Ananta Paine

Britta Eiz-Vesper

Rainer Blasczyk

Dr Stephan Immenschuh
email: immenschuh.stephan@mh-hannover.de

Signaling to Heme Oxygenase-1 and its Anti-Inflammatory Therapeutic Potential*

Keywords: Gene expression, heme oxygenase-1, immunomodulation, inflammation, oxidative stress, signaling

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. Induction of HO-1 protects against the cytotoxicity of oxidative stress and apoptotic cell death. More recently, HO-1 has been recognized to have major immunomodulatory and anti-inflammatory properties, which have been demonstrated in HO-1 knockout mice and a human case of genetic HO-1 deficiency. Beneficial protective effects of HO-1 in inflammation are not only mediated via enzymatic degradation of proinflammatory free heme, but also via production of the anti-inflammatory compounds bilirubin and carbon monoxide. The immunomodulatory role of HO-1 is associated with its cell type-specific functions in myeloid cells (including macrophages and monocytes) and in endothelial cells, as both cell types are crucially involved in initiating inflammatory responses. This review covers the molecular mechanisms and signaling pathways that are involved in HO-1 gene expression. In particular, it is discussed how key nuclear factors such as the redoxdependent transcriptional activators NF-E2 related factor 2 (Nrf2), NF-B and AP-1 along with the transcription repressor BTB and CNC homologue 1 (Bach1) mediate inducible HO-1 gene expression. The role of central pro-and anti-inflammatory cellular signaling cascades including p38 MAPK and phosphatidylinositol-3 kinase (PI3K)/Akt in HO-1 regulation is highlighted. Finally, we summarize emerging strategies that apply targeted pharmacological induction of HO-1 for therapeutic interventions in inflammatory conditions.

A c c e p t e d M a n u s c r i p t INTRODUCTION Heme oxygenase (HO) catalyzes the first and rate-limiting enzymatic step of heme degradation and produces carbon monoxide (CO), iron and biliverdin [START_REF] Tenhunen | The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase[END_REF][START_REF] Ryter | Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications[END_REF][START_REF] Maines | The heme oxygenase system: a regulator of second messenger gases[END_REF], which is converted into bilirubin (BR) via biliverdin reductase. Two genetically distinct HO isozymes, HO-1 and HO-2, are known. HO-2 represents the constitutive non-inducible isoform and is primarily expressed in brain and testis [START_REF] Trakshel | Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform[END_REF]. By contrast, the inducible isoform HO-1, which exhibits low basal expression levels in most cells and tissues, is highly up-regulated by a wide variety of oxidative stress stimuli. Due to its regulatory pattern, induction of HO-1 has generally been considered to be an adaptive cellular response against the toxicity of oxidative stress [START_REF] Keyse | Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts[END_REF][START_REF] Vile | Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts[END_REF][START_REF] Otterbein | Heme oxygenase: colors of defense against cellular stress[END_REF][START_REF] Choi | Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury[END_REF][START_REF] Immenschuh | Gene regulation of heme oxygenase-1 as a therapeutic target[END_REF][START_REF] Gozzelino | Mechanisms of cell protection by heme oxygenase-1[END_REF]. More recently, HO-1 has also been recognized to exhibit important immunmodulatory and anti-inflammatory functions. A potential link between HO-1 and inflammation has initially been shown in an animal model, in which specific up-regulation of HO enzyme activity attenuated complement-dependent inflammation [START_REF] Willis | Heme oxygenase: a novel target for the modulation of the inflammatory response[END_REF]. Shortly thereafter, it has been demonstrated in a HO-1 knockout mouse model that these animals develop a chronic inflammatory disease and are highly vulnerable to an experimental sepsis induced by the classical pro-inflammatory mediator endotoxin [START_REF] Poss | Reduced stress defense in heme oxygenase 1-deficient cells[END_REF]. Importantly, phenotypical alterations in the only known human case of genetic HO-1 deficiency are highly similar to those observed in HO-1 knockout mice [START_REF] Yachie | Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency[END_REF]. By contrast, HO-2 deficient mice have an intact immune system, but exhibit major neurological defects [START_REF] Burnett | Ejaculatory abnormalities in mice with targeted disruption of the gene for heme oxygenase-2[END_REF]. Independently, targeted overexpression of HO-1 has been shown to have beneficial effects in various experimental animal models of inflammation [START_REF] Ryter | Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications[END_REF][START_REF] Soares | Immunoregulatory effects of HO-1: how does it work?[END_REF]. Due to the critical role of HO-1 in immunological responses [START_REF] George | Suppression by CD4+CD25+ regulatory T cells is dependent on expression of heme oxygenase-1 in antigen-presenting cells[END_REF][START_REF] Remy | Carbon monoxide inhibits TLR-induced dendritic cell immunogenicity[END_REF][START_REF] Kapturczak | Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1-deficient mouse[END_REF], the mechanisms of its immunomodulatory functions are currently under intense investigation.

In this review, we discuss the current understanding of how HO-1 may mediate its antiinflammatory effects in myeloid and endothelial cells. Moreover, we summarize the regulatory role of major transcription factors (TFs) and signaling pathways that govern the inducible HO-1 gene expression. Finally, we highlight the therapeutic potential of targeted pharmacological induction of HO-1.

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Cell-specific immunomodulatory functions of HO-1 in myeloid and endothelial cells

Inflammation is a complex reaction of the immune system in vascularized tissues at sites of an infection, toxin exposure or cell injury. Although HO-1 is expressed in all cells and tissues the salutary anti-inflammatory effects of HO-1 appear to be critically dependent on its cell type-specific functions in myeloid and endothelial cells.

Myeloid cells

Myeloid cells comprise monocytes, macrophages and dendritic cells, which play crucial regulatory roles in the innate and adaptive immune system [START_REF] Geissmann | Development of monocytes, macrophages, and dendritic cells[END_REF]. For example, macrophages ingest and kill invading microorganisms as a first line of defense and are activated by various immunological stimuli such as microbial products and cytokines to initiate inflammatory immune responses [START_REF] Zhang | Macrophage activation by endogenous danger signals[END_REF]. In rodent macrophages, HO-1 has been shown to be up-regulated by lipopolysaccharide (LPS) [START_REF] Otterbein | Mechanism of hemoglobin-induced protection against endotoxemia in rats: a ferritin-independent pathway[END_REF][START_REF] Bauer | Expression pattern of heme oxygenase isoenzymes 1 and 2 in normal and stress-exposed rat liver[END_REF][START_REF] Immenschuh | Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells[END_REF], which then attenuates the expression of various proinflammatory genes including cyclooxygenase-2, inducible nitric oxide (NO) synthase (iNOS), tumor necrosis factor (TNF)- or interleukin (IL)-6 [START_REF] Kapturczak | Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1-deficient mouse[END_REF][START_REF] Otterbein | Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway[END_REF] (Figure 1). In contrast to the LPS-dependent induction of HO-1 in rodent macrophages, gene expression of HO-1 is down-regulated by treatment with LPS in human monocytes [START_REF] Miyazaki | Expression of heme oxygenase-1 in human leukemic cells and its regulation by transcriptional repressor Bach1[END_REF]. More recently, the myeloid cell-specific immunomodulatory functions of HO-1 have also been investigated in a conditional HO-1 knockout mouse model. Mice with myeloid cell-specific genetic HO-1 deficiency exhibited a defect of the interferon- pathway along with pathological immune responses in experimentally induced infections and an experimental autoimmune encephalomyelitis [START_REF] Tzima | Myeloid heme oxygenase-1 regulates innate immunity and autoimmunity by modulating IFN-beta production[END_REF]. Finally, HO-1 is also important for the function of dendritic cells, which are the main cell population for antigen-presentation and for intiation of adaptive immune responses. Specifically, targeted up-regulation of HO-1 has been shown to modulate maturation and cell-specific functions of dendritic cells in human and mouse models [START_REF] Remy | Carbon monoxide inhibits TLR-induced dendritic cell immunogenicity[END_REF][START_REF] Park | Heme Oxygenase-1 Expression in Murine Dendritic Cell Subpopulations. Effect on CD8+ Dendritic Cell Differentiation in Vivo[END_REF].

In conclusion, HO-1 appears to have versatile functions via immunomodulation of myeloid cells.

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Endothelial cells

Endothelial monolayers are intimately linked with inflammation, because they constitute a barrier between the peripheral blood stream and inflamed tissues. The endothelium regulates recruitment and transmigration of immunologically active blood cells such as polymorphonuclear leukocytes and T lymphocytes to the site of an inflammation [START_REF] Muller | Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response[END_REF][START_REF] Cook-Mills | Active participation of endothelial cells in inflammation[END_REF].

HO-1 has been shown to directly affect the cellular interactions of polymorphonuclear leukocytes with endothelial cells in an in vivo rat model, in which increased HO activity downregulated the adhesion of these cells during experimental oxidative stress conditions [START_REF] Hayashi | Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme[END_REF].

Independently, major pathological alterations of the endothelium have been observed in HO-1 knockout mice, in which endothelial cells were more susceptible to apoptotic cell death and denudation from the extracellular matrix [START_REF] True | Heme oxygenase-1 deficiency accelerates formation of arterial thrombosis through oxidative damage to the endothelium, which is rescued by inhaled carbon monoxide[END_REF]. Independently, anti-inflammatory endothelial protection via HO-1 has been shown to be mediated via its ability to down-regulate TNFinduced expression of various adhesion molecules [START_REF] Liu | Methemoglobin is a potent activator of endothelial cells by stimulating IL-6 and IL-8 production and E-selectin membrane expression[END_REF][START_REF] Soares | Heme oxygenase-1 modulates the expression of adhesion molecules associated with endothelial cell activation[END_REF]. More recently, it has been reported that HO-1 was involved in the recruitment of endothelial progenitor cells to the site of an experimental vascular injury in various animal models [START_REF] Wu | Heme oxygenase-1 increases endothelial progenitor cells[END_REF].

Thus, HO-1 counteracts inflammatory reactions via modulation of various endothelial cell functions.

Anti-inflammatory functions of HO-1: degradation of proinflammatory free heme and production of the anti-inflammatory compounds BR and CO

The mechanisms that mediate the anti-inflammatory effects of HO-1 are not understood in detail. It has been appreciated in recent years, however, that the enzymatic degradation of proinflammatory free heme and the production of the anti-inflammatory compounds biliverdin/BR and CO may play major roles to counteract inflammatory reactions.

Heme consists of a tetrapyrrole ring with a central iron ion and is an abundant compound in mammalians with contradictory biological functions. On the one hand, heme plays a physiological role for oxygen and mitochondrial electron transport as an essential prosthetic group of hemoglobin, myoglobin and cytochromes [START_REF] Wijayanti | Biology of heme in health and disease[END_REF][START_REF] Mense | Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases[END_REF]. On the other hand, non-protein A c c e p t e d M a n u s c r i p t 6 bound free heme is highly toxic as it may cause oxidative stress. Due to the prooxidant properties of free heme, which have been shown in various animal and cell culture models, the enzymatic synthesis and degradation of this molecule is tightly controlled [START_REF] Wijayanti | Biology of heme in health and disease[END_REF][START_REF] Ryter | The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro-and antioxidant properties[END_REF][START_REF] Ponka | Cell biology of heme[END_REF].

More recently, free heme has also been shown to have proinflammatory properties [START_REF] Gozzelino | Mechanisms of cell protection by heme oxygenase-1[END_REF][START_REF] Soares | Immunoregulatory effects of HO-1: how does it work?[END_REF][START_REF] Wagener | Different faces of the heme-heme oxygenase system in inflammation[END_REF]. For example, heme has been demonstrated to be responsible for the increased influx of leukocytes into organs during an experimental inflammation in vivo [START_REF] Wagener | Heme-induced cell adhesion in the pathogenesis of sickle-cell disease and inflammation[END_REF]. Detrimental proinflammatory effects of free heme have also been shown in an animal model of experimental cerebral malaria, in which the heme-dependent detrimental effects were more pronounced in HO-1 deficient mice [START_REF] Pamplona | Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria[END_REF]. Thus, it is conceivable that the enzymatic degradation of proinflammatory free heme via HO-1 plays a critical role for the antiinflammatory functions of HO-1 [START_REF] Gozzelino | Mechanisms of cell protection by heme oxygenase-1[END_REF][START_REF] Soares | Immunoregulatory effects of HO-1: how does it work?[END_REF][START_REF] Ryter | The heme synthesis and degradation pathways: role in oxidant sensitivity. Heme oxygenase has both pro-and antioxidant properties[END_REF][START_REF] Ponka | Cell biology of heme[END_REF][START_REF] Wagener | Different faces of the heme-heme oxygenase system in inflammation[END_REF].

The role of BR as a beneficial compound with potent antioxidant and anti-inflammatory effects has only been appreciated in recent years [START_REF] Stocker | Antioxidant activities of bile pigments[END_REF][START_REF] Kapitulnik | Pleiotropic functions of biliverdin reductase: cellular signaling and generation of cytoprotective and cytotoxic bilirubin[END_REF]. Protection against experimental inflammation via HO-1-derived biliverdin has been shown in animal models of proinflammatory cardiovascular [START_REF] Hayashi | Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme[END_REF] and gastrointestinal disorders [START_REF] Berberat | Heme oxygenase-1-generated biliverdin ameliorates experimental murine colitis[END_REF]. Independently, beneficial effects of BR have directly been implicated in the protection against endothelial activation and dysfunction in human aortic endothelial cells [START_REF] Kawamura | Bilirubin from heme oxygenase-1 attenuates vascular endothelial activation and dysfunction[END_REF]. Interestingly, BR has also been suggested to specifically reduce leukocyte transmigration to the site of an experimental inflammation via interaction with adhesion molecules [START_REF] Keshavan | Unconjugated bilirubin inhibits VCAM-1-mediated transendothelial leukocyte migration[END_REF].

Although CO is generally considered a toxic gas, it has been recognized to have major physiological functions as a signaling molecule [START_REF] Maines | The heme oxygenase system: a regulator of second messenger gases[END_REF][START_REF] Boczkowski | CO-metal interaction: Vital signaling from a lethal gas[END_REF][START_REF] Kim | CO as a cellular signaling molecule[END_REF]. Specifically, HO-1-derived CO has been shown to be involved in the regulation of apoptosis, vasodilation and inflammation. In an early report on the potential protective effects of this gas, administration of exogenous CO blocked the LPS-induced production of proinflammatory cytokines via modulation of p38

MAP kinase [START_REF] Otterbein | Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway[END_REF]. Similar to the signaling gas NO, CO up-regulates the production of cGMP and this mechanism has been implicated in other functions of CO such as vasodilation and blockage of smooth muscle cell proliferation. Major potential for future therapeutic A c c e p t e d M a n u s c r i p t 7 applications may have CO-releasing molecules (CORMs), which are compounds that deliver CO to its target sites without the toxicity of gaseous CO [START_REF] Motterlini | Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities[END_REF][START_REF] Motterlini | Therapeutic applications of carbon monoxidereleasing molecules[END_REF].

In summary, HO-1 counteracts inflammatory responses via metabolic conversion of proinflammatory free heme and production of the anti-inflammatory compounds BR and CO.

Redox-dependent TFs mediate the inducible HO-1 gene expression

Targeted modulation of HO-1 for potential anti-inflammatory therapeutic interventions not only requires detailed knowledge of the immunomodulatory effects of HO-1. To achieve this goal, it is also necessary to precisely understand the mechanisms that regulate HO-1 gene expression.

HO-1 is induced by a plethora of physiological and pathological stimuli including oxidative stress signals, cytokines, bacterial compounds and growth factors. HO-1 expression is primarily regulated on the transcriptional level and multiple cis-acting regulatory elements (REs) of the HO-1 promoter have been shown to mediate the basal and inducible HO-1 gene expression in different species (reviewed in [START_REF] Ryter | Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications[END_REF][START_REF] Choi | Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury[END_REF][START_REF] Immenschuh | Gene regulation of heme oxygenase-1 as a therapeutic target[END_REF][START_REF] Alam | How many transcription factors does it take to turn on the heme oxygenase-1 gene?[END_REF]). Two upstream enhancer regions, which are termed E1 and E2, play major functional roles for redox-dependent induction of HO-1 [START_REF] Alam | Multiple elements within the 5' distal enhancer of the mouse heme oxygenase-1 gene mediate induction by heavy metals[END_REF][START_REF] Alam | Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and inducer-dependent transcription enhancer[END_REF]. Both E1 and E2 enhancer regions contain several antioxidant response elements (AREs), which have also been identified in the promoters of other stress-inducible antioxidant and phase 2 detoxifying genes [START_REF] Nguyen | Regulatory mechanisms controlling gene expression mediated by the antioxidant response element[END_REF][START_REF] Kensler | Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway[END_REF]. An important difference between the rodent and human HO-1 genes with major biological relevance is a GT-microsatellite polymorphism, which is localized in the proximal human HO-1 gene promoter region. Lower numbers of GT repeats within this polymorphic sequence have been associated with higher inducibility of HO-1 gene expression in response to stress stimuli [START_REF] Yamada | Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to emphysema[END_REF] and individuals with this allele seem to be protected against cardiovascular disorders (reviewed in [START_REF] Exner | The role of heme oxygenase-1 promoter polymorphisms in human disease[END_REF]).

In the following, we highlight the critical role of the major redox-dependent TFs NF-E2-related factor 2 (Nrf2), BTB and CNC homologue 1 (Bach1), NF-B and AP-1 in regulating the inducible HO-1 gene expression.

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The Keap1/Nrf2 system

Redox-dependent transcriptional induction of HO-1 is primarily mediated through the cap'n'collar (CNC) TF Nrf2 [START_REF] Alam | Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene[END_REF], which was initially identified while screening for proteins that interact with the NF-E2 binding motif [START_REF] Moi | Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region[END_REF]. Nrf2 forms heterodimers with small Maf proteins and up-regulates a program of inducible protective genes via interaction with AREs [START_REF] Kensler | Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway[END_REF][START_REF] Motohashi | Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors[END_REF].

Activation of Nrf2 by oxidative stress is mainly controlled by the cytosolic inhibitor Kelch-like ECH-associated protein 1 (Keap1) [START_REF] Itoh | Keap1 represses nuclear activation of antioxidant response elements by Nrf2 through binding to the amino-terminal Neh2 domain[END_REF][START_REF] Kobayashi | Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation[END_REF], also termed inhibitor of Nrf2 [START_REF] Kaspar | Nrf2:INrf2 (Keap1) signaling in oxidative stress[END_REF](Figure 2).

Numerous prooxidant stimuli cause dissociation of Nrf2 from Keap1, which then permits subsequent nuclear translocation of Nrf2 [START_REF] Kaspar | Nrf2:INrf2 (Keap1) signaling in oxidative stress[END_REF][START_REF] Kimura | Molecular basis distinguishing the DNA binding profile of Nrf2-Maf heterodimer from that of Maf homodimer[END_REF]. In a zebrafish model it has been demonstrated that various chemicals activate Nrf2 in a compound-specific manner via the modulation of various regulatory sites of Keap1 [START_REF] Kobayashi | The antioxidant defense system Keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds[END_REF]. More recently, the Keap1/Nrf2 pathway has also been shown to be activated by the regulator protein p62 in experimental conditions that cause autophagy [START_REF] Komatsu | The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through inactivation of Keap1[END_REF]. Thus, the Keap1/Nrf2 system appears to be a central sensor for a broad spectrum of unfavourable cellular conditions.

It is important to point out, that the Keap1/Nrf2 module may not only be regulated by prooxidant stimuli, but also by stress-independent signals such as glycogen synthase kinase (GSK)-3-dependent phosphorylation (Figure 3). Finally, it is not clear whether Nrf2dependent induction of HO-1 is part of a general Nrf2-regulated antioxidant response that includes other Keap1/Nrf2-regulated genes such as NAD(P)H:oxidoreductase or thioredoxin reductase-1. A potential mechanism that could mediate Nrf2-specific induction of HO-1 involves the brahma-related gene 1 (BRG1). BRG1 has been shown to be necessary for the specific recruitment of Nrf2 to the promoter of the HO-1 gene, but not to that of other phase 2 detoxifying genes [START_REF] Zhang | BRG1 interacts with Nrf2 to selectively mediate HO-1 induction in response to oxidative stress[END_REF].

In conclusion, the redox-dependent Keap1/Nrf2 system plays a central role for HO-1 induction in response to oxidativ stress.

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The transcription repressor Bach1

The transcription repressor Bach1 has been recognized to be a key regulator of the inducible HO-1 gene expression (Figure 2). Bach1 was initially identified as a regulator of globin gene expression in erythroid cells [START_REF] Oyake | Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site[END_REF]. Similar to Nrf2, Bach1 belongs to the CNC family of TFs and forms heterodimers with small Maf proteins that bind to Maf recognition elements [START_REF] Motohashi | Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors[END_REF][START_REF] Igarashi | The heme-Bach1 pathway in the regulation of oxidative stress response and erythroid differentiation[END_REF] such as AREs of the HO-1 promoter. In contrast to Nrf2, Bach1 has six heme regulatory motifs, which are crucial for its regulatory functions. Bach1 has initially been shown to repress HO-1 gene expression in the presence of low levels of intracellular heme. When intracellular heme levels are elevated, Bach1 changes its conformation and dissociates from the HO-1 promoter, allowing Nrf2 to bind to the ARE and to activate HO-1 gene expression

[70] (Figure 2). More recently, heme has also been shown to control cellular Bach1 protein levels via a mechanism that involves proteasomal degradation of this protein [START_REF] Zenke-Kawasaki | Heme induces ubiquitination and degradation of the transcription factor Bach1[END_REF]. As free heme exhibits major proinflammatory effects, it is conceivable that Bach1 might play a regulatory role in inflammation. In line with this notion, a regulatory link between Bach1 and the proinflammatory cytokine IL-6 has recently been elucidated in Bach1 knockout mice in the context of hyperoxic lung injury [START_REF] Tanimoto | Genetic ablation of the Bach1 gene reduces hyperoxic lung injury in mice: role of IL-6[END_REF].

Bach1 does not only regulate HO-1 gene expression by heme, but also by other prooxidant compounds such as cadmium [START_REF] Suzuki | Cadmium induces nuclear export of Bach1, a transcriptional repressor of heme oxygenase-1 gene[END_REF], diamide [START_REF] Ishikawa | Redox regulation of the transcriptional repressor Bach1[END_REF] and sodium arsenite [START_REF] Reichard | BACH1 is a specific repressor of HMOX1 that is inactivated by arsenite[END_REF]. More recently, the regulatory role of Bach1 in stress-dependent HO-1 induction has also been shown to be dependent on the differentiation status of keratinocytes in cell culture [START_REF] Okada | Bach1-dependent and -independent regulation of heme oxygenase-1 in keratinocytes[END_REF].

The interplay between Bach1 and Nrf2 to regulate HO-1 gene expression is discussed controversially. As an example, sodium arsenite has been shown to cause Bach1-specific HO-1 induction independent from Nrf2, whereas sodium arsenite-dependent regulation via this mechanism was not observed for other ARE-regulated genes such as thioredoxin reductase-1 [START_REF] Reichard | BACH1 is a specific repressor of HMOX1 that is inactivated by arsenite[END_REF]. Similarly, it has recently been demonstrated that knockdown of Bach1 in human keratinocytes specifically up-regulated gene expression of HO-1, but not that of other Nrf2-regulated genes [START_REF] Macleod | Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds[END_REF]. In contrast, other investigators have reported that Bach1 induced HO-1 gene expression in coordination with a subset of other Nrf2-regulated genes [START_REF] Hintze | Bach1 repression of ferritin and thioredoxin reductase1 is heme-sensitive in cells and in vitro and coordinates expression with heme oxygenase1, beta-globin, and NADP(H) quinone (oxido) reductase1[END_REF][START_REF] Dhakshinamoorthy | Bach1 competes with Nrf2 leading to negative regulation of the antioxidant response element (ARE)-mediated NAD(P)H:quinone oxidoreductase 1 gene expression and induction in response to antioxidants[END_REF]. In conclusion, the interplay of Bach1 and Nrf2 is crucial for the redox-dependent HO-1 induction and might give this system a high range of plasticity to adapt to adverse cellular conditions.

NF-B

The NF-κB/Rel family of proteins comprises several TFs that regulate the inducible gene expression of various immunological and antioxidant protective responses including the upregulation of major proinflammatory cytokines, adhesion molecules and antioxidant stress proteins [START_REF] Perkins | Good cop, bad cop: the different faces of NF-kappaB[END_REF][START_REF] Gilmore | Introduction to NF-kappaB: players, pathways, perspectives[END_REF]. Under basal conditions NF-κB is contained in the cytoplasm by inhibitor of NF-κB (I-κB). In response to multiple signals the regulatory NF-B subunits p50 and p65 dissociate from I-κB and subsequently translocate to the nucleus [START_REF] Ghosh | New regulators of NF-kappaB in inflammation[END_REF][START_REF] Chen | Shaping the nuclear action of NF-kappaB[END_REF]. Although NF-κB has been shown to be induced by stimuli that are also known to up-regulate HO-1 gene expression, the role of this TF in HO-1 gene regulation has been discussed controversially.

This might be explained by the fact that functional B-elements have remained elusive for a long while, because indirect approaches including computer-based sequence predictions, treatment with pharmacological NF-B inhibitors and dominant negative mutants of I-B have been applied in most studies.

In two recent reports functional binding sites for NF-B of the promoters of the rat and mouse HO-1 genes have been identified. A κB element of the proximal rat HO-1 gene promoter region has been shown to control HO-1 up-regulation by the phorbol ester phorbol myrisate acetate (PMA), which is an activator of macrophages. PMA-dependent up-regulation of HO-1 gene expression was not observed in cells from mice, which were deficient for the NF-κB subunit p65 and was mediated via an I-κB-kinase-independent pathway [START_REF] Naidu | An atypical NF-kappa B-regulated pathway mediates phorbol ester-dependent heme oxygenase-1 gene activation in monocytes[END_REF]. Moreover, Li and colleagues have described a functional κB element in the mouse HO-1 promoter. These authors have shown that a mechanism involving the NF-κB subunits p50 and p65 as well as the inducible NO-synthase mediated HO-1 up-regulation in vivo [START_REF] Li | Gene transfer of inducible nitric oxide synthase affords cardioprotection by upregulating heme oxygenase-1 via a nuclear factor-{kappa}B-dependent pathway[END_REF].

In summary, NF-B appears to be directly involved in the induction of HO-1 gene expression.

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Activating protein-1 (AP-1)

The TF AP-1 is composed of structurally and functionally related members of the Jun (c-Jun, JunB and JunD), Fos (c-Fos, FosB, Fra1 and Fra2) and activating TF (ATF) protein families.

Dimers of these proteins regulate gene expression via interaction with AP-1 sites, which are also known as TPA-responsive elements (TREs). Inducible gene expression via AP-1 has been shown to be involved in a diverse range of cellular responses including immunological and antioxidant stress responses [START_REF]The regulation of AP-1 activity by mitogen-activated protein kinases[END_REF][START_REF] Hess | AP-1 subunits: quarrel and harmony among siblings[END_REF]. Similar to NF-B, AP-1 is up-regulated by a wide variety of prooxidant and pro-inflammatory stimuli. Alam and colleagues have initially reported that AP-1 played a crucial role for the induction of the mouse HO-1 gene [START_REF] Alam | Identification of a second region upstream of the mouse heme oxygenase-1 gene that functions as a basal level and inducer-dependent transcription enhancer[END_REF][START_REF] Alam | Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene[END_REF].

Subsequently, various functional AP-1 sites, which mediated inducer-dependent gene expression of HO-1, have been identified in the promoter regions of the rat and human HO-1

genes [START_REF] Alam | How many transcription factors does it take to turn on the heme oxygenase-1 gene?[END_REF][START_REF] Alam | Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene[END_REF][START_REF] Immenschuh | Transcriptional activation of the haem oxygenase-1 gene by cGMP via a cAMP response element/activator protein-1 element in primary cultures of rat hepatocytes[END_REF][START_REF] Hock | JunB and JunD regulate human heme oxygenase-1 gene expression in renal epithelial cells[END_REF][START_REF] Immenschuh | Transcriptional induction of the heme oxygenase-1 gene expression by okadaic acid in primary rat hepatocyte cultures[END_REF]. Elucidation of the molecular mechanisms that are involved in AP-1dependent HO-1 gene regulation has turned out to be challenging for two major reasons.

First, the classical AP-1 (TGATGCA) site is contained in the consensus sequence of AREs (TGCTGAGTCA), which are localized in the E1 and E2 regions of the HO-1 gene promoter and serve as major target sites for Nrf2 [START_REF] Alam | Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene[END_REF]. Cross-talk of Nrf2 with members of the AP-1 family, however, appears to be highly complex. As an example, c-Jun has recently been shown to directly interact with Nrf2 to activate the expression of the ARE-regulated genes NAD(P)H:quinone reductase and glutamate-cysteine ligase catalytic subunit by the chemical compound 4-hydroxy-2-nonenal [START_REF] Levy | The role of c-Jun phosphorylation in EpRE activation of phase II genes[END_REF]. Moreover, others have shown that Nrf2 might indirectly regulate the inducible expression of the glutamate-cysteine ligase catalytic subunit gene via activation of AP-1 [START_REF] Yang | Nrf1 and Nrf2 regulate rat glutamate-cysteine ligase catalytic subunit transcription indirectly via NF-kappaB and AP-1[END_REF]. Second, AP-1 sites in the HO-1 promoter mediate inducer-dependent HO-1 gene expression via mechanisms that involve synergistic cooperation of AP-1 with other TFs such as USF2 or SP-1. This has been shown for regulation of the rodent and human HO-1 genes in various cell culture models [START_REF] Wijayanti | Heme oxygenase-1 gene activation by the NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride via a protein kinase B, p38-dependent signaling pathway in monocytes[END_REF][START_REF] Samoylenko | Opposite expression of the antioxidant heme oxygenase-1 in primary cells and tumor cells: regulation by interaction of USF-2 and Fra-1[END_REF][START_REF] Wright | Human haem oxygenase-1 induction by nitro-linoleic acid is mediated by cAMP, AP-1 and E-box response element interactions[END_REF][START_REF] Deshane | Sp1 regulates chromatin looping between an intronic enhancer and distal promoter of the human heme oxygenase-1 gene in renal cells[END_REF].

In summary, we have highlighted the role of various redox-dependent TFs that mediate the complex regulation of inducible HO-1 gene expression. Detailed overviews on this issue have also been given by others [START_REF] Ryter | Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications[END_REF][START_REF] Choi | Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury[END_REF][START_REF] Alam | How many transcription factors does it take to turn on the heme oxygenase-1 gene?[END_REF].

Signaling cascades that mediate HO-1 gene regulation

In general, activation of TFs is regulated by intracellular signaling cascades, which are controlled by modules of kinases/phosphatases and redox reactions. In the following, we highlight major signalling cascades that mediate HO-1 induction and are involved in the regulation of inflammatory immune responses.

p38 MAPK

It has been known for many years that activation of MAPKs plays a central role for the induction of HO-1 gene expression [START_REF] Alam | How many transcription factors does it take to turn on the heme oxygenase-1 gene?[END_REF] (Figure 3). Three major subfamilies of MAPK are known: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38

MAPK [START_REF] Kyriakis | Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation[END_REF]. While ERK is primarily considered to be activated in response to hormones and growth factors, JNK and p38 are primarily induced by stress-related stimuli [START_REF] Kyriakis | Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation[END_REF][START_REF] Wagner | Signal integration by JNK and p38 MAPK pathways in cancer development[END_REF]. Due to space limitations, it is not intended to give a comprehensive overview on how various MAPKs might mediate HO-1 gene regulation, but rather discuss the specific role of p38 MAPK for HO-1 regulation.

Similar to HO-1, p38 MAPK has been shown to be involved in antioxidant and antiinflammatory responses. Not surprisingly, gene expression of HO-1 and p38 MAPK are activated by the same or similar stimuli. Numerous reports have demonstrated that inhibition of p38 activity by specific small molecule inhibitors, dominant negative mutants or gene silencing strategies blocked HO-1 induction in response to multiple stimuli [2, 9, 51] (Figure 3). Of note, the  and  isoforms of p38 play counter-regulatory roles to the p38  and  isoforms, which has been demonstrated for sodium arsenite-and LPS-dependent induction of HO-1 [START_REF] Kietzmann | Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes[END_REF][START_REF] Wijayanti | Role of NF-kappaB and p38 MAP kinase signaling pathways in the lipopolysaccharide-dependent activation of heme oxygenase-1 gene expression[END_REF]. Unexpectedly and contradictory to what is generally thought on the activating role of p38 MAPK on HO-1 gene expression, inhibition of p38 has been demonstrated to up-regulate HO-1 gene expression in human hepatoma cells via interaction with the TF Nrf2 [START_REF] Yu | p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens[END_REF][START_REF] Keum | Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element-mediated heme oxygenase-1 in human hepatoma HepG2 cells[END_REF]. These findings have essentially been confirmed in an independent report, in which pharmacological inhibition and genetic deficiency of p38 also up-regulated HO-1 gene expression [START_REF] Naidu | Inhibition and genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression via Nrf2[END_REF]. Increased levels of HO-1 gene expression in A c c e p t e d M a n u s c r i p t 13 p38-deficient cells might be explained by the fact that these cells contain increased levels of intracellular ROS as compared to wild type cells [START_REF] Dolado | p38alpha MAP kinase as a sensor of reactive oxygen species in tumorigenesis[END_REF]. Because p38 is a sensor of ROS, cross-talk between HO-1 and p38 may have additional yet unknown functions in the context of oxidative stress and inflammation. It is also important to point out that p38 is not only an upstream regulator of HO-1, but that p38 is also a downstream target of HO-1. Silva and colleagues have demonstrated that increased HO-1 activity degraded p38 in endothelial cells suggesting that these two molecules might form a physiological relevant feedback loop to regulate apoptosis [START_REF] Silva | The antiapoptotic effect of heme oxygenase-1 in endothelial cells involves the degradation of p38 alpha MAPK isoform[END_REF].

In summary, p38 and HO-1 make up a closely linked signaling module with major regulatory functions in antioxidant and anti-inflammatory cellular responses.

The phosphatidylinositol-3 kinase (PI3K)/Akt pathway

PI3K/Akt is an anti-apoptotic survival pathway and is regulated by a number of receptordependent mechanisms that are activated by growth factors and cytokines [START_REF] Koyasu | The role of PI3K in immune cells[END_REF]. Moreover, in models of PI3K genetic deficiency an essential role of this kinase has been implicated in the regulation of inflammatory reactions [START_REF] Ghigo | PI3K inhibition in inflammation: Toward tailored therapies for specific diseases[END_REF]. Accumulating experimental evidence has indicated that activation of PI3K/Akt not only up-regulates HO-1 gene expression, but that the protective effects of this signaling cascade might be intimately linked with the salutary effects of HO-1 [START_REF] Martin | Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol[END_REF]. HO-1 gene expression has been shown to be up-regulated via PI3K/Akt in immunological cells in response to various signals such as prostaglandins or the pharmacological compounds [START_REF] Ryter | Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications[END_REF][START_REF] Wijayanti | Heme oxygenase-1 gene activation by the NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride via a protein kinase B, p38-dependent signaling pathway in monocytes[END_REF][START_REF] Martin | Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol[END_REF][START_REF] Alvarez-Maqueda | 15deoxy-delta 12,14-prostaglandin J2 induces heme oxygenase-1 gene expression in a reactive oxygen species-dependent manner in human lymphocytes[END_REF]. A more recent report has shown that HO-1 induction was mediated via activation of this signaling cascade by a mitochondrial redoxdependent pathway in vascular endothelial cells [START_REF] Hamdulay | Celecoxib activates PI-3K/Akt and mitochondrial redox signaling to enhance heme oxygenase-1-mediated anti-inflammatory activity in vascular endothelium[END_REF]. Finally, it has been suggested that PI3K/Akt and GSK3 may have counter-regulatory functions in HO-1 gene regulation. The complex interplay between these two kinases appears to involve mechanisms that control the nuclear localization of Nrf2 and Bach1 [START_REF] Salazar | Glycogen synthase kinase-3beta inhibits the xenobiotic and antioxidant cell response by direct phosphorylation and nuclear exclusion of the transcription factor Nrf2[END_REF][START_REF] Kaspar | Antioxidant-induced phosphorylation of tyrosine 486 leads to rapid nuclear export of Bach1 that allows Nrf2 to bind to the antioxidant response element and activate defensive gene expression[END_REF] (Figure 3). Details on the underlying regulatory mechanisms, however, are largely unknown.

A c c e p t e d M a n u s c r i p t

In conclusion, the PI3K/Akt cascade is a key mediator of HO-1 induction in response to cytokines and growth factors.

IL-10 and the Jak-STAT pathway

The anti-inflammatory cytokine IL-10, which is a major inhibitor of activated macrophages and dendritic cells, has consistently been shown to induce HO-1 gene expression as reported by Lee and Chau [START_REF] Lee | Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice[END_REF] as well as by others [START_REF] Chen | Interleukin 10 attenuates neointimal proliferation and inflammation in aortic allografts by a heme oxygenase-dependent pathway[END_REF][START_REF] Ricchetti | Heme oxygenase 1 expression induced by IL-10 requires STAT-3 and phosphoinositol-3 kinase and is inhibited by lipopolysaccharide[END_REF][START_REF] Mandal | The anti-inflammatory effects of adiponectin are mediated via a heme oxygenase-1-dependent pathway in rat Kupffer cells[END_REF]. Although the signaling cascades that couple HO-1 gene expression to the IL-10 receptor are not clear, activation of STAT3 has repeatedly been suggested to be involved in this regulatory pathway [START_REF] Ricchetti | Heme oxygenase 1 expression induced by IL-10 requires STAT-3 and phosphoinositol-3 kinase and is inhibited by lipopolysaccharide[END_REF]. Jak-STAT signaling plays a key role in the immune system and mediates major cytokineactivated pathways. In endothelial cells STAT3 has been shown to be necessary to mediate the HO-1 dependent protection against hyperoxic lung injury [START_REF] Zhang | Endothelial STAT3 is essential for the protective effects of HO-1 in oxidant-induced lung injury[END_REF]. Functional STAT3 elements have recently been identified in the promoter regions of the rat [START_REF] Tron | Regulation of rat heme oxygenase-1 expression by interleukin-6 via the Jak/STAT pathway in hepatocytes[END_REF] and human

[120] HO-1 genes. Interestingly, a positive feedback circuit between IL-10 and HO-1 has been shown to be functional, which might amplify the anti-inflammatory effects of IL-10 in LPS-stimulated macrophages [START_REF] Lee | Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice[END_REF].

Taken together, the IL-10 pathway is an important regulator of HO-1 gene expression in mononuclear cells.

The toll-like receptor (TLR)-4 pathway

HO-1 induction by LPS is mediated via (a) TLR-4 coupled pathway(s) and has been extensively studied in macrophages [START_REF] Otterbein | Mechanism of hemoglobin-induced protection against endotoxemia in rats: a ferritin-independent pathway[END_REF][START_REF] Immenschuh | Nitric oxide mediates the lipopolysaccharide dependent upregulation of the heme oxygenase-1 gene expression in cultured rat Kupffer cells[END_REF]. Interestingly, Figueiredo and colleagues have recently demonstrated that heme led to HO-1 gene expression via direct interaction with TLR-4 and suggested that this interaction could at least partially explain the proinflammatory LPS-like effects of heme [START_REF] Figueiredo | Characterization of heme as activator of Toll-like receptor 4[END_REF]. HO-1 gene expression by LPS in macrophages is not only induced via a TLR-4-dependent mechanism, but increased HO-1 activity has also been shown to have inhibitory effects on intracellular signaling, that is initiated by TLR-4-activation.

The inhibitory effect of HO-1 on TLR-4 signaling could be regulated via HO-1-derived CO,

A c c e p t e d M a n u s c r i p t 15 which blocked translocation of TLR-4 into lipid rafts [START_REF] Nakahira | Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts[END_REF] and/or the interaction of TLR-4 with the structural membrane protein caveolin-1 [START_REF] Wang | The heme oxygenase-1/carbon monoxide pathway suppresses TLR4 signaling by regulating the interaction of TLR4 with caveolin-1[END_REF]. This regulatory interplay between TLR-4

and HO-1 appears to form a negative feedback loop, which might inhibit excessive activation of macrophages by LPS.

Taken together, activation of the central proinflammatory TLR-4 cascade leads to HO-1 gene activation and a negative feedback circuit in macrophages might be of major significance for the regulation of inflammatory responses.

Pharmacological induction of HO-1 as an anti-inflammatory therapeutic target

A promising near term approach to apply HO-1 for therapeutic interventions is the targeted induction of this inducible enzyme via pharmacological compounds. In the following, we discuss how pharmacological induction of HO-1 might be applicable for specific antiinflammatory therapeutic interventions.

Metalloporphyrins such as cobalt protoporphyrin IX, which are prototypical inducers of HO-1 and are commonly used in experimental cell culture and animal models, do not seem to be applicable for clinical interventions, because they lack cell-specificity and are severely toxic.

Similarly, the HO substrate heme, which is one of the most potent inducers of HO-1 and has been approved for the treatment of acute intermittent porphyria (heme arginate), only has limited potential for the treatment of inflammatory disorders. By contrast, a growing number of currently available pharmacologic compounds, which induce HO-1 and are applied in standard therapies, might be useful for clinical interventions in inflammatory disorders. As an example, statins, which have initially been introduced to prevent atherosclerosis via their cholesterol-lowering effects, have recently also been recognized to exhibit anti-inflammatory effects via HO-1 induction [START_REF] Lee | Simvastatin induces heme oxygenase-1: a novel mechanism of vessel protection[END_REF][START_REF] Grosser | The antioxidant defense protein heme oxygenase 1 is a novel target for statins in endothelial cells[END_REF]. Moreover, treatment with 5-aminosalicylic acid (5-ASA), which is one of the pharmacologic standard therapies of inflammatory bowel disease, has been shown to mediate its protective anti-inflammatory effects at least in part through up-regulation of HO-1 in an animal model of colitis [START_REF] Horvath | The involvement of heme oxygenase-1 activity in the therapeutic actions of 5-aminosalicylic acid in rat colitis[END_REF]. Finally, various polyphenols, which are a group of antioxidant compounds and are currently investigated for their anti- Due to the cell-type specific immunomodulatory effects of HO-1, targeted up-regulation of HO-1 in myeloid and endothelial cells seems to be a straightforward anti-inflammatory therapeutic option. In fact, accumulating evidence has demonstrated that specific HO-1 induction in these cells protects against inflammatory reactions. As an example, it has recently been demonstrated in an in vivo rat model that cell-specific up-regulation of HO-1 in liver tissue macrophages via the cytokine adiponectin protects against experimental ethanoldependent inflammation [START_REF] Mandal | The anti-inflammatory effects of adiponectin are mediated via a heme oxygenase-1-dependent pathway in rat Kupffer cells[END_REF]. Other compounds, which have been shown to mediate myeloid cell-specific induction of HO-1, are the cardiovascular hormone atrial natriuretic peptide [START_REF] Kiemer | Kupffer-cell specific induction of heme oxygenase 1 (hsp32) by the atrial natriuretic peptide-role of cGMP[END_REF] and the clinically applied antiprotease compound 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) [START_REF] Wijayanti | Heme oxygenase-1 gene activation by the NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride via a protein kinase B, p38-dependent signaling pathway in monocytes[END_REF]. Clearly, these compounds might be applicable to specifically induce HO-1 in myeloid cells for therapeutic interventions. In endothelial cells, quercetin and theaflavin have been shown to provide specific HO-1-dependent antiinflammatory protection in an ApoE knockout mouse model of atherosclerosis [START_REF] Loke | Specific dietary polyphenols attenuate atherosclerosis in apolipoprotein E-knockout mice by alleviating inflammation and endothelial dysfunction[END_REF].

Moreover, it has recently been demonstrated that the nonsteroidal anti-inflammatory drug celecoxib provided specific anti-inflammatory effects in endothelial cells via induction of HO-1 [START_REF] Hamdulay | Celecoxib activates PI-3K/Akt and mitochondrial redox signaling to enhance heme oxygenase-1-mediated anti-inflammatory activity in vascular endothelium[END_REF].

Finally, it is important to point out that targeted up-regulation of HO-1 has failed to provide anti-inflammatory protection, when induced after the onset of inflammation. This has been shown in animal models of inflammatory bowel disease and pancreatitis, respectively. In either case HO-1 was only protective when induced before the onset of experimental inflammation [START_REF] Paul | Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis[END_REF][START_REF] Nakamichi | Heminactivated macrophages home to the pancreas and protect from acute pancreatitis via heme oxygenase-1 induction[END_REF]. These findings indicate that anti-inflammatory protection via HO-1 induction is questionable in established inflammation, but might be useful as a preventive measure.

In summary, targeted HO-1 induction in myeloid and endothelial cells has major antiinflammatory therapeutic potential. Therefore, identification and characterization of A c c e p t e d M a n u s c r i p t pharmacological compounds that induce HO-1 in a cell-specific and cell context-specific manner deserve further attention.

Conclusions

1.

HO-1 plays key immunomodulatory and anti-inflammatory roles via its cell typespecific effects in myeloid and endothelial cells.

2.

Critical functions of HO-1 are degradation of proinflammatory free heme and enzymatic production of the anti-inflammatory compounds CO and BR.

3.

The inducible HO-1 gene regulation is mediated via an interplay of redoxdependent activating TFs and the transcription repressor Bach1, which are under the control of a complex network of signaling cascades.

4.

Targeted induction of HO-1 in myeloid and endothelial cells has major therapeutic potential for the treatment of inflammatory disorders.

HO-1 expression

A c c e p t e d M a n u s c r i p t 10

 10

A c c e p t e d M a n u s c r i p t 12

 12

A c c e p t e d M a n u s c r i p t 16 inflammatory

 16 and anticancer activities, have been shown to provide anti-inflammatory protection via the induction of HO-1[START_REF] Rahman | Regulation of inflammation and redox signaling by dietary polyphenols[END_REF].

Figure 1 :

 1 Figure 1: Regulatory role of HO-1 in the inflammatory response of macrophages

Figure 2 :

 2 Figure 2: Regulation of HO-1 gene expression via the redox-dependent TFs Nrf2 and

Figure 3 :

 3 Figure 3: Signaling cascades that target Keap1/Nrf2 and Bach1 to regulate HO-1 gene expression

Figure 1 A

 1 Figure 1

Figure 2

 2 Figure 2

* Supported by grant SFB547 A8 from the Deutsche Forschungsgemeinschaft (SI).


Florence Folmer

Marcel Jaspars

Marc Schumacher

Mario Dicato

Dr Marc Diederich
email: marc.diederich@lbmcc.lu

Marine natural products targeting phospholipases A2

Keywords: anti-inflammatory, arachidonic acid, eicosanoids, marine natural product, membrane phospholipids, PLA 2

Phospholipases A 2 (PLA 2 s) form a family of enzymes catalyzing the hydrolysis of membrane phospholipids into arachidonic acid, which is the major precursor of pro-inflammatory eicosanoids. As a result, PLA 2 s have been considered as potential targets in anti-inflammatory drug discovery.

Marine natural products are a rich source of bioactive compounds, including PLA 2 inhibitors. Here, we review the properties of marine PLA 2 inhibitors identified since the first discovery of PLA 2 inhibitory activity in the marine natural product manoalide in the mid 1980's.

Introduction

Inflammation is the response of vascular tissues to harmful stimuli such as injury, pathogens, or irritants. While inflammation normally functions as a defense mechanism in higher animals, deregulated inflammation is implicated in a large number of diseases such as autoimmune diseases, allergies, asthma, rheumatoid arthritis, inflammatory bowel diseases, pelvic inflammatory diseases, glomerulonephritis, atherosclerosis, myocardial ischemia, and cancer [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF][START_REF] Yedgar | Inhibition of phospholipase A 2 as a therapeutic target[END_REF][START_REF] Chang | Phospholipase A 2 : Function and pharmacological regulation[END_REF]. The process of inflammation is controlled by a group of substances called chemical mediators [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF]. Endogenous chemical mediators consist of vasoactive amines, cytokines, bradikinin, fibrin, complement components, eicosanoids, platelet activating factor (PAF), nitric oxide (NO), and neuropeptides [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF]. Eicosanoids, in particular, play a critical role in virtually every step of inflammation. Eicosanoids, which comprise prostaglandins, prostacyclins, thromboxanes, and leukotrienes, are a family of oxygenated fatty acid metabolized by cyclooxygenases (COX) and lipoxygenases (LOX) from arachidonic acid [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF]. Despite the extensive efforts invested in developing drugs that suppress the conversion of arachidonic acid into pro-inflammatory eicosanoids, the latter approach has been unsuccessful. Undesired side-effects resulting from the lack of specificity of COX and LOX are responsible for the failure of the concept [START_REF] Yedgar | Inhibition of phospholipase A 2 as a therapeutic target[END_REF]. As an alternative, the quest for inhibitors of phospholipases A 2 (PLA 2 s), the enzymes that catalyze the hydrolysis of membrane phospholipids into arachidonic acid, has opened up a new research avenue in anti-inflammatory drug discovery [START_REF] Yedgar | Inhibition of phospholipase A 2 as a therapeutic target[END_REF][START_REF] Lombardo | Cobra venom phospholipase A 2 inhibition by manoalide[END_REF][START_REF] Glaser | Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2[END_REF][START_REF] Burke | Phospholipase A 2 structure/function, mechanism, and signaling[END_REF]. As a matter of fact, PLA 2 s isolated from snake venom have been shown to induce all the inflammatory symptoms of snakebite such as acute pain, oedema, hypotension, hemorrhage, and neuromuscular junction blockage. Furthermore, rheumatoid arthritis, asthma, psoriasis, myocardial ischemia, and pancreatis have all been shown to be associated with elevated levels of serum PLA 2 .

Lysophospholipids produced by PLA 2 s have also been shown to induce gastric ulceration in rats, and to induce an inflammation similar to acute cholecystitis in the gall bladder mucosa [START_REF] Chang | Phospholipase A 2 : Function and pharmacological regulation[END_REF]. Here, we review the properties of marine PLA 2 inhibitors identified since the first discovery of PLA 2 inhibitory activity in the marine natural product manoalide [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF], by research groups lead by Edward Dennis [START_REF] Lombardo | Cobra venom phospholipase A 2 inhibition by manoalide[END_REF] and by Robert Jacobs [START_REF] Glaser | Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2[END_REF] at the universities of San Diego and Santa Barbara, respectively, in the mid 1980's. They are grouped into four families: secreted PLA 2 s (sPLA 2 s), cytosolic PLA 2 s (cPLA 2 s), lipoprotein associated PLA 2 s ((Lp)PLA 2 ), and calcium-independent PLA 2 s (iPLA 2 s) [START_REF] Yedgar | Inhibition of phospholipase A 2 as a therapeutic target[END_REF][START_REF] Glaser | Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2[END_REF][START_REF] Burke | Phospholipase A 2 structure/function, mechanism, and signaling[END_REF]. The calcium-dependent sPLA 2 s are commonly found in snake, scorpion, and bee venom. They are of low molecular weight (13-15 kDa) and characteristically contain a histidine residue in their catalytic site [START_REF] Yedgar | Inhibition of phospholipase A 2 as a therapeutic target[END_REF][START_REF] Burke | Phospholipase A 2 structure/function, mechanism, and signaling[END_REF]. The mode of action of sPLA 2 s involves a nucleophilic attack onto the phospholipid's sn-2 bond.

While the role of sPLA 2 s in inflammation remains poorly understood, it has been suggested that sPLA 2 s induce an increase in cPLA 2 -dependent eicosanoid release, and that they synergize with other pro-inflammatory mediators [START_REF] Yedgar | Inhibition of phospholipase A 2 as a therapeutic target[END_REF][START_REF] Burke | Phospholipase A 2 structure/function, mechanism, and signaling[END_REF]. cPLA 2 s are 85 kDa enzymes containing a serine and an aspartic acid residue in the active site.

Noteworthy, cPLA 2 s are the only PLA 2 s with specificity for arachidonic acid at the phospholipase sn-2 position. cPLA 2 s are calcium-dependent enzymes activated by extra-cellular stimulations from pathogens, tissue injury, or physical or chemical stresses. The cytolic concentrations of calcium required for PLA 2 activation result from the cleavage of phospholipids into inositol 1,4,5-triphosphate (IP 3 ) by phospholipase C (PLC), followed by the binding of IP 3 to calcium channels in the endoplasmic reticulum [START_REF] Yedgar | Control of phospholipase A 2 activities for the treatment of inflammatory conditions[END_REF]. Because of their central role in mediating the generation of eicosanoids and of PAFs, and hence in mediating inflammation, cPLA 2 s have been recognized as very attractive targets in drug discovery, despite some rare side-effects including the formation of intestinal ulcers, and several pharmaceutical companies, such as Pfizer have started to develop promising cPLA 2 -specific drug candidates [START_REF] Yedgar | Control of phospholipase A 2 activities for the treatment of inflammatory conditions[END_REF][START_REF] Cummings | Phospholipase A 2 as targets for anti-cancer drugs[END_REF][START_REF] Niknami | Molecules in focus: Cytosolic phospholipase A 2 -α[END_REF]. Unlike cPLA 2 s, (Lp)PLA 2 s, or platelet aggregation factor acetylhydrolases (PAF-AHs), have anti-inflammatory properties, as they are able to degrade the proinflammatory signaling molecules PAFs by cleaving their acetyl group at the sn-2 position. However, (Lp)PLA 2 s have become an important target in PLA 2 inhibitory drug discovery, as they are known to lead to coronary heart diseases [START_REF] Burke | Phospholipase A 2 structure/function, mechanism, and signaling[END_REF]. iPLA 2 s have complex and still poorly understood implications in signalling pathways. iPLA 2 s play a role in bone formation, apoptosis, insulin secretion, sperm development , and axon regeneration [START_REF] Yedgar | Inhibition of phospholipase A 2 as a therapeutic target[END_REF][START_REF] Burke | Phospholipase A 2 structure/function, mechanism, and signaling[END_REF]. The present review focuses only on inhibitors of sPLA 2 s and A c c e p t e d M a n u s c r i p t -5 -cPLA 2 s. The latter two are present in most types of cells, and both of them are known to be implicated in inflammation through eicosanoid biosynthesis [START_REF] Burke | Phospholipase A 2 structure/function, mechanism, and signaling[END_REF][START_REF] Niknami | Molecules in focus: Cytosolic phospholipase A 2 -α[END_REF][START_REF] Yang | Semivioxanthin isolated from marine-derived fungus regulates tumor necrosis factor-α, cluster of differentiation (CD) 80, CD86, and major histocompatibility complex class II expression in RAW264.7 cells via nuclear factor-κB and mitogen-activated protein kinase signaling pathways[END_REF][START_REF] Zarai | A novel hepatopancreatic phospholipase A 2 from Hexaplex trunculus with digestive and toxic activities[END_REF].

As illustrated in Figure 1, PLA 2 s initiate the pro-inflammatory signaling cascade by catalyzing the hydrolysis of the sn-2 acyl ester bond of membrane phospholipids, leading to the release of the -6 fatty acid arachidonic acid and of lysophospholipids [START_REF] Yedgar | Control of phospholipase A 2 activities for the treatment of inflammatory conditions[END_REF]. Next, arachidonic acid is oxygenated into prostaglandin (PG) PGH 2 by COX, or into 5-hydroperoxyeicosatetraenoic acid (5-HPTETE) by LOX.

The conversion of arachidonic acid to PGH 2 by COX occurs in two steps. First, two molecules of O 2 are added as two peroxide linkages, and a 5-membered carbon ring is formed near the middle of the fatty acid chain, leading to an unstable intermediate prostaglandin G (PGG 2 ). One of the peroxide linkages then sheds a single oxygen atom to form the PGH 2 [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF]. PGH 2 is the unstable precursor of PGD 2 , PGE 2 , PGI 2 , and thomboxane A 2 (TXA 2 ) [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF]. PGE 2 and PGI 2 enhance edema formation and leukocyte infiltration by promoting blood flow in the inflamed region, and they stimulate the pain-inducing activity of bradykinin and autacoids. PGE 2 induces pain, heat, and fever. TXA 2 triggers platelet aggregation [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF]. LOX converts arachidonic acid into lipid hydroxyperoxides that exert relevant functions as mediators of inflammation: 5hydroperoxyeicosatetraenoic acid (5-HPTETE) is spontaneously reduces to 5hydroxyeicosatetraenoic acid (5-HETE), which is further converted by 5lipoxygenase to leukotriene A 4 . LTA 4 may be converted to LTB 4 . LTB 4 is a potent chemoattractant for polymorphonuclear leukocytes. It activates neutrophil functional responses, leading to the generation of free oxygen free radicals and to the release of lysosomal enzymes. LTB 4 also causes the adhesion and chemotaxis of leukocytes, it stimulates aggregation, enzyme release, generation of superoxide in neutrophils, and it makes blood vessels more permeable [START_REF] Yang | Semivioxanthin isolated from marine-derived fungus regulates tumor necrosis factor-α, cluster of differentiation (CD) 80, CD86, and major histocompatibility complex class II expression in RAW264.7 cells via nuclear factor-κB and mitogen-activated protein kinase signaling pathways[END_REF]. Eosinophils, mast cells, and alveolar macrophages use LTC 4 synthase to conjugate glutathione with LTA 4 to make LTC 4 , which is transported outside the cell where a glutamic acid moiety is removed to make LTD 4 . LTD 4 is then cleaved by dipeptidases to make LTE 4 . LTC 4 , LTD 4 , and LTE 4 play an important role in atherosclerosis, in asthma, in allergenic rhinitis, and in inflammatory gastrointestinal diseases. Eicosanoids also activate the production of pro-inflammatory reactive oxygen species (ROS), nitric oxide (NO), and cytokines [START_REF] Chang | Phospholipase A 2 : Function and pharmacological regulation[END_REF][START_REF] Niknami | Molecules in focus: Cytosolic phospholipase A 2 -α[END_REF][START_REF] Yang | Semivioxanthin isolated from marine-derived fungus regulates tumor necrosis factor-α, cluster of differentiation (CD) 80, CD86, and major histocompatibility complex class II expression in RAW264.7 cells via nuclear factor-κB and mitogen-activated protein kinase signaling pathways[END_REF][START_REF] Monti | The molecular mechanism of human group IIA phospholipase A2 inactivation by bolinaquinone[END_REF][START_REF] Haefner | Drugs from the deep: marine natural products as drug candidates[END_REF]. The lysophospholipids produced during the conversion of membrane phospholipids to arachidonic acid are a precursor for PAF. In addition,

Marine PLA 2 inhibitors

PLA 2 activity has been reported in several marine organisms, including hard and soft corals, jellyfish, starfish, sea anemones, and soft corals, and marine snails [START_REF] Zarai | A novel hepatopancreatic phospholipase A 2 from Hexaplex trunculus with digestive and toxic activities[END_REF][START_REF] Nevalainen | Phospholipase A2 in porifera[END_REF]. Hence, from an ecological perspective, it is not surprising that marine organisms have developed potent PLA 2 inhibitors, which may be used as chemical defences in their natural environment. Marine PLA 2 inhibitors reported to date are primarily terpenoids isolated from sponges, nudibranchs, and algae. Their chemical and biological properties are described below and summarized in Table 1. The chemical structures of the compounds are shown in Figure 2.

PLA 2 inhibiting sesquiterpenes

One of the most investigated marine PLA 2 inhibitors is the merosesquiterpene bolinaquinone (1) isolated from the sponge Dysidea sp. Bolinaquinone (1) has been shown to inhibit the enzymatic activity of sPLA 2 with an IC 50 value of 100 nM [START_REF] Monti | The molecular mechanism of human group IIA phospholipase A2 inactivation by bolinaquinone[END_REF].

While the inhibition of sPLA 2 by bolinaquinone (1) is very potent, it is not selective against this enzyme. Bolinaquinone 1 is known to also affects cPLA 2 [START_REF] Monti | The molecular mechanism of human group IIA phospholipase A2 inactivation by bolinaquinone[END_REF][START_REF] Haefner | Drugs from the deep: marine natural products as drug candidates[END_REF][START_REF] Busserolles | Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M[END_REF][START_REF] Lucas | Modulatory effect of bolinaquinone, a marine sesquiterpenoid, on acute and chronic inflammatory processes[END_REF][START_REF] Mayer | Marine pharmacology in 2003-2004: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, -17 -antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action[END_REF][START_REF] Nakao | Enzyme inhibitors from marine invertebrates[END_REF][START_REF] Keyzers | Anti-inflammatory metabolites from marine sponges[END_REF].

Bolinaquinone (1) is known to reduce LTB 4 production in neutrophils and NO and PGE 2 production in macrophages [START_REF] Monti | The molecular mechanism of human group IIA phospholipase A2 inactivation by bolinaquinone[END_REF][START_REF] Haefner | Drugs from the deep: marine natural products as drug candidates[END_REF][START_REF] Busserolles | Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M[END_REF][START_REF] Lucas | Modulatory effect of bolinaquinone, a marine sesquiterpenoid, on acute and chronic inflammatory processes[END_REF][START_REF] Mayer | Marine pharmacology in 2003-2004: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, -17 -antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action[END_REF][START_REF] Nakao | Enzyme inhibitors from marine invertebrates[END_REF][START_REF] Keyzers | Anti-inflammatory metabolites from marine sponges[END_REF]. Another, closely related sesquiterpenoid quinone, ilimaquinone (2) isolated from the sponge Hippiospongia metachromia [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF], has also been shown to inhibit PLA 2 (IC 75 = 270 M against bee venom sPLA 2 ) [START_REF] Keyzers | Anti-inflammatory metabolites from marine sponges[END_REF].

The anti-psoriasis sesquiterpene hydroquinone avarol (3) and the sesquiterpene quinones avarone (4) and dysidine (5) isolated from the sponge Dysidea avara inhibit sPLA 2 activity and PGE 2 release in keratinocytes and in monocytes (IC 50 = 2 M).

Furthermore, avarol has been shown to reduce eicosanoid release and ROS generation in stimulated leukocytes [START_REF] Mayer | Marine pharmacology in 2003-2004: Marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, -17 -antiplatelet, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action[END_REF][START_REF] Nakao | Enzyme inhibitors from marine invertebrates[END_REF][START_REF] Keyzers | Anti-inflammatory metabolites from marine sponges[END_REF]. Dysidiotronic acid (6) isolated from Dysidea sp. also inhibits sPLA 2 (IC 50 = 2.6 M) [START_REF] Nakao | Enzyme inhibitors from marine invertebrates[END_REF][START_REF] Keyzers | Anti-inflammatory metabolites from marine sponges[END_REF]. The sesquiterpene lactone cavernolide [START_REF] Yedgar | Control of phospholipase A 2 activities for the treatment of inflammatory conditions[END_REF] isolated from the sponge Fasciospongia cavernosa inhibits sPLA 2 activation (IC 50 = 8.8 M), as well as iNOS and COX-2 gene expression [START_REF] Nakao | Enzyme inhibitors from marine invertebrates[END_REF][START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF][START_REF] Mayer | Phospholipase A 2 inhibitors from marine algae[END_REF]. Amongst sesquiterpenes isolated from algae, rhipocephalin (8) extracted from the green alga

PLA 2 inhibiting diterpenes

The diterpenes gracilin A (10), aplyroseol 1 [START_REF] Zarai | A novel hepatopancreatic phospholipase A 2 from Hexaplex trunculus with digestive and toxic activities[END_REF], and 12acetoxytetrahydroaplysulphurin 1 (12) isolated from Aplysilla sp. sponges [START_REF] Nirmal | Modelling studies on phospholipase A 2 -inhibitor complexes[END_REF], and dendrillolide A (13) and norrisolide ( 14) isolated from the sponge Dendrilla sp. inhibit bee venom sPLA 2 with IC 50 values around 5 M [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF]. They all contain a masked 1,4-dialdehyde function, which has been suggested to play a key role in their bioactivity [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF]. The meroditerpne epitaondiol (15) isolated from the brown alga Stypopodium flabelliforme inhibits TXB 2 production by potently inhibiting human sPLA 2 (IC 50 = 3.8 M) [START_REF] Keyzers | Anti-inflammatory metabolites from marine sponges[END_REF][START_REF] König | Marine natural products research: current directions and future potential[END_REF]. The tetra-and bicyclic diterpenes phomactins A-C (16-18) isolated from the marine fungus Phoma sp. are potent PAF antagonists. While the precise mode of action of 16-18 remains poorly understood, it is likely that these three compounds may act as PAF antagonists by inhibiting cPLA2s or by activating (Lp)PLA 2 [START_REF] Niknami | Molecules in focus: Cytosolic phospholipase A 2 -α[END_REF][START_REF] König | Marine natural products research: current directions and future potential[END_REF][START_REF] Sugano | Phomactin A: A novel PAF antagonist from a marine fungus Phoma sp[END_REF]. The arabidose-containing diterpene fuscoside B (19) isolated from the gorgonian Eunicea fusca has not been reported as a PLA 2 inhibitor, but it has been shown to inhibit the conversion of arachidonic acid to LTB 4 by inhibiting 5-LO (IC 50 = 18 M) [START_REF] Nakao | Enzyme inhibitors from marine invertebrates[END_REF][START_REF] Dunlap | Biomedicinals from the phytosymbionts of marine invertebrates: A molecular approach[END_REF].

PLA 2 inhibiting sesterterpenes

Sesterterpenes have an outstanding potential as anti-inflammatory compounds.

The sesterterpene manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF], which was isolated for the first time in the early 1980s from the sponge Luffariella variabilis by Scheuer et al. [START_REF] Silva | Manoalide, an antibiotic sesterterpenoid from the marine sponge Luffariella variabilis[END_REF], became the first marine natural product reported as PLA 2 inhibitor, and it remains, to date, the most investigated marine PLA 2 antagonist. The PLA 2 inhibiting properties of manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF] were discovered simultaneously by research groups lead by Edward Dennis [START_REF] Lombardo | Cobra venom phospholipase A 2 inhibition by manoalide[END_REF] and by Robert Jacobs [START_REF] Glaser | Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2[END_REF] at the universities of San Diego and Santa Barbara, respectively, in the mid 1980's. Both groups confirmed that PLA 2 inhibition was responsible for the previously observed potent anti-inflammatory properties of manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF] [START_REF] Cummings | Phospholipase A 2 as targets for anti-cancer drugs[END_REF][START_REF] Haefner | Drugs from the deep: marine natural products as drug candidates[END_REF][START_REF] Busserolles | Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M[END_REF][START_REF] Nakao | Enzyme inhibitors from marine invertebrates[END_REF][START_REF] Auria | Dysidotronic acid, a new and selective human phospholipase A 2 inhibitor from the sponge Dysidea sp[END_REF][START_REF] Gross | Terpenoids from marine organisms: unique structures and their pharmacological potential[END_REF]. Like bolinaquinone [START_REF] Teixeira | Inflammatory effects of snake venom myotoxic phospholipases A 2[END_REF], manoalide ( 20) is a non-specific inhibitor of PLA 2 s [START_REF] Auria | Dysidotronic acid, a new and selective human phospholipase A 2 inhibitor from the sponge Dysidea sp[END_REF]. Manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF] phospholipase C [START_REF] Auria | Dysidotronic acid, a new and selective human phospholipase A 2 inhibitor from the sponge Dysidea sp[END_REF]. Mechanistic studies revealed that the PLA 2 inhibitory activity of manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF] results from the irreversible binding of two of the compound's masked aldehyde groups (the -hydroxydihydropyran ring and the hydroxybutenolide ring) to lysine residues at the active site of PLA 2 [START_REF] Busserolles | Protection against 2,4,6-trinitrobenzenesulphonic acid-induced colonic inflammation in mice by the marine products bolinaquinone and petrosaspongiolide M[END_REF][START_REF] Gross | Terpenoids from marine organisms: unique structures and their pharmacological potential[END_REF][START_REF] Uddin | Deoxymanoalides from the nudibranch Chromodoris willani[END_REF][START_REF] Ebada | Bioactive sesterterpenes and triterpenes from marine sponges: Occurence and pharmacological significance[END_REF].

Manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF] was licensed to Allergan Pharmaceuticals and reached Phase II clinical trials as a topical antipsoriatic, its development was however, discontinued due to formulation problems [START_REF] Haefner | Drugs from the deep: marine natural products as drug candidates[END_REF][START_REF] Gross | Terpenoids from marine organisms: unique structures and their pharmacological potential[END_REF]. In addition to manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF], several analogues of the molecule have been isolated from sponges belonging to the genus Luffariella, as well from other sponges. The major manoalide analogues include secomanoalide [START_REF] Mayer | Phospholipase A 2 inhibitors from marine algae[END_REF], which has the same potency as manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF], luffariellolide [START_REF] Nirmal | Modelling studies on phospholipase A 2 -inhibitor complexes[END_REF] (IC 50 = 230 nM against bee venom sPLA 2 ), luffariellins A (23) and B (24) (IC 50 = 60 nM against bee venom sPLA 2 ), and luffolide (25) (IC 50 = 40 nM against bee venom sPLA 2 ) [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF]. Manoalide analogues have also been isolated from nudibranchs of the Chromodoris genus, which prey primarily on Luffariella sp. sponges [START_REF] Uddin | Deoxymanoalides from the nudibranch Chromodoris willani[END_REF].

Noteworthy, the nudibranch derived compounds, which include luffariellins C [START_REF] Silva | Manoalide, an antibiotic sesterterpenoid from the marine sponge Luffariella variabilis[END_REF] and D [START_REF] Auria | Dysidotronic acid, a new and selective human phospholipase A 2 inhibitor from the sponge Dysidea sp[END_REF], and deoxymanoalide (28) (IC 50 = 0.2 M against snake venom PLA 2 ) and deoxysecomanoalide (29) (IC 50 = 0.5 M against snake venom PLA 2 ), are all reduced (deoxy) counterparts of spongean manoalide analogues, and their PLA 2 inhibitory activity is a ten-fold weaker than the ones observed in the sponges [START_REF] Uddin | Deoxymanoalides from the nudibranch Chromodoris willani[END_REF][START_REF] Ebada | Bioactive sesterterpenes and triterpenes from marine sponges: Occurence and pharmacological significance[END_REF] Petrosaspongiolide M (34) also inhibits the expression of iNOS and COX-2, and, as a result, the production of NO and PGE 2 , respectively, and NF-B activation [START_REF] Ebada | Bioactive sesterterpenes and triterpenes from marine sponges: Occurence and pharmacological significance[END_REF][START_REF] Dal Piaz | Molecular basis of phospholipase A2 inhibition by petrosaspongiolide M[END_REF][START_REF] Monti | Scalaradial, a dialdehyde-containg marine metabolite that causes an unexpected noncovalent PLA 2 inactivation[END_REF][START_REF] Monti | The inactivation of phospholipase A 2 by scalaradial: a biomimetic study by electrospray mass spectrometry[END_REF][START_REF] Monti | Further insights on the structural aspects of PLA(2) inhibition by gamma-hydroxybutenolidecontaining natural products: a comparative study on petrosaspongiolides M-R[END_REF]. Studies performed by Monti et al. have revealed that, although scalaradial [START_REF] Monti | Further insights on the structural aspects of PLA(2) inhibition by gamma-hydroxybutenolidecontaining natural products: a comparative study on petrosaspongiolides M-R[END_REF] does bind covalently to bee venom PLA 2 , the key step in the PLA 2 inhibitory activity of scalaradial ( 35) is, as observed with petrosaspongiolide M (34), its nonvalent binding to the enzyme's active site [START_REF] Monti | Scalaradial, a dialdehyde-containg marine metabolite that causes an unexpected noncovalent PLA 2 inactivation[END_REF]. The furanosesterterpene palinurin [START_REF] Miyaoka | PLA 2 inhibitory activity of marine sesterterpenoids cladocorans, their diastereomers and analogues[END_REF] isolated from the sponge Ircinia echinata has been shown to inhibit TXB 2 (IC 50 = 5 M), and the furan ring is thought to be the pharmacophore of the molecule [START_REF] Monti | The binding mode of petrosaspongiolide M to the human group IIA phospholipase A 2 : exploring the role of covalent and noncovalent interactions in the inhibition process[END_REF]. The Interestingly, studies on diastereoisomers of cladocoran A (40) and B (41) revealed that the presence of a -hydroxybutenolide moiety itself is not sufficient for PLA 2 inhibitory activity, and that the size and shape of the molecule also play critical roles towards the compounds' potency [START_REF] Miyaoka | PLA 2 inhibitory activity of marine sesterterpenoids cladocorans, their diastereomers and analogues[END_REF].

Non-terpenoid marine PLA 2 inhibitors

The bromohydroquinones cymopol (42) and cyclocymopol (43) isolated from the green alga Cymopolia barbata inhibit bee venom sPLA 2 activity with IC 98 values of 4.7 and 3.4 M, respectively [START_REF] Mayer | Phospholipase A 2 inhibitors from marine algae[END_REF]. The pyridinium alkaloids spongidines A-D (44-47) isolated the sponge Spongia sp. inhibit human sPLA 2 (IC 50 = 10 ) [START_REF] Marino | New pyridinium alkaloids from a marine sponge of the genus Spongia with a human phospholipase A(2) inhibitor profile[END_REF], and the bromophenols vidalol A (48) and B (49) isolated from the red alga Vidilia obtusaloba inhibit bee venom sPLA 2 (IC 50 = 1.6 g/mL) despite lacking a -hydroxybutenolide or masked 1,4-dialdehyde group [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF]. Finally, one of the most recently discovered marine PLA 2 inhibitors, namely the methoxylated fatty acid 7-methoxy-9methylhexadeca-4,8-dienoic acid (MMHDA) (50) isolated from the brown alga Ishige okamurae has been shown to inhibit bacterial PLA 2 (IC 50 = 2 g/mL) [START_REF] Cho | A methoxylated fatty acid isolated from the brown seaweed Ishige okamurae inhibits bacterial phospholipase A 2[END_REF]. isoforms, and to establish the specificity of the compounds for their target PLA 2 .

Future perspectives and concluding remarks

Specific PLA 2 inhibitors are indeed more likely to be bioactive at lower concentrations than non-specific inhibitors, and they are less prone to induce undesired side-effects [START_REF] Cummings | Phospholipase A 2 as targets for anti-cancer drugs[END_REF]. Amongst the marine natural products included in the present review article, bolinaquinone (1) and manoalide (20) and its analogues have been shown to potently inhibit PLA 2 s, but in a non-specific manner. Manoalide [START_REF] Potts | Phospholipase A2 inhibitors from marine organisms[END_REF] has been valued as a potential drug candidate, and it has been taken forward to clinical trials, but it had to be dropped due to formulation problems. To our knowledge, the sesterterpenes palauolol [START_REF] Marino | New pyridinium alkaloids from a marine sponge of the genus Spongia with a human phospholipase A(2) inhibitor profile[END_REF] and palauolide [START_REF] Cho | A methoxylated fatty acid isolated from the brown seaweed Ishige okamurae inhibits bacterial phospholipase A 2[END_REF] have only been The lysophospholipids produced during the conversion of membrane phospholipids to arachidonic acid are a precursor for PAF.

2 .

 2 The PLA 2 -mediated inflammation signaling cascade PLA 2 s are lipolytic enzymes found in almost all types of cells. They specifically hydrolyze the 2-acyl ester bond of 1,2-diacyl-sn-3-glycerophospholipids such as arachidonic acid. Fifteen different PLA 2 s have been characterized to date.

  activation and extravasion of pro-inflammatory leukocytes and activate the secretion of pro-inflammatory histamine by mast cells[START_REF] Yedgar | Control of phospholipase A 2 activities for the treatment of inflammatory conditions[END_REF].

  Rhipocephalus phoenix has been shown to inhibit bee venom sPLA 2 (IC 100 = 4.1 M),

  A c c e p t e d M a n u s c r i p t -7 -and caulerpyne (9) produced by the green alga Caulerpa prolifera inhibits bee venom sPLA 2 activity with an IC 92 value of 4.2 M [21].

  inhibits human sPLA 2 (IC 50 = 1.7 M); snake venom sPLA 2 (IC 50 = 0.03 M); and cPLA 2 (IC 50 = 10 M) [8, 14, A c c e p t e d M a n u s c r i p t -8 -15, 18, 27, 28]. Manoalide (20) has been shown to inhibit cPLA 2 (IC 50 = 10 M) and

A c c e p t e d M a n u s c r i p t - 9 -

 9 hydroxybutenolide ring of petrosapongiolide M (34)[START_REF] Ebada | Bioactive sesterterpenes and triterpenes from marine sponges: Occurence and pharmacological significance[END_REF][START_REF] Dal Piaz | Molecular basis of phospholipase A2 inhibition by petrosaspongiolide M[END_REF][START_REF] Monti | Further insights on the structural aspects of PLA(2) inhibition by gamma-hydroxybutenolidecontaining natural products: a comparative study on petrosaspongiolides M-R[END_REF][START_REF] Monti | The binding mode of petrosaspongiolide M to the human group IIA phospholipase A 2 : exploring the role of covalent and noncovalent interactions in the inhibition process[END_REF].

  sesterterpenes cladocoran A (40) and B (41) isolated from the coral Cladocora cespitosa inhibit sPLA 2 (IC 50 = 0.78 M and 1.95 M, respectively) [37]. Cladocoran A (40) and B (41) caught the attention of Miyako et al. because of their possession of a -hydroxybutenolide moiety as in manoalide (20) and cacospongiolide B (30).

A c c e p t e d M a n u s c r i p t - 10 -

 10 Given the critical role of inflammation in diseases, identifying and developing novel anti-inflammatory drug candidates is of great importance in drug discovery. PLA 2 s play a very important role in inflammation, and they are hence regarded as an interesting target for anti-inflammatory drugs. Fifty marine natural products and counting have been identified as potent PLA 2 inhibitors. Although the quest for novel marine PLA 2 inhibitors faded a little during the 1990's, the last three years have been associated with a fresh spark of enthusiasm into this field of research. Additionally, significant progress has been made recently in the classification and characterization of the different families of phospholipases, and in the understanding of the biochemistry and biology of PLA 2 s. We can therefore expect a high number of novel, highly promising PLA 2 inhibitors to be developed over the next few years, from marine sources, as well as from terrestrial organisms or synthetically produced.Researchers working in this field of research are still facing some major challenges, as they need to find compounds that express high levels of specificity to the PLA 2 s that they are inhibiting. The development of a thorough understanding of the chemical and biological properties of the various types of PLA 2 s, and of their specificity to various diseases, is also a critical point that needs to be addressed, as is the precise understanding of the mechanism of action of PLA 2 -targeting drug candidates. Only recently have the specific biological roles of the different classes of PLA 2 s, and of the different isoforms within these classes, started to become understood, and even though a relatively large number of marine natural products have been tested for their PLA 2 inhibitory effects, most of them have only been screened against a single class of PLA 2 s. For the tested compounds to become potential drug candidates, or to become useful research tools in fundamental biology, it is absolutely critical to screen their bioactivity against each one of the four PLA 2 classes, and against various PLA 2

A c c e p t e d M a n u s c r i p t - 11 -Figure 1 .

 111 Figure 1. The PLA 2 -mediated inflammation signaling cascade cPLA 2 s are calcium-dependent enzymes activated by extra-cellular stimulations from pathogens, tissue injury, or physical or chemical stresses. The cytolic concentrations of calcium required for PLA 2 activation result from the cleavage of phospholipids into IP 3 by PLC, followed by the binding of IP 3 to calcium channels in the endoplasmic reticulum. PLA 2 s hydrolyze the sn-2 acyl ester bond of membrane phospholipids, which leads to the release of arachidonic acid and lysophospholipids. Arachidonic acid is oxygenated into PGH 2 by COX, or into 5-

Figure 2 .

 2 Figure 2. Molecular structure of marine PLA 2 inhibitors.

Figure 1 A

 1 Figure 1

Figure 2 A

 2 Figure 2

  acid is oxygenated into PGH 2 by COX, or into 5-HPTETE by LOX. PGH 2 is the unstable precursor of PGD 2 , PGE 2 , PGI 2 , and thomboxane A 2 (TXA 2 ). PGE 2 and PGI 2 enhance edema formation, pain induction, and fever development. TXA 2 triggers platelet aggregation. LOX converts arachidonic acid into 5-HPTETE, which is spontaneously reduces to 5-HETE, and then to leukotriene A 4 . LTA 4 may be converted to LTB 4 , a potent chemoattractant for polymorphonuclear leukocytes. Eosinophils, mast cells, and alveolar macrophages conjugate glutathione with LTA 4 to make LTC 4 , which is transported outside the cell where a glutamic acid moiety is removed to make LTD 4 . LTD 4 is then cleaved by dipeptidases to make LTE 4 . LTC 4 , LTD 4 , and LTE 4 play an important role in atherosclerosis, asthma, allergenic rhinitis, and inflammatory gastrointestinal diseases.

Table 1 . Bioactivity of marine PLA2 inhibitors.

 1

	Compound	Source organism		IC 50	References
			target PLA 2	( M)
	Sesquiterpenes
	bolinaquinone 1	Dysidea sp. (S)	non-specific	0.1	[13]
	ilimaquinone 2	Hippiospongia	bee venom
		metachromia (S)	sPLA 2	< 270	[19, 20]
	avarol 3	D. avara (S)	sPLA 2	2	[17-19]
	avarone 4	D. avara (S)	sPLA 2	2	[17-19]
	dysidine 5	D. avara (S)	sPLA 2	2	[16-19]
	dysidiotronic acid 6	D. avara (S)	sPLA 2	2.6	[18, 19]
	cavernolide 7	Fasciospongia			[16, 18, 20,
		cavernosa (sponge) sPLA 2	8.8	21]
	rhipocephalin 8	Rhipocephalus	bee venom
		phoenix (GA)	sPLA 2	> 4.0	[21]
	caulerpyne 9	Caulerpa prolifera	bee venom
		(GA)	sPLA 2	> 4.0	[21]
	Diterpenes
	gracilin A 10	Aplysilla sp. (S)	bee venom
			sPLA 2	5	[20]
	aplyroseol 1 11	Aplysilla sp. (S)	bee venom
			sPLA 2	5	[20]
	12-acetoxytetrahydro-	Aplysilla sp. (S)	bee venom
	aplysulphurin1 12		sPLA 2	5	[20]
	dendrillolide A 13	Dendrilla sp. (S)	bee venom
			sPLA 2	5	[20]
	norrisolide 14	Dendrilla sp. (S)	bee venom
			sPLA 2	5	[20]
	epitaondiol 15	Stypopodium	human
		flabelliforme (BA)	sPLA 2	3.8	[19, 23]
	Sesterterpenes
	manoalide 20	Luffariella	human
		variabilis (S)	sPLA 2 :	1.7
			snake venom
			sPLA 2 :	< 0.1	[4, 5, 27,
			cPLA 2 :	10	29, 30]
	secomanoalide 21	L. variabilis (S)	snake venom		[20, 29,
			sPLA 2 :	< 0.1	30]
	luffariellolide 22	L. variabilis (S)	bee venom		[20, 29,
			sPLA 2	0.2	30]
	luffariellin A-B 23-24 L. variabilis (S)	bee venom		[20, 29,
			sPLA 2	0.06	30]
	luffolide 25	L. variabilis (S)	bee venom		[20, 29,
			sPLA 2	0.04	30]

-15 -BA, brown alga; C, coral, F, fungus; GA, green alga; N, nudibranch; N.A., not available; RA, red alga; S, sponge prostaglandins (PG) induce vasodilatation; TXA 2 triggers platelet aggregation; eicosanoids also activate the production of ROS, NO, and cytokines.

Acknowledgements

The authors thank C. Cerella for a critical reading. M. Diederich's research at the Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC) is financially supported by "Recherche Cancer et Sang " foundation, by «Recherches Scientifiques Luxembourg» asbl, by «Een Häerz fir Kriibskrank Kanner» association, the Action Lions "Vaincre le Cancer" Luxembourg, The Fonds National de la Recherche Luxembourg, Televie Luxembourg and the Foundation for Scientific Cooperation between Germany and Luxemburg is thanked for additional support.

Further support was received from the European Union (ITN "RedCat" 215009 and Interreg IVa project "Corena").

Abbreviations 5-HPTETE, 5-hydroperoxyeicosatetraenoic acid; COX, cyclooxygenase; cPLA 2 , cytosolic PLA 2 ; IP 3 , inositol 1,4,5-triphosphate; iPLA 2 , calcium independent PLA 2 ; LOX, lipoxygenase; (Lp)PLA 2 , lipoprotein-associated PLA 2 ; LT, leukotriene; NO, nitric oxide; PAF, platelet-activating factor; PG, prostaglandin; PLA 2 , phospholipase A 2 ; PLC, phospholipase C; ROS, reactive oxygen species; sPLA 2 , secretory PLA 2 ; TX, thromboxane