genodermatosis[END_REF]. Interestingly, many patients develop symptoms like webbing of the fingers, pseudoainhum, ectropion, oesophageal or urethral stenosis (Has, et al., 2008a;[START_REF] Mansur | Novel and recurrent KIND1 mutations in two patients with Kindler syndrome and severe mucosal involvement[END_REF], which reflect mucocutaneous fibrosis. So far, > 40 distinct FERMT1 mutations have been reported including large deletions, splice site, nonsense, and frameshift mutations, all of them leading to premature termination codons and, consequently, to complete lack of kindlin-1 in epithelial cells [START_REF] Lai-Cheong | Kindler syndrome[END_REF].

Although all patients have null mutations, the severity of the symptoms spans a broad spectrum suggesting a contribution of secondary processes guided by modifying factors. In KS skin and in the kindlin-1 knockout mouse, disorganized basal keratinocytes lose their proper architecture, polarization and the boundary to the dermis and exhibit minimal proliferation [START_REF] Herz | Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes[END_REF]Ussar, et al., 2008). These abnormalities correlate strongly with in vitro findings on kindlin-1 deficient keratinocytes [START_REF] Has | Kindlin-1 Is required for RhoGTPase-mediated lamellipodia formation in keratinocytes[END_REF][START_REF] Herz | Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes[END_REF]Ussar, et al., 2008). However, since kindlin-1 is not expressed in fibroblasts, the mechanisms of the significant dermal part of the KS phenotype, i.e. the progressive soft tissue fibrosis in the skin and the oral, ophthalmic and urogenital mucosa remain unexplained.

The skin is a prime example of an organ where epithelial-mesenchymal communication pathways are essential for tissue development, homeostasis and regeneration. The epidermis, which is purely epithelial and avascular, and the mesenchymal dermis, which contains blood vessels, nerves and adnexal structures embedded in connective tissue, communicate via soluble mediators, such as cytokines and growth factors, to maintain physiological tissue functions [START_REF] Werner | Keratinocyte-fibroblast interactions in wound healing[END_REF].

Against this background, we addressed the pathogenesis of dermal changes in KS by exploring cytokine profiles of KS keratinocytes, by characterizing KS skin fibroblasts in vitro, and by validating the findings in KS skin in vivo. We show that kindlin-1 negative keratinocytes upregulate the expression of interleukin (IL)-24, IL-20, transforming growth factor-β2 (TGF-β2), interleukin-1 family member 5 (IL1F5), platelet derived growth factor B (PDGFB) and connective tissue growth factor (CTGF), and that KS fibroblasts exhibit an activated phenotype. These findings correlate with the presence of macrophages and mediators of fibrosis, like α-smooth muscle actin (SMA), TGF-β1, IL-6 and CTGF, in KS skin. Based on this we predict that mutations in the FERMT1 gene cause epithelial cell stress and, as a stress response, secretion of cytokines that mediate local inflammation and fibrosis.

MATERIAL AND METHODS

Patients and Mutation Detection

Nine KS patients with known FERMT1 mutations were included in this study (Table 1). After informed consent, EDTA-blood and skin samples were obtained for diagnostic purposes. Mutation detection was described in detail elsewhere (Has, et al., 2008a). The GenBank reference sequence was NM_017671.4. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence according to the HGVS guidelines. The study was approved by the ethics committee of the University of Freiburg and was conducted in accordance to the Helsinki protocols.

Cell Culture and Cell Treatments

Cell lines derived from normal human keratinocytes (NK) and KS patient 4 (KSK), as well as primary keratinocytes of KS patients 1 and 8, and of control individuals have been characterized previously [START_REF] Has | Kindlin-1 Is required for RhoGTPase-mediated lamellipodia formation in keratinocytes[END_REF]Has, et al., 2008a;[START_REF] Kern | Chronic colitis due to an epithelial barrier defect: the role of kindlin-1 isoforms[END_REF].

Keratinocytes were cultured in defined keratinocyte growth medium (KGM, Invitrogen, Karlsruhe, Germany). Cell lines were used for experiments in passages 5 to 18, and primary cells in passages 2 to 4. Primary dermal fibroblasts were isolated from biopsies of patients 1, 2 and 3 and of three age-matched controls, and grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10% fetal calf serum (FCS) and 2mM glutamine, penicillin G, streptomycin and amphotericin B. They were used for experiments in passages 2 to 5.

For cytokine treatment, primary normal fibroblasts (NF) were seeded in 6 well plates, and allowed to adhere overnight. The next day the medium was replaced with DMEM with F o r P e e r R e v i e w 6 1% FCS. Ten ng/ml of recombinant human IL-20 and IL-24, with or without 5 µg/ml of blocking antibodies to rhIL20 and rhIL24 (both from R&D Systems, Wiesbaden, Germany) were added. As a vehicle control the same amount of PBS was used. At different time points cells were harvested for RNA and protein analysis.

To evaluate potential paracrine mechanisms, NF were treated with keratinocyteconditioned media, and the expression of extracellular matrix proteins, collagen I, tenascin C and fibronectin, was assessed in NF media and lysates [START_REF] Ghaffari | Keratinocyte-conditioned media regulate collagen expression in dermal fibroblasts[END_REF]. To collect keratinocyte conditioned media, NK and KSK (in passages 10-15) were cultured in KGM without supplements for 24 hours. NF from two control individuals (passages 5) were treated with the above conditioned media for 24 and 48 hours in triplicate experiments. Media and lysates of NF were collected, and total protein was quantified as described below under "Protein Extraction and Immunoblotting". Normalized amounts of media and lysates were analyzed by immunoblotting.

Induction of Cell Stress

Cell stress was induced in vitro using three different experimental systems, cellular hypoxia, oxidative stress and ultraviolet (UV) irradiation. To mimic cellular hypoxia, confluent NK and KSK in KGM were treated with 1000 µM CoCl 2 [START_REF] Chen | IL-20 is regulated by hypoxia-inducible factor and up-regulated after experimental ischemic stroke[END_REF], or with vehicle for 1 hour. To induce oxidative stress, NK and KSK were incubated with 1000 µM H 2 O 2 or vehicle for 4 hours. For evaluation of effects of UV-irradiation, NK and KSK were treated with 30 or 60 mJ/cm 2 of UVB (Waldmann GmbH, VS-Schwenningen, Germany). Controls were treated identically but not irradiated. For each condition, three experiments were performed. RTqPCR reactions were done in duplicate with three different RNAs. For ELISA, keratinocyte lysates and media were collected from three different cultures. To ensure equal loading in semiquantitative RT-PCR, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was simultaneously amplified. The PCR products were assessed on 1.5% agarose gels. Real-time quantitative PCR (RTqPCR) was carried out using the Real-Time PCR Detection System (BioRad CFX96). The reactions were performed in 20 µl volume containing 15 ng of cDNA and ready to use iQ-SYBR Green Supermix (BioRad, München, Germany). The data were analyzed using the BioRad CFX Manager Software (version 1.5). The expression levels were calculated relative to those of hypoxanthine phosphoribosyltransferase (HPRT1) and of 18s RNA. Efficiencies were determined for each marker and shown to be close to the efficiency of the normalizing marker. Relative expression was determined as 2 -∆∆CT . Primer sequences are summarized in Supplementary Table 1. For each experiment, three different RNAs were used and each reaction was performed in duplicate.

Microarray Processing and Data Analysis

RNA concentration was determined using a UV-Vis spectrophotometer NanoDrop® ND-1000 (NanoDrop Technologies, Wilmington, USA). RNA integrity and quality were estimated on Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and RNA integrity number index was calculated for each sample using the Agilent 2100 Expert software. This provides a numerical assessment of the integrity of RNA that facilitates the standardization of the quality interpretation; for microarray processing, only RNAs with For the expression analyses we used the human gene expression microarrays from Agilent (Design ID 014850) based on the 4 x 44k design format. Sample labeling and hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol. Briefly, for the hybridization of the microarrays, 0.5 µg of total RNA was reverse transcribed into cDNA and labeled with Cy3 using the one-color Quick Amp Labeling Kit (Agilent). Hybridization was performed with 1.65 µg of labeled cDNA per array at a temperature of 65°C overnight. An Agilent G2565CA DNA microarray scanner was used to scan the arrays at a resolution of 5 µm. The Feature Extraction Software 9.5.3.1 was then used to process and analyze array images. The software returns a series of spot quality measures to evaluate the quality and reliability of spot intensity estimates. The raw data were analyzed using GeneSpring GX 10.0 (normalization: shift to 75.0 percentile, baseline transformation: median of all samples). The normalized data were filtered to exclude probes flagged "absent" in all samples. The remaining probes were tested for statistical significance of expression using a two sample t-test (unpaired) with an asymptotic p-value computation and a p-value cut-off of 0.05. Multiple testing correction was performed according to Benjamini-Hochberg (Benjamini and Hochberg, 1995; p-value cut off of 0.05). The cut off for the fold change is ≥2 (1 log 2 ) up-or down-regulation.

Protein Extraction and Immunoblotting

Confluent cell monolayers were lysed and homogenized in 25 mM Tris-HCl, pH 7,5, containing 0,1 M NaCl, 1% NP-40, 10 mM EDTA, 1 mM PEFA-Bloc, 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Calbiochem, Darmstadt, Germany). The culture media were collected and 10 mM EDTA, 1 mM PEFA-Bloc were added. The protein content was determined with a micro Lowry assay (DC Protein Assay, Bio-Rad, Munich, Germany). For immunoblotting, equal amounts of proteins were separated on 8 %, 10 % or 12% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies (Supplementary Table 2) for 2 hours. Visualization was with the ECL Plus system (Amersham, Freiburg, Germany). Band intensities were quantified with Gel-Pro Analyzer software (MediaCybernetics, Inc., Bethesda, MD, USA).

ELISA

For analysis of IL-20 and IL-24 levels secreted into media by NK and KSK under normal culture conditions and after UVB irradiation, a sandwich ELISA was performed. The human IL-20 ELISA Development Kit (Peprotech,Hamburg,Germany) was used according to the manufacturer's instructions. For IL-24 ELISA, the monoclonal capture antibody (clone 283161) and the biotinylated detection antibody from R&D Systems were used. Briefly, Maxisorp 96 well flat bottom plates (Nunc, Roskilde, Denmark) were coated with 0.5 µg/ml of the capture antibodies overnight. All steps were performed at room temperature and between all steps, the wells were washed four times with washing buffer (0.05% Tween in PBS). Coated wells were blocked for one hour with blocking buffer (1% BSA in PBS), and thereafter incubated for 3 hours with 100 µl of sample and standard (serial dilutions of recombinant IL-20 and IL-24, respectively). Detection was performed with 0.25 µg/ml of the detection antibodies, followed by avidin-HRP-conjugate and OPD-substrate. Optical density was measured at 450 nm with a reference at 650 nm over 30 min in 5 min intervals in a Tecan reader (Tecan, Austria). IL-20 and IL-24 levels in media were calculated based on the standard curves and normalized to the total protein amount in the corresponding lysates. Dermatohistopathology, immunohistochemistry and indirect immunofluorescence (IIF) were performed using standard techniques [START_REF] Herz | Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes[END_REF]. Briefly, for IIF, skin cryosections were fixed with acetone, incubated with 0.1% BSA / TBS for 30 minutes and then with primary antibodies over night (Supplementary Table 2).

For morphological analysis, fibroblasts were grown on 4 well glass chamber slides (Nunc, Langenselbold, Germany) coated with 40 µg/ml collagen I overnight. They were fixed with 2 % paraformaldehyde in PBS for 15 minutes, permeabilized with 0.1 % Triton X-100 and subjected to IIF staining with antibodies to SMA conjugated with Cy3 and TRITCconjugated phalloidin. The chamber slides were mounted in DAKO medium (Dako Cytomation, Hamburg, Germany), observed with epifluorescence microscopy (Zeiss Axio Imager, Zeiss, Germany), and the images were captured using Zeiss internal software. The fluorescence signal was quantified using the image processing software Image J (http://rsbweb.nih.gov/ij/docs/index.html), after background subtraction.

Contraction of Collagen Gels

The collagen gel contraction assays were performed as described [START_REF] Zhang | Interactions of primary fibroblasts and keratinocytes with extracellular matrix proteins: contribution of alpha2beta1 integrin[END_REF], with minor modifications. Briefly, NF and KSF were seeded at a density of 1.5 x 10 5 cells/ml into 60 mm bacteriological plates (4 ml/dish) in DMEM supplemented with 10% FCS, sodium ascorbate (50 µg/ml) and containing 0.60 mg/ml of acid-extracted collagen I from rat tails. The cultures were placed at 37°C to allow collagen polymerization, and gradual contraction was monitored by measuring gel diameter of triplicate setups at successive time points up to 72 hours.

Statistical Analysis

Results

Soft Tissue Fibrosis in Adults with KS

Nine patients with known FERMT1 mutations were included in this study. Their age, mutations and phenotypic features indicating fibrosis in skin and mucous membranes are summarized in Table 1. Patients 1 -4, 6 -8 have been described in detail before (Has, et al., 2008a;Has, et al., 2006;Has, et al., 2008b;[START_REF] Herz | Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes[END_REF][START_REF] Kern | Chronic colitis due to an epithelial barrier defect: the role of kindlin-1 isoforms[END_REF][START_REF] Mansur | Novel and recurrent KIND1 mutations in two patients with Kindler syndrome and severe mucosal involvement[END_REF]. Importantly, at the time of presentation and skin biopsy, none had blisters, wounds, or skin fragility. Three individuals were children of < 10 years of age (patients 1, 4 and 5).

Interestingly, they showed no clinical signs of fibrosis of the skin or mucous membranes. In contrast, adults aged 20 -32 years (patients 2, 3, 6-9) exhibited symptoms indicative of soft tissue fibrosis, including webbing and contractures of the fingers, pseudoainhum (Fig. 1A), poikiloderma (Fig. 1B,C), microstomy (Fig. 1B), esophageal stenosis, ectropion, urethral stenosis, and fusion of labia majora and minora. Patient 6 had been previously diagnosed with systemic sclerosis, but was reclassified as KS after disclosure of the FERMT1 mutation.

Dermatopathology of a biopsy obtained from sun protected skin on the trunk of patient 8 revealed changes characteristic of KS: epidermal atrophy, micro-blisters, pigmentary incontinence, occasional perivascular melanin deposits and dilated blood vessels (not shown).

Elastica van Gieson staining demonstrated a dramatic reduction of elastic fibres in the dermis and their replacement with non-elastic connective tissue (Fig. 1D andE), a finding typical of soft tissue fibrosis.

KS Keratinocytes Express Increased Amounts of Cytokines and Growth Factors

To dissect the particular contribution of keratinocytes to the initiation of fibrosis, oligonucleotide microarrays were used to compare the expression patterns in NK and KSK. These revealed significant upregulation of IL-24 (23.4 fold), IL-20 (17.4 fold), TGFB2 (6.5 fold) and IL1F5 (2.9 fold) in KSK. The upregulation was confirmed with both semiquantitative RT-PCR and RTqPCR using two different RNAs extracted from independent NK and KSK cultures, and RNA from primary keratinocytes of patients 1 and 8 (Fig. 2A andB). In addition, the mRNA levels of PDGFB and CTGF were analyzed by the above PCR techniques; both were increased in immortalized and primary KSK, as compared to their controls (Fig. 2A). The expression of TGFB1 was also analyzed, but no significant difference was observed.

KS Keratinocytes Respond to Cell Stress by Expressing IL-20 and IL-24

Both IL-20 and IL-24 are constitutively expressed in keratinocytes and, therefore, may have a role in epidermal homeostasis [START_REF] He | IL-24 transgenic mice: in vivo evidence of overlapping functions for IL-20, IL-22, and IL-24 in the epidermis[END_REF]. In KS, the homeostasis is presumably perturbed by fragility of basal epidermal keratinocytes and subsequent cell stress. Therefore, we used three methods to induce cellular stress in vitro and tested its effects on the expression of the two cytokines. Treatment of cultured keratinocytes with CoCl 2 , which mimics hypoxia and with UVB irradiation clearly induced expression of IL-20 and IL-24 in both NK and KSK, whereas H 2 O 2 , which causes oxidative stress increased IL-20 expression in KSK (Fig. 3A). Intriguingly, the induction was stronger in KSK than in NK, and IL-20 was stronger influenced than IL-24. To confirm these results on protein level, IL-20 and IL-24 were quantified in keratinocyte media by ELISA (Fig. 3B). IL-20 was 2.6 fold (156 ± 4 versus 60 ± 9 pg/100µg protein, p<0.001), and IL-24 was 5.3 fold (41 ± 12 versus 219 ± 42 pg/100µg protein) increased in KSK media compared to NK media, under normal cell culture conditions. Moreover, after UVB irradiation, both cytokines were significantly elevated in KSK, compared to NK media (IL-20: 276 ± 13 versus 86 ± 11, p<0.001; IL-24: 351 ± 35 versus 74 ± 37, p<0.05). [START_REF] Commins | The extended IL-10 superfamily: IL-10, IL-19, IL-20[END_REF]. Now we show that also fibroblasts express the type I receptor complex (Fig. 4A). The complex is functional, since stimulation of both NF and KS fibroblasts (KSF) with IL-20 and IL-24 led to phosphorylation of STAT3 (Fig. 4B). In a control experiment, blocking antibodies to both cytokines prevented the phosphorylation (not shown). These observations indicated that IL-20 and IL-24 secreted by keratinocytes may act both in an autocrine and in a paracrine manner. The paracrine epithelial-to-mesenchymal communication was also suggested by the fact that treatment of NF with IL-20 and IL-24 led to 3-4 fold upregulation of COL1A1 mRNA in these cells (Fig. 4C), thus directly connecting these interleukins to synthesis of collagen I in dermal fibroblasts.

KS Fibroblasts Exhibit Characteristics of Activated Fibroblasts

Next, primary early passage KSF from patients 1, 2 and 3 were compared with primary NF from age-matched controls. Semiquantitative RT-PCR analysis demonstrated that both cell types expressed similar amounts of kindlin-2 and α1, α2 and β1 integrin subunits, but had no detectable expression of kindlin-1 (not shown). Similarly, adhesion and proliferation of both cell types did not differ significantly (not shown). However, KSF were morphologically distinguishable in that they had larger surface areas than NF (mean surface area of all NF 2145 µm 2 ± 1233 versus all KSF 4796 µm 2 ± 3308, p<0.0001), and exhibited increased SMA positive stress fibres (not shown). Subsequently, immunoblot analysis 15 demonstrated that KSF contained 2.5-4 fold higher SMA levels and secreted significantly more collagen I and tenascin C than NF (Fig. 5A-C), both findings indicating an activated fibroblast phenotype consistent with the differentiation to myofibroblasts [START_REF] Kalluri | Fibroblasts in cancer[END_REF][START_REF] Tomasek | Myofibroblasts and mechano-regulation of connective tissue remodelling[END_REF].

Functionally, KSF contracted collagen gels stronger than NF (Fig. 5D). The fibroblastpopulated collagen gels represent a well accepted in vitro model for cell-matrix interactions during wound contraction and fibrotic processes [START_REF] Dallon | A review of fibroblast-populated collagen lattices[END_REF]. They were used to delineate the behaviour of the cells in a more in vivo-like environment. Gel contraction generally depends on cell contraction, cell tractional forces related to cell locomotion, and initial cell elongation and spreading [START_REF] Dallon | A review of fibroblast-populated collagen lattices[END_REF][START_REF] Ehrlich | Physiological variables affecting collagen lattice contraction by human dermal fibroblasts[END_REF]. In accordance with their activated phenotype, fibroblasts of all three KS patients reduced the size of collagen lattices stronger than their normal counterparts, as seen in fibrotic processes [START_REF] Tse | Enhanced Dupuytren's disease fibroblast populated collagen lattice contraction is independent of endogenous active TGF-beta2[END_REF][START_REF] Clark | Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract collagen matrices[END_REF].

KSK Conditioned Medium Induces Collagen I and Tenascin C Expression in NF

To assess the effects of soluble factors secreted by KSK on NF in vitro, NF were cultivated for up to 48 h in the presence of conditioned media collected either from KSK or NK. Interestingly, NF treated with KSK media, secreted about 1.5 fold more collagen I than NF treated with NK media, whereas tenascin C, which was present at low levels in NF treated with NK media, was strongly induced by KSK media (Fig. 6). Fibronectin expression and secretion were not influenced. This relatively small increase in collagen I, and the presence of tenascin C are consistent with a slowly progressive fibrosis, as observed in KS. To validate the in vitro data, we analysed skin samples of nine KS patients and found similar abnormalities in all of them. In addition to the general increase of non-elastic connective tissue in the dermis (Fig. 1E), there was strong deposition of tenascin-C beneath the basement membrane, irrespective of age of the patient or the site of the skin biopsy (Fig. 7A,B). Further, SMA was present in the papillary dermis of KS patients, not only in the blood vessels but also in dermal cells, confirming the presence of myofibroblasts (Fig. 7A,B).

Inflammation and Dermal Fibrosis in KS Skin in vivo

Inflammatory mediators, such as CD68, F4/80 and IL-10 positive macrophages were present in the dermis of all KS patients irrespective of age and site of the biopsy (Fig. 7C,D).

Quantification demonstrated that SMA expression increased with age, whereas the infiltration with CD68 positive macrophages was similar in all patients (not shown). In contrast, number and distribution of lymphocytes and granulocytes was similar in KS and control skin, as shown by staining with several cell-type specific antibodies (not shown). Importantly, three key markers of fibrosis, IL-6, TGF-β1, and CTGF were found in the dermis of KS patients, but not of controls (Fig. 7E-J). Interestingly, there was a strong expression of TNFa in KS skin (Fig. 7K,L).

Discussion

By demonstrating that dermal changes in KS are a consequence of primary kindlin-1 deficiency in keratinocytes we suggest paracrine epithelial-mesenchymal signals as novel phenotype modifiers in this disease (Fig. 8). FERMT1 mutations lead to upregulation of several cytokines in keratinocytes, similar to cytokine secretion induced by certain keratin mutations in epidermolysis bullosa simplex [START_REF] Coulombe | Epidermolysis bullosa simplex: a paradigm for disorders of tissue fragility[END_REF][START_REF] Roth | Cytokines as genetic modifiers in K5-/-mice and in human epidermolysis bullosa simplex[END_REF]. In both diseases the mutated proteins, kindlin-1 and keratin 5, are expressed only in epidermal keratinocytes and are associated with maintenance of functional cytoskeletal structures. As a consequence, their pathological alterations cause cell fragility and stress.

In the present study, expression array data disclosed IL-20 and IL-24 as major upregulated genes in KS keratinocytes, and the findings were validated with RTqPCR and confirmed on protein level by ELISA. Thus far, little has been known about the functions of these cytokines in the skin. A recent study reported their induction after skin injury [START_REF] Roupe | Injury is a major inducer of epidermal innate immune responses during wound healing[END_REF], and previous investigations suggested regulation of these interleukins through stress induced pathways [START_REF] Chen | IL-20 is regulated by hypoxia-inducible factor and up-regulated after experimental ischemic stroke[END_REF][START_REF] Hunt | Ultraviolet B light stimulates interleukin-20 expression by human epithelial keratinocytes[END_REF][START_REF] Otkjaer | The p38 MAPK regulates IL-24 expression by stabilization of the 3' UTR of IL-24 mRNA[END_REF][START_REF] Otkjaer | IL-20 gene expression is induced by IL-1beta through mitogen-activated protein kinase and NF-kappaB-dependent mechanisms[END_REF]. To corroborate this in KS, we used three different well-established methods -hypoxia, oxidative stress and UVB irradiation -to induce cell stress in normal and KS keratinocytes and showed that stress enhances expression and secretion of IL-20 and IL-24 by KS cells. The upregulation was stronger in KS keratinocytes, presumably because their pre-existing cytoskeletal abnormalities aggravated the stress. These results extend observations on stimulation of IL-20 upon hypoxia [START_REF] Chen | IL-20 is regulated by hypoxia-inducible factor and up-regulated after experimental ischemic stroke[END_REF] and UVB irradiation [START_REF] Hunt | Ultraviolet B light stimulates interleukin-20 expression by human epithelial keratinocytes[END_REF]. Both IL-20 and IL-24 expression is dependent on p38 MAPK [START_REF] Otkjaer | The p38 MAPK regulates IL-24 expression by stabilization of the 3' UTR of IL-24 mRNA[END_REF][START_REF] Otkjaer | IL-20 gene expression is induced by IL-1beta through mitogen-activated protein kinase and NF-kappaB-dependent mechanisms[END_REF], which is increased in KS cells (own unpublished results). Our data suggest that dermal fibroblasts are targets for IL-20 and IL-24. They have functional receptors for both cytokines, as shown by increased STAT3 phosphorylation in fibroblasts after treatment with IL-20 and IL-24. Interestingly, the cytokines also directly induced collagen I expression in fibroblasts. However, this is likely to be only one contributor to the dermal changes in KS, since fibrosis is a complex, orchestrated process with many players in vivo. Little is known about IL1F5, a member of the interleukin 1 cytokine family.

This cytokine has been shown to specifically inhibit the activation of NF-kappaB induced by interleukin 1 family, member 6 [START_REF] Blumberg | Opposing activities of two novel members of the IL-1 ligand family regulate skin inflammation[END_REF], but its contribution to KS phenotypes remains to be established.

A major mediator of fibrotic processes, TGF-β2, was among the factors upregulated in KS keratinocytes. Its role in KS skin may be similar to that in airway epithelium, where epithelial-derived TGF-β2 modulates the subepithelial extracellular matrix and mediates repair responses to epithelial injury [START_REF] Thompson | Epithelial-derived TGF-beta2 modulates basal and wound-healing subepithelial matrix homeostasis[END_REF]. Epithelial-mesenchymal interactions mediated by soluble factors can also occur in the other direction. An example of mesenchyme-to-epithelium signals is modulation of collagen VII synthesis in epidermal keratinocytes. During tissue repair, the expression of this essential dermal-epidermal adhesion protein is stimulated by mesenchyme-derived TGF-β [START_REF] Konig | Epithelial-mesenchymal interactions enhance expression of collagen VII in vitro[END_REF].

Interestingly, dermal fibrosis similar to KS has been observed in mice with keratinocyte-restricted deletion of integrin beta-1, which exhibited basement membrane defects, macrophage infiltration, increased expression of TGF-β1, CTGF and tenascin-C, and dermal fibrosis [START_REF] Brakebusch | Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes[END_REF]. In that case the molecular mechanisms were not explored. In light of our present findings and the fact that kindlin-1 is an intracellular ligand and modulator of β1 integrin, it is likely that β1 integrin deficiency causes epidermal fragility and cell stress leading to perturbed epithelial-mesenchymal interactions and fibrosis in the dermis. A significant contribution to soft tissue fibrosis comes from chronic inflammatory processes, which are known to be the cause of fibrosis in various tissues and organs [START_REF] Wynn | Cellular and molecular mechanisms of fibrosis[END_REF]. Our prediction is that in KS chronic mild inflammation is followed by progressive fibrosis in the dermis, presumably as a result of the expression and activation of TGF-β, CTGF and IL-6 [START_REF] Hinz | Formation and function of the myofibroblast during tissue repair[END_REF]. These cytokines play an important role in the induction of extracellular matrix synthesis by fibroblasts and thus in the pathogenesis of the fibrotic diseases (reviewed by [START_REF] Hinz | Formation and function of the myofibroblast during tissue repair[END_REF][START_REF] Wynn | Cellular and molecular mechanisms of fibrosis[END_REF]. In KS, the inflammation seems to have two components: on one hand, the secretion of cytokines by kindlin-1 null keratinocytes and, on the other hand, the infiltration with macrophages. The massive upregulation of tenascin C, which is not seen in normal skin [START_REF] Schenk | Dermo-epidermal separation is associated with induced tenascin expression in human skin[END_REF], is likely to reflect continuous tissue and basement membrane remodelling processes in KS. It is difficult to establish the precise contribution of each pathogenic factor. Probably, cumulative effects maintain profibrotic loops leading to a slowly progressive fibrosis. The clinical consequences are extensive skin and mucous membrane lesions and slight stiffening of the skin, similar to scleroderma, a complex, acquired connective tissue disorder characterized by excessive extracellular matrix deposition in the dermis [START_REF] Abraham | Overview of pathogenesis of systemic sclerosis[END_REF][START_REF] Varga | Systemic sclerosis: a prototypic multisystem fibrotic disorder[END_REF]. Incidentally, patient 6 of the present study had received immunosuppressive drugs as scleroderma treatment for several years, before KS was diagnosed. A similar situation was reported recently in an other KS patient [START_REF] Has | Mild Clinical Phenotype of Kindler Syndrome Associated with Late Diagnosis and Skin Cancer[END_REF]. The fact that cytokine-mediated alterations of epithelial-mesenchymal signals have now been identified as phenotype modifiers in KS will improve understanding of the molecular disease mechanisms, prevent unnecessary therapeutic exposures and lay a basis for design of novel biological therapies. These target fibroblasts and activate them to express SMA and to increase the production of collagen I. Macrophages are recruited and express IL-10, a marker for regulatory macrophages [START_REF] Mosser | Exploring the full spectrum of macrophage activation[END_REF]. TGF-β1, CTGF and IL-6 stimulate fibroblasts to differentiate into myofibroblasts and to synthesize more extracellular matrix proteins, including tenascin-C and collagen I. As epithelial-mesenchymal interactions are bidirectional, it is likely that the altered dermal environment also influences keratinocyte functions, thus contributing to the development of the fibrotic skin phenotype over many years, as seen in adult KS patients. E, F: Abundant IL-6 in the dermis in KS skin, but not in control skin. G, H: The presence of active TGF-β1 is increased in KS skin, as compared to normal skin. I, J: The downstream effector of TGF-β1, CTGF, is found in KS skin but not in normal skin. In panels D, F, H and J, insets represent 2-fold magnifications of the areas marked with a dotted line. K, L: TNFα is not detected in normal human skin, but is strongly expressed in KS skin, mainly in the epidermis. Nuclei are stained with DAPI (blue). Bar for all panels = 50 µm.

  -Transcription PCR and Real-Time Quantitative PCRTotal RNA was extracted with QIAmp RNA blood mini kit (Qiagen, Hilden, Germany) from subconfluent cells. Reverse transcription (RT) was performed with Advantage RT-for-PCR Kit from 500 ng total RNA (BD Biosciences, Heidelberg, Germany).

  .0 were further processed to reduce experimental biases due to poor RNA quality.

  differences was determined using Student's test or ANOVA (for Fig.5) with a statistical software in GraphPad Prism. In figures 2B, 3A, 4C and 5A and B, control samples (untreated NK or NF, respectively) were set to 1, all other samples were indicated as fold change compared to control.

Fibroblasts

  Are Target Cells for IL-20 and IL-24 It was unclear whether dermal cells can respond to signals mediated by keratinocytederived IL-20 and IL-24. The common receptors for IL-20 and IL-24, type I composed of IL20R1 / IL20R2 and type II composed of IL22R1 / IL20R2 chains are expressed in keratinocytes, but not in immune cells

  supported by a grant from DFG (HA 5663/1-1) to C.H. and L.B.-T., by the International Kindler Syndrome Network Kindlernet grant from ERANET, project Nr. 01GM0812, by the Network Epidermolysis bullosa grant from the Federal Ministry for Education and Research (BMBF), project 9 (L.B.-T.), by the Excellence Initiative of the German Federal Governments and the Freiburg Institute for Advanced Studies FRIAS (H.B., M.B., L.B.-T.).

Figure 1 .Figure 2 .

 12 Figure Legends

Figure 3 .

 3 Figure 3. Cell stress triggers upregulation of IL-20 and IL-24 in keratinocytes. A: NK and

Figure 4 .

 4 Figure 4. Fibroblasts are target cells for IL-20 and IL-24. A: Semiquantitative RT-PCR

Figure 5 .

 5 Figure 5. KSF have an activated phenotype consistent with differentiation to myofibroblasts,

Figure 6 .

 6 Figure 6. Deposition of collagen I and production of tenascin C are induced by treatment of

Figure 7 .

 7 Figure 7. KS skin in situ is marked with inflammation and fibrosis. IIF analysis of normal

Figure 8 .

 8 Figure 8. A schematic model for the pathogenesis of fibrosis in KS. As the background to

Figure 1 .Figure 2 .

 12 Figure 1. The fibrotic phenotype in KS skin. A: Dorsal aspect of the left hand of the 20 year-old patient 6 showing skin atrophy, erythema and scaling, webbing of the fingers, severe nail dystrophy and pseudoainhum (fibrotic ring) of the fifth finger. B: The face of patient 6 resembles "scleroderma face" with tight skin, pointed nose, microstomy and teleangiectasia. C: Poikiloderma of the neck of the 28 year-old patient 9. D: Elastica van Gieson staining of control skin reveals abundant dark elastic fibers. E: Elastica van Gieson staining of the skin of the 27 year-old patient 8 demonstrates drastic reduction and abnormal aggregation of elastic fibres in the upper dermis. Bar for D and E = 100 µm

Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .

 34567 Figure 3. Cell stress triggers upregulation of IL-20 and IL-24 in keratinocytes. A: NK and KSK cell lines were treated with 1000µM CoCl2 (+) or with PBS (-) for 1h; 1000µM H2O2 (+) or with PBS (-) for 4h; irradiated with 60mJ/cm2 UVB (+) or not irradiated (-). Gene expression was quantified by RTqPCR. For each condition three RNAs isolated and transcribed independently were submitted in duplicate to the RTqPCR reaction. Values of untreated NK were arbitrarily set to 1, all other samples were indicated as fold change compared to control. B: The graphs show the amount of IL-20 (left) and IL-24 (right) in the culture media of NK and KSK, under normal culture conditions, and after UVB irradiation as measured by sandwich ELISA.

  Figure8. A schematic model for the pathogenesis of fibrosis in KS. As the background to delineate the microanatomical structures, immunohistochemical staining of KS skin with anti-CD68 antibodies was used. CD68 positive macrophages are labelled in red and nuclei in blue (E, epidermis; D, dermis). Kindlin-1 deficient keratinocytes respond to cell stress by secreting cytokines and growth factors, including IL-20, IL-24, IL1F5, TGF-β2, CTGF and PDGFB. These target fibroblasts and activate them to express SMA and to increase the production of collagen I. Macrophages are recruited and express IL-10, a marker for regulatory macrophages[START_REF] Mosser | Exploring the full spectrum of macrophage activation[END_REF]. TGF-β1, CTGF and IL-6 stimulate fibroblasts to differentiate into myofibroblasts and to synthesize more extracellular matrix proteins, including tenascin-C and collagen I. As epithelial-mesenchymal interactions are bi-directional, it is likely that the altered dermal environment also influences keratinocyte functions, thus contributing to the development of the fibrotic skin phenotype over many years, as seen in adult KS patients.

Table 1 .

 1 Mutations and main clinical features indicating fibrosis of skin and mucous membranes in KS patients investigated in this studyLegend: y, years; F, female; M, male; 1 if one mutation is mentioned, it is in a homozygous state; if two mutations are mentioned, the constellation is compound heterozygous; 2 with respect to GenBank AL118505.17. The GenBank reference sequence was NM_017671.4. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence according to the HGVS guidelines.Supplemetary Table 1. Primers used in this studySupplementary Table 2. Antibodies used in this study

	Patient Age(y),	Mutations	Mutations	Fibrosis in skin	Fibrosis in mucous membranes	Reference
		Gender	DNA 1	Protein
	1.	7, F	c.676dup	p.Gln226ProfsX17	Not present	Not present	Kern et al., 2007
	2.	28, F	g.80929_89169del 2	C-terminal truncation	Webbing and contractures of the fingers	Congenital vaginal and anal atresia, fusion	Has et al., 2008b
						of labia majora and minora
	3.	32, M	g.80929_89169del	C-terminal truncation	Webbing and contractures of the fingers,	Ectropion, urethral stenosis	Has et al., 2008b
					pseudoainhum
	4.	7, F	g.70250_74168 2 /	p.[Pro381Argfs+	Not present	Not present	Has et al., 2006
			c.910G>T	Glu304X]
	5.	10, M	c.328C>T	p.Arg110X	Not present	Not present	Has et al., 2009
	6.	20, F	c.676dup	p.Gln226ProfsX17	Webbing and contractures of the fingers,	Microstomy, esophageal stenosis, fusion	Mansur et al., 2007
					pseudoainhum	of labia majora and minora
	7.	25, F	g.70250_74168del	p.Pro381Argfs	Webbing of fingers	Microstomy, esophageal stenosis,	Has et al., 2006
						ectropion
	8.	27, M	c.1718+1G>A	C-terminal truncation	Webbing and contractures of the fingers,	Esophageal strictures, urethral meatal	Has et al., 2008
					pseudoainhum	stenosis
	9.	28, M	c.456dup	p.Asp153ArgfsX4	Webbing and contractures of the fingers	Urethral meatal stenosis, anal strictures	Has et al., 2009

Legend: IB, immunoblotting; IF, immunofluorescence

John Wiley & Sons, Inc.Human Mutation

Acknowledgments