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Inflammatory Signaling in Cartilage: MAPK and NF- B Pathways in Chondrocytes and the Use of Inhibitors for Research into Pathogenesis and Therapy of Osteoarthritis
Jeremy Saklatvala*
Kennedy Institute of Rheumatology, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH
Abstract: Osteoarthritis is characterised by degeneration of articular cartilage. It is thought to be primarily a disease of cartilage. Inflammatory response genes, such as proteinases, cyclooxygenase, and cytokines are implicated in its pathogenesis. The evidence for expression of these genes in articular cartilage in osteoarthritis is reviewed. The expression of inflammatory response genes is controlled by four major intracellular signalling pathways. These lead to activation of the three mitogen-activated protein kinases (MAPK) and the transcriptional regulator nuclear factor kappa (NF)-B. The current state of knowledge of the structure of these pathways is summarized. Pharmacological inhibitors of the protein kinases of the pathways in current use are described, and insights into chondrocyte gene expression obtained with them are discussed. Very limited use of these inhibitors has yet been made in animal models of osteoarthritis. The main use of the inhibitors in the near future will be in investigation of pathogenetic mechanisms in osteoarthritis, both in experimental animals and in vitro, with a view to identifying therapeutic targets. Prospects for using signalling pathway inhibitors for therapy in osteoarthritis are distant.

Key Words: Chondrocyte, MAP kinase, NF-B, matrix metalloproteinase, protein kinase, protein kinase inhibitor, inflammatory response, osteoarthritis. INTRODUCTION The intracellular signalling pathways that control expression of genes of the inflammatory response have become therapeutic targets in a wide range of diseases. A key step in the pathophysiology of osteoarthritis is breakdown of the extracellular matrix of articular cartilage by tissue proteinases, enzymes whose expression is upregulated by inflammatory stimuli. There is evidence of increased production of these and other proteins of the inflammatory response in osteoarthritic cartilage. It is therefore possible that blocking the intracellular signalling pathways, for example by inhibiting protein kinases, could be used therapeutically in the disease to prevent the production of proteinases and other inflammatory mediators. Development of inhibitors of inflammatory signal transduction pathways is still at an early stage. It remains to be seen if they are therapeutically useful, but nevertheless they are proving invaluable for investigating molecular mechanisms of disease, and for identifying which pathways are important for key genes in a particular pathophysiological situation. In this article the evidence for inflammatory gene expression in osteoarthritis is critically reviewed. The organisation of the core signal transduction pathways inducing inflammatory genes is summarized and the protein kinase inhibitors in use and development are described. The insights that these inhibitors are giving into chondrocyte gene regulation, as well as their effects on the course of experimental arthritis, are then discussed. It is important to bear in mind that osteoarthritis is probably not a single disease entity: it is clinically heterogeneous and there are likely to be many factors underlying its origin and determining its course. The cartilage disintegration may be a final common pathway that follows tissue failure which may have a number of causes. EVIDENCE FOR INFLAMMATORY GENE EXPRESSION IN OSTEOARTHRITIC CARTILAGE (a) Defining Inflammation The characteristic loss of cartilage that is the hallmark of osteoarthritis is widely believed to be caused by an excess of intrinsic tissue proteinase activity due to the matrix metalloproteinases and aggrecanases. These enzymes are inducible by inflammatory stimuli such as the primary inflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF). However their production is not restricted to inflammatory sites or in any way specific to inflammation. They are also induced by growth factors, and may be expressed during developmental processes and tissue remodelling. Is the cartilage lesion of osteoarthritis inflammation or, as has been argued in the past, simply degeneration? Inflammation can be defined at three levels. Macroscopically it is the four cardinal signs of heat, swelling, redness and pain which reflect vascular changes. At a histopathological level it is the infiltration of leucocytes, while at a molecular level it is a pattern of gene expression induced by activating cells via either the IL-1 receptor or TNF receptor families. Many genes are induced, and the pattern varies with cell type. Broadly, they comprise a large number of cytokines, cell-surface molecules and enzymes. Macroscopically, at the level of the clinical signs, osteoarthritis is not an overtly inflammatory disease when compared with rheumatoid arthritis for example, although it is often punctuated by inflammatory episodes possibly reflecting synovitis. At a histological level, it is not characterized by prominent leucocyte infiltration, although small inflammatory foci are often seen in the synovial membrane. Synovitis could be involved in both initiation and progression of the disease, but is generally considered secondary to changes in the cartilage. The assumption that osteoarthritis is a primary disease of cartilage is a reasonable one, but has to be made with caution. At a molecular level, osteoarthritic cartilage appears to be expressing some genes typical of an inflammatory response. Whether or not this is a result of activation of the inflammatory signal transduction pathways by inflammatory cytokines such as IL-1, and whether blocking such activation is likely to be therapeutically practical and beneficial, are the questions at issue.

*Address correspondence to this author at the Kennedy Institute of Rheumatology, Imperial College London, 1 Aspenlea Road, Hammersmith, London W6 8LH; Tel: +44 (0) 20 8383 444; Fax +44 (0) 20 8383 4496; E-mail: j.saklatvala@imperial.ac.uk

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(b) Proteinases in Osteoarthritic Cartilage Cartilage from osteoarthritic joints contains transcripts of the two major collagenases, MMP-1 and MMP-13 [1] and the general proteinase stromelysin (MMP-3) [2]. MMP-1 and MMP-3 proteins have been detected in osteoarthritic synovial fluid [3,4], although the levels were considerably less than in rheumatoid arthritis [4]. There is strong evidence that collagenase is active in osteoarthritic cartilage because the specific cleavage of collagen occurs when the tissue is cultured [5]. The evidence of these early studies has been supplemented by that of more recent investigations in which cDNA arrays or polymerase chain reaction (PCR) have been used to measure mRNA. Analysis of osteoarthritic knee cartilage early and late in the disease with a cDNA array [6] showed increases in transcripts of MMP-2, MMP-11 and MMP-13 in late samples, while MMP-3 mRNA, which was present in early disease and normal tissue, fell. More recent investigations from the same laboratory confirmed by real time PCR that MMP-13 mRNA was increased in late disease, but little significant difference between normal and diseased tissue was found in mRNA levels of MMP-1, or of the candidate aggrecanases ADAMTS-4 and ADAMTS-5 [7]. A comprehensive analysis of metalloproteinase and proteinase inhibitor mRNA in osteoarthritic femoral head cartilage [8] found increases in MMP-13 and MMP-3, and also in MMP-28 and ADAMTS-16. Little is known concerning the function of the latter two enzymes, but MMP-28 is also expressed in cartilage from rheumatoid joints [9] so may be an inflammatory response protein. This study also did not find up-regulation of ADAMTS-4 and ADAMTS-5, although there has been one report of increased aggrecanase activity and ADAMTS-4 mRNA in osteoarthritic cartilage [10]. ADAMTS-5 is a mediator of experimental OA and inflammatory arthritis in the mouse [11,12]. (c) Intracellular Enzymes of Inflammation The mRNA of COX-2, a typical inflammatory response gene, was found to be overexpressed in osteoarthritic cartilage [13]. The diseased tissue produced more prostaglandin (PG)E2 in culture than normal. The function of PGE2 in cartilage is not known. It elevates cAMP which can negatively regulate inflammatory response genes and inhibit cell proliferation. It has not been reported to have marked effects on synthesis of extracellular matrix components. Osteoarthritic cartilage also makes more nitric oxide than normal [14], but whether this is due to increased expression of iNOS, a well-characterized inflammatory response gene, or another NOS is unclear [14,15]. Osteoarthritic cartilage showed increased expression of iNOS by immunohistochemistry in one investigation [16] but not in another [17]. Nitric oxide has been implicated as a potential inhibitor of cartilage matrix synthesis and cause of chondrocyte apoptosis [18]. (d) Cytokines The cytokine that has been most widely investigated in osteoarthritic cartilage is IL-1. This is because it is the most strongly catabolic agent for cartilage as judged by its ability to cause extracellular matrix degradation [19], and to inhibit proteoglycan and collagen synthesis [20-22]. TNF is less potent but has similar effects [23], as does IL-17 [24,25]. The cartilage-catabolic effects of IL-1 and TNF are enhanced by IL-6-type cytokines [26]. In an early report of immunostaining of IL-1 in osteoarthritic cartilage [27] its presence was, paradoxically, inversely correlated with histological severity. Subsequent studies have reported immunostaining of IL-1 in diseased areas along with staining of TNF, iNOS [16] and various other cytokines [28,29]. A more recent investigation by quantitative PCR found increased mRNA of IL-1, IL-6 and IL-8 in osteoarthritic cartilage [30]. Cultured osteoarthritic cartilage also secreted more of these cytokines than normal together with more IL-18, TNF and monocyte chemoattractant protein (MCP)-1.

Cytokines produced in response to an inflammatory stimulus need not necessarily have adverse effects on the cartilage matrix. BMP-2 expression is increased in osteoarthritic cartilage [31]. It is inducible in chondrocytes by IL-1 or TNF but would be expected to have an anabolic effect on cartilage. THE INTRACELLULAR SIGNALLING PATHWAYS MEDIATING INFLAMMATORY RESPONSE GENE EXPRESSION (a) Receptors that Trigger Inflammatory Responses There is therefore evidence for expression in osteoarthritic cartilage of genes which are characteristic of an inflammatory response. This is strongest for the MMPs, particularly MMP-13, where data on mRNA levels are supported by biochemical studies of the cartilage and synovial fluid, and relevant proteolytic cleavage has been shown in the tissue. Inflammatory response genes are generally induced by activation of members of either IL-1 receptor or TNF receptor families. The former includes the two IL-1 receptors (only the type 1 signals), the nine or ten Toll-like receptors (TLRs) which recognise a wide range of microbial molecules, and the IL-18 receptor. It is also called the TIR (standing for Toll and IL-1 receptor) family. The TNF receptor family includes the type I and II TNF receptors, the lymphotoxin receptors, the receptor activator for nuclear factor (NF)-kB (RANK), CD40 and a number of other proteins important in immune cell signalling. An inflammatory response usually originates from leucocytes (particularly macrophages) being triggered by primary stimuli such as TIR or TNFR family ligands, or by an immune reaction. The initial stimulation produces a range of cytokines, some of which may synergise with the primary stimulus to amplify the response: for example, -interferon synergises with TIR and TNFR family ligands in the induction of iNOS, while IL-6-type cytokines synergise in production of acute phase response proteins and MMPs. IL1 and TNF are themselves important secondary amplifiers as well as primary stimuli, since they are produced by mononuclear phagocytes in response to TIR and TNFR family ligands. IL-1 and TNF are generated through signalling in the intracellular pathways which they themselves activate. If osteoarthritis is an inflammatory disease it is difficult, in the absence of leucocytes, to understand where the pro-inflammatory drive originates. If there is autocrine production of IL-1 and TNF how is this initiated? (b) MAP Kinases Four pathways form the essential core of intracellular signalling through which TIR and TNFR family ligands induce inflammatory response genes. These are the system leading to activation of the transcriptional regulator NF-B, and the phosphorylation cascades of each of the three types of mitogen-activated protein kinase (MAPK). Extensive recent reviews of these pathways [32,33] and the control of inflammatory gene expression [34] can be found elsewhere, so only the main features are illustrated in Fig. (1). The c-Jun N-terminal kinases (JNKs) and p38 MAPKs are strongly activated by inflammatory stimuli (ie TIR and TNFR family ligands) and by experimental cell stress (eg heat shock, UV irradiation, hyperosmolar conditions). They are sometimes called stressactivated kinases. The extracellularly regulated kinases (ERKs) 1 and 2 are activated by a wide range of stimuli, including growth factors, many cytokines and microbial products. In contrast to JNK and p38 they are only weakly activated, if at all, by cell stress. No pathway is specific for inflammation. Fig. (1A) shows the upstream mechanisms activated by the IL-1 receptor. Similar signalling complexes form in the case of TLRs, except that adaptor proteins additional to MyD88 may be used, and there is an extra pathway promoting induction of interferons [35]. Key steps in the stimulation of the MAPK cascades by IL-1 are the activation of Tpl2, MEKK3

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and TGF-activated kinase (TAK)1 by TNF-receptor activated factor (TRAF)6. MEKK3 and TAK1 activate JNK, p38 and NF-B, while Tpl2 activates the ERK pathway [36,37]. The TNFR signalling complex activates the same MKKs as the TIRs but by a different mechanism Fig. (1B). TRAF2 (and probably TRAF5) is coupled to the TNFR signalling complex and activates TAK1, MEKK3 and Tpl2 [37,38]. MEKK3, together with RIP, is important for NF-B activation, while Tpl2 is needed for both JNK and ERK activation by TNF. There is still uncertainty as to which MKKKs are important for signalling by the different individuals of the TIR and TNFR families, and usage varies with cell type [34]. The major physiological difference between signalling via TNFR and TIRs is that the TNFR also activates apoptotic pathways via death-domaincontaining adaptor proteins. In most, but not all conditions or cells, the anti-apoptotic effects of NF-B dominate. Some negative regulators of apoptosis are NF-B-dependent [39]. Fig. (1A ) also shows the signalling cascades below the level of the MKKs. The MKKs are specific, and phosphorylate the MAPKs on both hydroxyamino acids of a Thr-X-Tyr motif. They are dual specificity protein kinases. The intervening amino acid residue defines the type of MAPK: in ERKs it is glutamyl, in JNKs prolyl and in p38 glycyl. The ERKs are ubiquitously expressed, as are JNK1 and JNK2. JNK3 is more restricted, but is highly expressed in the nervous system. The p38 MAPK ,  and  forms are also widely expressed, while the  form is limited to muscle. The MAPKs are serine and threonine kinases and phosphorylate an overlapping set of substrates, some of which are themselves protein kinases, while others are transcription factors Fig. (1A). Transcription of inflammatory response genes is controlled by a limited group of transcription factors, which bind to the 5' upstream promoter sequences. Sites for NF-B, activator protein (AP)-1,

C/EBP and Ets family members are all common. The MAPKs phosphorylate these factors, making them transcriptionally active. While all three MAPK systems affect transcription, the p38 pathway also plays an important role in stabilising the mRNAs and enabling their translation. Many inflammatory response protein mRNAs are unstable. Stabilising them amplifies expression and reversing the stabilisation provides a mechanism for rapid removal of the mRNA at the end of a response. The mRNA instability is directed by AU-rich elements in the 3' untranslated regions of the mRNAs. The effect of the instability elements is countered by the MAPK-activated protein kinase (MAPKAPK)-2, which phosphorylates RNA-binding proteins and probably other substrates involved in the control [40]. MAPKAPK-2 is activated by p38 Fig. (1A). The MAPK pathways not only regulate inflammatory gene expression, they also affect apoptotic mechanisms. For example, inhibiting JNK reduces ischaemia-related apoptosis [41]. Both p38 and ERK are generally thought to be anti-apoptotic. The biochemical pathways shown in Fig. (1) have been elucidated mainly in cultured cell lines and much has been inferred from cells where genes are overexpressed, and from altered signalling in mice where genes have been selectively silenced. The physiological functions of the pathways have also been explored with pharmacological inhibitors, and some inhibitors have entered clinical trials for therapy in inflammatory disease. The MAPK pathways in chondrocytes have not been investigated in detail but appear to be very similar to those of other cells [42]. The relative expression of the different forms of p38 and JNK is not known. (c) The NF- B System The transcription factor NF-B comprises hetero and homodimers of the NF-B/Rel family of proteins: p65 (RelA), RelB, c-Rel,

Fig. (1). A: Signalling pathways activated by the IL-1 receptor type I. The major putative kinases and substrates of the MAPK systems are shown. Many downstream targets of ERK have been omitted. Protein kinases are shown in bold type. B: Signalling from the TNF receptor 1.

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p50 (NF-B1) and p52 (NF-B2). Only the Rel proteins have transcription activating domains. Homo and heterodimers of p50 and p52 are generally repressive with the exception of only very few promoters. The predominant mechanism of transcriptional inhibition by p50 homodimers appears to be the recruitment of histone deacetylases [43]. The abundant form of NF-B is a heterodimer of p65/RelA and p50. In the resting state this is bound to the inhibitory protein IB of which  or  are the major forms [32]. The trimeric complex shuttles between nucleus and cytoplasm, but is mainly cytoplasmic. When a TIR or TNFR signalling complex forms it activates the IB kinase (IKK) component of the IKK complex Fig. (2 ). The IKK complex is also a trimer and comprises IKK and , together with NEMO/IKK . NEMO/IKK  is an important adaptor enabling the receptor signalling complexes to activate the IKK. IKK phosphorylates IB  at Ser32 and Ser36. The phosphorylations target the IB for rapid ubiquitination and degradation. This removes the nuclear export signal of the inhibitor and unmasks a nuclear localisation signal on the p65 subunit. The NF-B dimer therefore locates mainly in the nucleus where it binds to specific sites in the promoter regions of sensitive genes and promotes transcription. The activity of the NF-B subunits is also controlled by their phosphorylation and possibly by other modifications. For example, the MAPK- and stress kinase-activated protein kinase (MSK), a downstream kinase activated by ERK or p38 Fig. (1A), increases p65 transcriptional activity by phosphorylating Ser276 [32], while multiple protein kinases phosphorylate Ser536 [44]. This classical pathway is activated not only by TIR and TNFR signalling complexes, but also by B cell receptor and T cell receptor activation [32].

A second form of latent NF-B B comprises p100, the precursor of p52 (NF-B2), complexed with RelB [45]. Activation of this referred to as the alternative pathway Fig. (2). Upon cell activation p100 is phosphorylated by IKK. This results in ubiquination of p100 and it is processed to p52 by the proteasome. A nuclear localization signal is unmasked and the p52/RelB dimer translocates to the nucleus. This alternative pathway is activated in response to signalling by some TNFR members:CD40, lymphotoxin  receptor and the B-cell activation factor receptor of the TNF family (BAFFR). The MKKK called NF-B-inducing kinase (NIK) is involved in the activation of IKK  Fig. (2). Whether IKK affects the classical pathway is uncertain. NF-B activation in chondrocytes in response to inflammatory stimuli appears to be very similar to other cell types [46,47] although the expression levels of the different components and the relative roles of the two pathways have not been investigated. PHARMACOLOGICAL INHIBITORS OF INFLAMMATORY SIGNALLING PATHWAYS (a) ERK Pathway PD098059 Fig. (3A) [48,49] and U0162 Fig. (3B) [50,51] are synthetic small molecules which inhibit the activators of ERK, MKK/MEK 1 and 2. Their mode of action is unclear. They do not compete with either ATP or substrate for binding to the MKK. They have been widely used in experiments with cells, and are reasonably selective for the ERK pathway. U0162 is the more potent compound and has an IC50 ~ 0.1 M on cells. While ERK signalling is important for inflammatory gene expression, the pathway

Fig. (2). The classical and alternative pathways of NF-B. The classical pathway involves activation of NF-B complexes bound to IB. The most abundant involve p65 (RelA) and the NF-B1, p50, which is automatically processed from the precursor NF-B1 p105. The pathway is triggered by TIR, TNFR1/2, T and B cell receptors, IKK is activated in the signalsome complex and phosphorylates IB on Ser32 and Ser36. This is a signal for ubiquitination and proteasomal degradation of IB. In the alternative pathway the NF-B2 precursor, p100, is processed to p52 in a manner similar to the degradation of IB in the classical pathway. The pathway is important in lymphoid tissue development and is activated by certain TNFR family members: CD40, lymphotoxin  receptor and B cell-activating factor of TNF family (BAFF) receptor. IKK, activated by the NF-B-inducing kinase (NIK), phosphorylates p100 targeting it for ubiquitination and limited proteolysis by the proteasome to p52.

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is not thought to be a good target for therapeutic inhibition because it is involved in the function of so many cellular regulators in addition to inflammatory stimuli. (b) JNK Inhibitors The JNKs are attractive drug targets for pharmaceutical companies because inhibiting them may not only inhibit inflammation, but may also protect cells against ischaemia-related apoptosis and reperfusion injury. Two approaches have been used for their inhibition: synthetic small molecules and cell-permeable peptides. The most widely used and best-characterized inhibitor is the anthropyrazolone derivative SP600125 Fig. (3C) [52]. It competes with ATP for the binding site in the kinase and although it has a Ki ~ 200 nM for all three JNKs, it only has an IC50 ~ 5-10 M in cellbased assays [51]. The difference in the concentrations needed for inhibition in biochemical and cell culture experiments is presumably due to the high intracellular ATP concentration. Concerns have been expressed over its specificity when it is used at 10 M [53]. Two other recently reported JNK inhibitors are AS601245, a benzothiazol derivative which is competitive with ATP [54] and AS600292, a sulfonamide [55]. A 20 amino acid peptide from the JNK-binding domain of the JNK-interacting protein (JIP), a scaffold protein, inhibits JNKs [56]. This was fused to a 10-amino acid sequence of the HIV transactivator protein TAT that directs cellular import, and was used to inhibit intracellular JNK activity [41,57]. The peptide inhibitor has been used in animals to protect against neuronal death caused by experimental transient ischaemia [41]. Another JNK inhibitory peptide comprises the c-Jun  domain fused to the protein transduction domain of HIV1-TAT [58]. (c) p38 MAPK Inhibitors The imidazole pyrimidine inhibitors of p38 such as SB203580 Fig. (3D) and SB202190 Fig. (3E) have been used for about 10 years and are very well characterized [40,59]. They inhibit the  and  forms of p38 and are reasonably specific if used at concentrations up to 1 M. At higher concentrations (1-10 M) they inhibit JNKs and PKB [60]. They are anti-inflammatory in experimental animals but their toxicity has prevented clinical use. Many newer p38 inhibitors are in development in the pharmaceutical industry
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and some are in clinical trials for treatment of inflammatory diseases such as rheumatoid arthritis [40]. (d) Inhibitors of NF- B Activation Various approaches have been taken to inhibiting the NF-B system. Proteasomal proteinase inhibitors such as Z-Leu-Leu-LeuCHO (also known as MG-132) and lactacystin have been widely used. These block the proteasomal degradation of the ubiquitinated IB which is the crucial step allowing nuclear translocation of the transcription factor upon cell activation. However, interfering with proteasomal function has many cellular effects, so these inhibitors are not specific to the inflammatory response. The major mechanism by which inflammatory stimuli activate NF-B is through IKK. Some natural products and non-steroidal anti-inflammatory drugs (eg aspirin) are weak IKK inhibitors, and more potent synthetic inhibitors are being developed. BMS-345541, an imidazoquinoxaline, is an allosteric inhibitor with IC50 ~ 0.3 M for IKK, and ~ 4 M for IKK  [61]. It has been used to suppress collageninduced arthritis [62], experimental colitis [63] and graft rejection [64] in mice. AS602868 is an anilino pyrimidine derivative with an IC50 20 nM for IKK  and 14 M for IKK [65]. It is competitive with ATP. Other less well-characterized synthetic IKK inhibitors are a diathiazole, SC-514 [66], -carbolines [67], a thiophenecarboxamide [68] and S1627 [69]. Inhibition of IKK activity in cells has also been achieved with various cell permeable peptides. One contains the NEMO/IKK-binding domain of IKK linked to an antennapedial homeo-domain which confers cell permeability. This peptide prevents the interaction between IKK and IKK  required for the latters activation [70]. Other reported inhibitors are a peptide representing the nuclear localisation sequence of NF-B p50 linked to the signal peptide of Kaposi FGF [71], and a peptide based on the phosphorylation sites in p65 fused to the antennapedial homeo-domain [72]. EFFECTS OF INHIBITORS OF INFLAMMATORY SIGNAL TRANSDUCTION PATHWAYS ON CHONDROCYTE FUNCTION AND EXPERIMENTAL ARTHRITIS The signalling pathway inhibitors have been used on cartilage and chondrocytes mainly to explore the dependence of expression of particular genes on individual pathways. They have been tested in animal models of inflammatory arthritis, but there is little inforE N NH N
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O PD098059 NH2 SP600125 F SB202190 B D N H2N NC CN NH NH2 N F U0126 H2N SB203580 S O


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Fig. (3). Structure of some protein kinase inhibitors. A and B are MKK/MEK 1 and 2 inhibitors, C is a JNK inhibitor, D and E are p38 MAPK inhibitors.

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mation regarding their effects on the course of experimental osteoarthritis. The effect of inhibiting p38 MAPK in cartilage was investigated in early experiments carried out with SB203580. Collagen breakdown in cartilage explants cultured with IL-1 was inhibited, which was consistent with inhibition of MMP-1 and MMP-3 expression in cell culture [73]. Interestingly there was no effect on IL-1-stimulated proteoglycan degradation, which is thought to be dependent upon aggrecanases such as ADAMTS-4 and ADAMTS5. The p38 inhibitor did not reverse the suppressive effect of IL-1 on proteoglycan synthesis. The p38 inhibitors ameliorate experimental inflammatory arthritis [59], but there have been no reports of their effects on experimental osteoarthritis progression. The role of the ERK pathway has been investigated in experimental osteoarthritis. The MKK/MEK1/2 inhibitor PD198306 was tested in rabbits in osteoarthritis induced by anterior cruciate ligament section [74]. The inhibitor was administered systemically and it reduced cartilage lesion size and histological damage seen after 8 weeks. There was less synovial inflammation, and less immunostaining of active ERK and MMP-1 in the cartilage of treated animals. The JNK inhibitor SP600125 inhibited radiographic damage [75], and modestly reduced swelling in adjuvant-induced arthritis in rats, and the IKK inhibitor BMS-345541 reduced inflammation and destruction in collagen-induced arthritis in mice [62]. There are no reports of the effects of JNK or NF-B inhibitors on the course of experimental osteoarthritis. USE OF PROTEIN KINASE INHIBITORS TO DEFINE ROLE OF PATHWAYS IN EXPRESSION OF SPECIFIC GENES IN CHONDROCYTES The inhibitors of signalling are proving very valuable for dissecting the dependence of expression of particular genes on individual pathways. If we knew which genes were crucial to development of osteoarthritis, and which pathways their expression was most dependent on, we could choose therapeutic targets logically. Many studies have investigated the sensitivity of proteinase gene expression to signalling inhibitors. An early investigation [76] found that while both MMP-1 and MMP-13 protein expression induced by IL-1 in a human chondrosarcoma line was blocked by a p38 inhibitor, only MMP-13 was strongly affected by a MKK/ MEK1/2 inhibitor (PD98059). MMP-13 was also more strongly dependent on JNK and NF-B than MMP-1. Slightly different results were obtained with isolated rabbit articular chondrocytes stimulated with IL-1. MMP-1 expression was unaffected by either the p38 or the MEK inhibitor, while MMP-13 expression was reduced by p38 blockade. The failure of a MKK/MEK1/2 inhibitor to inhibit MMP-1 expression in IL-1 treated rabbit chondrocytes was unexpected in view of the suppression it caused in experimental osteoarthritis [74]. Of course, the stimulus for MMP-1 expression in experimental arthritis may be something other than IL-1, and the expression may therefore be more dependent on the ERK pathway. In experiments on human and bovine chondrocytes in which IL-1, TNF or IL-17 was used as stimulus, inhibition of p38, JNK (with SP600125), and of MKK/MEK1/2 (with PD98059 or U01625), all suppressed mRNA for MMP-13, MMP-3 and aggrecanase-1 [7779]. Generally in these studies protein levels of the enzymes were reduced in accordance with the reduction of steady state mRNA levels. The level of the regulation of the expression of the proteinases by the signalling pathways has been investigated in chondrocytes. MMP-13 transcription depends upon NF-B and AP-1 activation [76], as well as upon Runx-2 which may be activated by the p38 pathway [80]. MMP-1 transcription is controlled through AP-1 factors and NF-B [81]. The MMPs are also controlled posttranscriptionally. MMP-1 and MMP-3 mRNAs are stabilised by the p38 pathway via their 3' untranslated regions (3'-UTR) [82]and MMP-13 has a functional AU-rich destabilising element in its 3'

UTR [76]. Thus the expression of MMP-1, MMP-3 and MMP-13 induced by inflammatory cytokines depends on all four pathways. It is not clear which pathway in human chondrocytes would have to be blocked for most effective MMP suppression in osteoarthritis. Less information is available concerning the effect of inflammatory signalling pathway inhibitors on expression of chondrocyte genes other than MMPs. COX-2 expression in IL-1 stimulated chondrocytes is sensitive to p38 MAPK and MKK/MEK1/2 inhibitors [83]. Interestingly, production of IL-1-inducible PGE synthase was also blocked by SB203580, which inhibits p38 and , but not by a specific p38 inhibitor. This is the first report of implicating a p38 specific effect [83]. Expression of iNOS induced by IL-1 is sensitive to p38 inhibition in bovine, but not human, articular chondrocytes [84]. Production of NO and expression of iNOS have been implicated in causing cell death in osteoarthritic cartilage. Cartilage explants from dogs with experimental osteoarthritis had a reduced number of apoptotic cells after treatment with inhibitors of p38 or MKK/MEK1/2 [85]. The inhibitors also reduced expression of iNOS and COX-2 in the explants. An NF-B inhibitor (the nuclear localisation sequence peptide) reduced COX-2 expression in the canine cartilage, but did not affect apoptosis. The effects of the inhibitors on chondrocyte survival were surprising. One would expect that inhibiting either NF-B or ERK would increase apoptosis. Inhibiting the ERK pathway in isolated chondrocytes caused apoptosis [86]. The relationship of the pathways to apoptotic mechanisms in chondrocytes may be complex. The effects of the pharmacological signal transduction inhibitors have been intensively investigated in many cell types besides chondrocytes. Mostly the aim has been to suppress expression of inflammatory response genes, and note the immediate physiological consequences. Little is known about what effects blocking the signalling pathways may have on normal cartilage metabolism in the longer term, or on natural repair processes. While the NF-B, JNK and p38 MAPK systems are thought to function primarily as sensors of cell stress and inflammatory mediators, they may also play a role in chondrogenesis [87] and normal cartilage metabolism. There is evidence that ERK and p38 may be involved in cartilage development and homeostasis [88]. The ERK pathway is activated by growth factors such as IGF-1, insulin and FGFs. Basic FGF is present in normal cartilage and induces Sox9, a master regulator of chondrocyte differentiation and expression of cartilage-specific genes [89]. The induction of Sox9 is inhibited by the MKK/MEK1/2 inhibitor U0126, suggesting that the ERK pathway is important for the expression of chondrocytic phenotype [89]. FGF-induced growth arrest of chondrocytes requires ERK and p38 activity [90]. The actions of connective tissue growth factor, which may also play a role in cartilage development and normal homeostasis, are affected by MAPK pathway inhibitors [91]. A p38 inhibitor prevented it stimulating proteoglycan production, and a MEK inhibitor abrogated its effect on DNA synthesis. If osteoarthritis is an inflammatory disease then interfering with the JNK and NF-B systems might be less likely to have adverse effects than inhibiting ERK and p38. THE NATURE OF THE INFLAMMATORY STIMULI INITIATING AND MAINTAINING OSTEOARTHRITIS Although there is evidence for inflammatory response gene expression in osteoarthritic cartilage, the importance of any of the gene products for the pathogenesis of the disease remains to be established. There is stronger evidence for the proteinases playing a role, than for example, cytokines, COX-2 or iNOS. There is no direct evidence that inflammatory cytokines are causing expression of the MMPs in osteoarthritic cartilage. As stressed earlier, MMPs and COX-2, can be induced by growth factors, as well as by inflammatory agents. Although immunostaining for IL-1 and TNF has been reported in osteoarthritic cartilage, it remains to be established that they actually have a primary role in driving proteinase

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expression and tissue degradation in the disease. If proteinase production was being driven by bFGF, for example, then inhibiting JNK and NF-B would probably have little effect on its expression. It is thought that any inflammatory response of the chondrocytes is likely to be deleterious to the extracellular matrix, so therapy should be aimed at blocking such a response. However, the changes in osteoarthritic chondrocytes while resembling an inflammatory response may nevertheless be an attempt at repair, and be delaying cartilage loss. One feature of osteoarthritic cartilage which is not consistent with an inflammatory phenotype of the chondrocyte is that it is often synthesising type II collagen, in contrast to normal adult cartilage, which synthesises very little [92,93]. Inflammatory cytokines such as IL-1 tend to suppress, rather than increase, collagen production [22]. IL-1 and TNF have been the most widely investigated potential mediators of cartilage catabolism in osteoarthritis. However other possible inducers of cartilage damage, for example degradation products of extracellular matrix molecules, need to be considered. Fragments of fibronectin cause production of MMPs and degradation of cartilage very similar to that induced by IL-1 [94]. Both the 29 kDa N-terminal fragment [95] and a 120 kDa fragment [96,97] activate the MAP kinases and induce MMP expression. Fibronectin itself does not have these effects. The 120 kDa portion contains the binding site for the 51 integrin, the main fibronectin receptor of chondrocytes. Integrin engagement is therefore implicated in the action of this fragment [96], while the 29 kDa moiety probably acts via a different mechanism [95]. Osteoarthritic synovial fluid contains active fibronectin fragments [98], and it has been postulated they could arise from MMP-3 action. Tetra-and hexasaccharides of hyaluronic acid, which are generated by hyaluronidase action, stimulate inflammatory intracellular signalling in dendritic cells by activating TLR4 [99], but their effects on chondrocytes and other connective tissue cells have not been reported. Much effort has been expended searching for mediators in osteoarthritis that drive inflammatory signalling. The role of inflammatory cytokines remains uncertain, and whether or not crucial mediators are intrinsic to the cartilage, or arise from the synovium or synovial fluid is still unknown. Activation of inflammatory signalling pathways may not need an extrinsic stimulus. They can be activated directly by mechanical damage to the tissue. Simply cutting cartilage activates the MAPKs and induces inflammatory gene expression [100]. Very little is yet known about how the tissue responds to mechanical damage of the matrix, but given the well known relationship of osteoarthritis to physical stress and damage these responses could be important in the genesis of the disease. CONCLUSIONS The prospect of using inflammatory signalling pathway inhibitors for therapy in osteoarthritis is uncertain and distant. We do not know whether genes currently implicated in pathogenesis, such as metalloproteinases, are expressed in osteoarthritis as a result of inflammatory signalling, and we do not know how the putative signalling is being driven. Much of the work referred to in this review has examined the effect of inhibitors on IL-1 stimulated chondrocytes and there is a widespread assumption that the stimulus in osteoarthritis is IL-1, or IL-1-like. The currently used protein kinase inhibitors, although proving very valuable for pharmacological experiments, are probably not highly specific and are likely to have toxic effects which will rule out their use in a chronic non-life threatening disease. An alternative approach to using protein kinase inhibitors might be to antagonise catabolic stimuli by delivering an anti-catabolic cytokine such as TGF- [101,102] by local gene therapy in badly affected joints. Given the uncertainty regarding the molecular mechanisms that underly cartilage degeneration, the main contribution of inhibitors will be to help define in animal models of osteoarthritis which genes and signalling pathways are crucial for tissue degeneration. More specific inhibitors are badly

needed, and with these it should be possible to unravel mechanisms and identify therapeutic targets with more confidence than is presently possible. ABBREVIATIONS ATF = Activating transcription factor C/EBP = CAAT enhancer binding protein CREB = c-AMP response element binding protein eIF = Eukaryotic initiation factor ERK = Extracellular signal-regulated kinase H3 = Histone H3 Hsp = Heat shock protein IB = Inhibitor of NF-B IKK = IB kinase IL-1RacP = IL-1R accessory protein IRAK = IL-1R associated kinase JNK = c-jun N-terminal kinase MAPK = Mitogen-activated protein kinase MAPKAPK = MAPK-activated protein kinase MEF = Myocyte enhancer factor MEKK = MAPK or ERK kinase kinase MKK = MAP kinase kinase MKKK = MKK kinase MNK = MAPK signal integrating protein kinase MSK = MAPK and stress kinase-activated protein kinase NF-B = Nuclear factor kappa B (p65/RelA heterodimer with p50) NF-Bp65 = p65 subunit of NF-B(RelA) p38 = p38 MAPK PRAK = p38-regulated/activated kinase SAP = SRF accessory protein SRF = Serum response factor TAK = TGF- activated protein kinase TLR = Toll-like receptor TRAF = TNF receptor associated protein FADD = Fas-associated death domain -containing protein RIP = Receptor interacting protein kinase TRADD = TNFR-associated death domain-containing protein REFERENCES
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Received: September 30, 2005

Accepted: April 20, 2006