Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism

EXTRACELLULAR MATRIX OF ADULT ARTICULAR CARTILAGE AND ALTERATIONS DUE TO OSTEOARTHRITIS (OA)

Osteoarthritis (OA) is a chronic disease characterised by slowly progressing destruction of the articular cartilage, accompanied by changes in synovium and subchondral bone.^1,2 The cellular component of the cartilage is the chondrocyte, a specialised mesenchymal cell that synthesises matrix proteins such as collagens and proteogly-cans, which are responsible for the tensile strength and compressive resistance, respectively, to mechanical loading.³ In adult articular cartilage, the chondrocytes maintain a low turnover rate of replacement of cartilage matrix proteins. The turnover of collagen has been estimated to occur with a half-life of >100 years,⁴ whereas the glycosaminoglycan constituents on the aggrecan core protein are more readily replaced and the half-life of aggrecan subfractions has been estimated to be in the range of 3–24 years.⁵

The adult articular chondrocyte, although quiescent in normal cartilage, can respond to mechanical injury, joint instability due to genetic factors and biological stimuli such as cytokines and growth and differentiation factors.^1,6 In young subjects without genetic abnormalities, biomechanical factors due to trauma are strongly implicated in initiating the OA lesion.^7–9 Mechanical disruption of cell–matrix interactions may lead to aberrant chondrocyte behaviour that is reflected in the appearance of fibrillations, cell clusters and changes in quantity, distribution or composition of matrix proteins.¹⁰ In the early stages of OA, a transient increase in chondrocyte proliferation and increased metabolic activity are associated with a local loss of proteoglycans occurring initially at the cartilage surface and followed by cleavage of type II collagen.^11,12 This results in increased water content and decreased tensile strength of the matrix as the lesion progresses.

A number of studies have demonstrated enhanced biosynthesis and increased global gene expression of aggrecan and type II collagen in human OA compared with normal cartilage.^13,14 The increased levels of factors such as bone morphogenetic protein-2 (BMP-2) and inhibin bA/activin suggest a possible mechanism for the anabolic response.^12,14–16 However, controversy exists, based on studies in animal models ^17–19 and analysis of cartilage samples or body fluids from patients with OA,^20–22 about whether type II collagen gene (COL2A1) expression is increased or suppressed, appearing to depend upon the zone of cartilage analysed and the stage of OA. Aigner and coworkers have shown that expression of the COL2A1 is suppressed in the upper zones of early OA cartilage associated with the catabolic phenotype, but increased in late-stage OA cartilage compared with normal and early degenerative cartilage.^23,24 Recent large-scale expression profiling, using full-thickness cartilage, showed that many collagen genes, including COL2A1, are upregulated in late-stage OA.^25,26 This applies predominantly to those chondrocytes in the middle and deep zones, as found by laser capture micro-dissection, whereas the anabolic phenotype was less obvious in the degenerated areas of the upper regions.²⁷

A mechanism involving phenotypic modulation has been proposed with expression of collagens normally absent in adult articular cartilage or at atypical sites in OA cartilage.²⁸ Type X collagen, a marker of the hypertrophic chondrocyte that is normally absent in adult articular cartilage, as well as type III collagen and type VI collagen, which are present at low levels in normal cartilage, and the chondroprogenitor splice variant of the type II collagen gene, type IIA, have been detected during certain stages of OA or in atypical sites within OA cartilage. In addition to COL10A1 and matrix metalloproteinase-13 (MMP-13), other chondrocyte terminal differentiation-related genes, such as MMP-9 and Indian hedgehog (Ihh) are detected in the vicinity of early OA lesions.²⁹ Surprisingly, decreased levels of Sox9 mRNA are detected near the lesions,²⁹ and the expression of this factor, which is required for activation of COL2A1 transcription and its interacting partners, l-Sox5 and Sox6, does not always localise with COL2A1 mRNA in adult articular cartilage.^27,30 Once the cartilage is severely degraded, the chondrocyte is unable to replicate the complex arrangement of collagen laid down during development. Furthermore, the chondrocyte stress response may result in the loss of viable cells due to apoptosis or senescence.³¹ Our recent study indicates that intracellular stress response genes are upregulated in early OA, whereas a number of genes encoding cartilage-specific and non-specific collagens and other matrix proteins are upregulated in late-stage OA cartilage.²⁶ Asporin, a transforming growth factor β (TGFβ) binding protein, is increased in late OA cartilage^26,32 and a polymorphism of this gene is associated with disease severity.³³ Runx2, a hypertrophic chondrocyte marker, is also increased in OA cartilage and may contribute to the loss of appropriate anabolic responses in adult articular chondrocytes.³⁴

SIGNALLING AND TRANSCRIPTIONAL REGULATION BY PROINFLAMMATORY MEDIATORS

IL1 and TGFα share the ability to activate a diverse array of intracellular signalling pathways, although the cell surface receptors and associated adaptor molecules are distinct. In chondrocytes, the JNK, p38 MAPK and NF-κB signalling pathways predominate in the regulation of IL1- and TGFα-induced genes. These pathways are also involved in the inhibition of COL2A1 expression by these cytokines,^67,68 and non-canonical NF-κB signalling via IKKα may also contribute to the abnormal phenotype of OA chondrocytes.⁶⁹ Along with ERK1/2, the key protein kinases in these signalling cascades are activated, particularly in the upper zones of OA cartilage.⁷⁰ Injurious mechanical stress and cartilage matrix degradation products can stimulate the same signalling pathways as those induced by IL1 and TGFα.^71–75 Since these pathways also induce or amplify the expression of cytokine genes, it remains controversial whether inflammatory cytokines are primary or secondary regulators of cartilage damage and defective repair mechanisms in OA. Nevertheless, physiological loading on cartilage may protect against cartilage loss by inhibiting IKKβ activity in the canonical NF-κB cascade and attenuating NF-κB transcriptional activity,⁷⁶ as well as by inhibiting phosphorylation of TAK1.⁷⁷

Cytokine-activated transcription factors of the NF-κB, C/EBP and ETS families, as well as EGR-1, are involved in repressing, directly or indirectly, COL2A1 promoter activity.^50,59,78 During trauma or inflammation, the chondrogenic transcription factors may be inactivated by direct or indirect interactions with cytokine-induced transcription factors. It has been proposed that cytokine-mediated repression of COL2A1 transcription is dependent upon inhibition of Sox9 expression,^44,79,80 although expression of Sox9 and COL2A1 does not always correlate.^50,81–83 However, Sox9 overexpression in chondrocytes may either increase or decrease COL2A1 transcription depending upon its concentration and the differentiation state of the cells.⁸⁴ During chondrocyte hypertrophy the suppression of Sox9 expression and activity by transcription factors such as Runx2 has been proposed as a mechanism essential for endochondral ossification.^85,86 Although Runx2 is required for IL1 induction of MMP-13 gene transcription in articular chondrocytes,⁸⁷ its role in the suppression of chondrocyte phenotype by inflammatory cytokines has not been defined. IL1 may also induce chondrocytes to synthesise other cytokines such as IL6, which downregulates COL2A1, aggrecan and link protein via the JAK/STAT pathway in association with suppression of Sox9.⁸⁸

COL2A1 is one of the cytokine-responsive genes without functional binding sites for NF-κB or AP-1, indicating that downregulation of COL2A1 promoter activity by IL1 cannot be ascribed to direct interaction of these transcription factors with DNA elements. A recent study also showed that NF-κB is not required for modulation of Sox9-dependent COL2A1 promoter activity by Bcl-2.⁸⁹ Despite previous findings,⁷⁹ we observe that IL1β treatment does not decrease the constitutive levels of Sox9, l-Sox5 and Sox6 mRNA,⁵⁰ similar to our findings regarding the inhibition of COL2A1 transcription by interferon γ.⁸¹ Sox9 and related HMG factors are architectural proteins that act to maintain the nucleosomes in an open configuration, thereby exposing the endogenous, chromatin-integrated COL2A1 promoter to regulatory binding proteins.⁹⁰ In differentiated chondrocytes at a post-developmental stage of low matrix turnover, the COL2A1 promoter cannot respond to negative regulation by cytokines unless it is activated. In support of this concept are findings that the integration of the responses of cytokine-induced promoters requires the assembly of higher-order nucleoprotein complexes orchestrated by HMG-I(Y) factors.^91,92

In addition to members of the AP-1 (Jun/Fos) family, EGR-1 is an immediate early growth response factor that plays a role in IL1β-regulated transcriptional events. We reported that EGR-1 inhibits COL2A1 promoter activity by binding to the –131/+125 bp core promoter and displacing Sp1 from at least one of the GGGCG boxes that overlap with the EGR-1 binding site.⁵⁰ A similar mechanism may account for the increased ratio of Sp3 to Sp1 binding to the Sp1 sites observed in response to IL1.⁹³ Since overexpression of CBP reverses the inhibition, it is probable that activation of EGR-1 and its binding to DNA disrupts the interactions among Sp1, CBP and TATA-binding proteins.⁵⁰ This early response appears to be transient, suggesting that complete transcriptional repression of COL2A1 promoter activity may be dependent upon the binding to DNA of other IL1β-induced factors, such as C/EBPβ and C/EBPδ.⁵⁹ In contrast to EGR-1, C/EBPβ and δ are induced by IL1β in a time-dependent manner over 48 h, suggesting that they are late regulators.⁹⁴ Both IL1β and TGFα can act together to decrease expression of the cartilage-characteristic proteins, but the response may be additive owing to the involvement of different combinations of C/EBP sites.^51,95 However, C/EBP proteins act as negative regulators of chondrogenesis during skeletal development, during which IL1β is not known to play a role, and their presence in muscle and in other non-cartilage tissues may contribute to the lack of expression of cartilage-characteristic genes.^94,95

CARTILAGE DESTRUCTION IN OA

During ageing and joint disease, this equilibrium between the synthesis and the degradation of cartilage matrix is disrupted and the rate of loss of proteoglycans from the matrix may exceed the rate of deposition of newly synthesised molecules, exposing the collagen network to irreversible destruction. In contrast, in rheumatoid arthritis, cartilage is degraded primarily in areas contiguous with the synovial pannus owing to the direct action of synovial proteinases and cytokines.¹⁰⁶ However, synovitis is common in advanced OA involving infiltration of mononuclear cells, and the expression of proinflammatory mediators is seen in early and late OA.^107,108 Recent evidence indicates that IL1β and TGFα are produced independently in OA synovial cells associated with differential expression of matrix-degrading enzymes.¹⁰⁹ Cytokines originating from the cartilage, synovium and other surrounding tissues can increase the gene expression levels and activities of MMPs and ADAMTS (a disintegrin and MMP with thrombospondin motifs) in chondrocytes. These include MMP-1, MMP-3, MMP-8, MMP-13, MMP-14 and the aggrecanases, ADAMTS-4 and –5. MMP-13 has a prominent role in degrading the collagen network in cartilage owing to its particularly potent ability to cleave type II collagen.^110,111 Furthermore, MMP-13-specific type II collagen cleavage products have been immunolocalised in OA cartilage,^56,112 and constitutive expression of MMP-13 in cartilage in mice produces OA-like changes in knee joints.¹¹³

SIGNALLING MECHANISMS INVOLVED IN INTERACTIONS OF CHONDROCYTES WITH CARTILAGE MATRIX

Alteration of chondrocyte metabolic responses may also result from mechanical disruption of chondrocyte–matrix associations.¹³⁵ More rapid matrix turnover may occur in the immediate pericellular zones compared with the interterritorial zones of cartilage.^112,136,137 This suggests roles for chondrocyte cell surface receptors such as integrins and discoidin domain receptor 2 (DDR2) in the response to mechanical stress that may result in the disruption of normal remodelling of matrix components.^138–141 The engagement of integrin receptors by fibronectin or collagen fragments activates focal adhesion kinase signalling and transmits signals intersecting with ERK, JNK and p38 signalling pathways, which converge on AP-1 and ETS sites to transactivate the MMP-13 promoter.^{124,142–144} The upregulation of MMP-13 due to upregulation and activation of DDR2 is somewhat distinct, since it requires direct interaction with native type II collagen fibrils rather than fragments and is not associated with upregulation of other MMP or ADAMTS genes.¹⁴¹ This mechanism was discovered in the type XI collagen haploinsufficient Cho/+ mouse with chondrodysplasia and the Col9a–/– mouse, both of which increase DDR2 at 6 months associated with MMP-13 gene expression and activity. In these mice, the collagen fibrils are modified and are present in increased amounts in the pericellular matrix compared with the relatively low amounts in wild-type cartilage.^141,145,146 This mechanism is not restricted to genetic abnormalities, since DDR2 expression associated with MMP-13-dependent collagen destruction is seen in human OA, as well as in surgical mouse OA, where loss of proteoglycans would permit interaction of the chondrocyte cell surface with denuded collagen fibrils.¹⁴⁷ The activation of DDR2 by intact type II collagen fibrils requires the PTK core and tyrosine phosphorylation at the Y740 site and leads to increased MMP-13 gene expression via Ras/Raf/MEK/ERK pathway and p38 signalling. Neither IL1-mediated nor integrin-mediated signalling is involved.¹⁴⁷

GADD45β AND MMP-13 RECAPITULATE DEVELOPMENTAL EVENTS IN OA

During a microarray study to identify BMP-2-induced early genes involved in signalling and transcriptional regulation in chondrocytes, we identified growth arrest and DNA damage-inducible (GADD)45β as one of the most highly expressed genes at 1 h.¹⁴⁸ GADD45β (MyD118) is member of a family of stress-induced proteins inducible by NF-κB,^149,150 but its role in cartilage had not been explored previously. We also found that Runx2 plays a synergistic role in both the induction and activity of GADD45β by enhancing BMP-2-induced Smad1 signalling and AP-1 (JunB or D/Fra2)-dependent MMP-13 gene expression.¹⁴⁸ The GADD45β gene product has been implicated as an anti-apoptotic factor during genotoxic stress and cell cycle arrest in other cell types. In a microarray analysis, we observed upregulation of GADD45β in normal cartilage and in early OA compared with late OA, whereas anabolic genes, including COL2A1, were upregulated in late OA.²⁶ Intracellular staining for GADD45β was present in chondrocytes throughout normal adult articular cartilage and in chondrocyte clusters and deep-zone chondrocytes with hypertrophic morphology in OA cartilage. Consistent with a potential role for GADD45β as a survival factor, knockdown of GADD45β enhances TGFα-induced apoptosis. In contrast, IL1β is a less effective inducer of chondrocyte apoptosis, suggesting the ESE-1 may not have a role in this process. Similar to ESE-1, however, GADD45β is induced by NF-κB and its overexpression downregulates COL2A1 promoter activity and mRNA levels.²⁶

CONCLUSION

Despite our increasing knowledge of the mechanisms regulating the production and activities of anabolic and catabolic factors involved in OA, the application of the principles of cell and molecular biology, such as those described in this review, to the development of disease-modifying treatments has been elusive.¹⁵¹ Diverse stimuli induce MMP-13 expression and activity in chondrocytes and both similar and distinct signalling and transcription events are involved. Some of these events also downregulate COL2A1 and other anabolic genes that might be recruited for cartilage repair. However, targeting these events in vivo may be difficult, since they occur at different times and at localised sites in cartilage, as well as in other joint tissues.¹⁵² Nevertheless, specific approaches should take into account these events, given the failure of broad-spectrum MMP inhibitors against osteoarthritic processes in clinical trials and their unexpected side effects on muscle and the skeleton.¹⁵³ We also need to consider other mediators of these events, such as Toll-like receptors,^154–157 intermediate cytokines and chemokines^158,159 and alternate signalling kinases, as well as the crosstalk between anabolic and catabolic signals. The role of epigenetics in the “unsilencing” of genes not expressed normally in cartilage and other joint tissues might also be a fruitful area of investigation.¹⁶⁰