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Rheumatology Advance Access originally published online on February 8, 2006
Rheumatology 2006 45(8):958-965; doi:10.1093/rheumatology/kel024
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Retinoic acid and oncostatin M combine to promote cartilage degradation via matrix metalloproteinase-13 expression in bovine but not human chondrocytes

W. D. Shingleton*, D. Jones, X. Xu1, T. E. Cawston and A. D. Rowan

Musculoskeletal Research Group and 1 Bio-Imaging Unit, University of Newcastle upon Tyne, Newcastle upon Tyne, UK.

Correspondence to: D. Rowan, Musculoskeletal Research Group, School of Clinical Medical Sciences, Medical School, Cookson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK. E-mail: a.d.rowan{at}ncl.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. Retinoic acid (RetA) and oncostatin M (OSM) have both been shown to mediate potent effects with respect to extracellular matrix integrity. This study assesses the effects of a RetA + OSM combination on cartilage catabolism.

Methods. Animal and human cartilage samples were used to assess the ability of RetA + OSM to promote the release of collagen and proteoglycan fragments, which was determined by measuring glycosaminoglycan and hydroxyproline, respectively. Total collagenolytic and tissue inhibitor of metalloproteinases (TIMP) inhibitory activities were determined by bioassay, whilst gene expression of matrix metalloproteinases (MMPs) and TIMP-1 were determined by northern blotting. Immunohistochemistry was used to assess the presence of MMP-1 and -13 in resorbing cartilage explants.

Results. Both agents alone induced proteoglycan release from bovine cartilage, whilst RetA-induced collagen release was variable. Reproducible and synergistic collagenolysis was observed with RetA + OSM, which appeared to be due to MMP-13. Similar collagen release was observed from porcine cartilage. Conversely, no collagen release was seen with human articular cartilage. In primary human chondrocytes, RetA + OSM failed to induce MMP-1 or -13 but caused a significant increase in TIMP-1 expression.

Conclusions. These novel observations show that the combination of RetA + OSM has profound effects on cartilage matrix turnover, but these effects are species-specific. A better understanding of the mechanism by which this combination differentially regulates MMP and TIMP expression in human chondrocytes could provide valuable insight into new therapeutic strategies aimed at the prevention of cartilage destruction.

KEY WORDS: Oncostatin M, Retinoic acid, Cartilage, MMP-13, TIMP-1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Extracellular matrix (ECM) remodelling is mediated by matrix metalloproteinase (MMPs), an enzyme family that collectively can degrade all ECM components. The degradative potential of MMPs is controlled at several key points, including the stimulation of synthesis and secretion by cytokines and growth factors, the production of latent pro-enzymes and inhibition by naturally occurring inhibitors [1]. The tissue inhibitors of metalloproteinases (TIMPs) are important regulators that inhibit all active MMPs, forming tight binding complexes [2].

Excessive, uncontrolled matrix catabolism is a characteristic of arthritic diseases, and a specific MMP subfamily (the collagenases) has been strongly implicated: MMP-1 is present in rheumatoid synovial fluid [3], has been localized in rheumatoid joints [4] and is up-regulated by cytokines such as interleukin-1 (IL-1) [5–7]; MMP-8 is co-expressed with IL-1 by osteoarthritic chondrocytes [8]; and MMP-13 is present in both rheumatoid and osteoarthritic cartilages [4, 9]. Expression of MMP-13 is highly significant since it is the most efficient collagenase against type II collagen [9, 10], the predominant cartilage collagen.

Cartilage is sparsely populated by chondrocytes, which maintain tissue integrity. Proteoglycan is rapidly released from cartilage in response to cytokines such as IL-1 [5], but is replaced relatively quickly [11]. In contrast, collagen is much less readily released, but when collagenolysis does occur structural integrity is irreversibly lost [12]. Collagenolysis, therefore, represents a key step in cartilage destruction.

Oncostatin M (OSM) is a cytokine of the IL-6 family, all of which bind a common receptor, glycoprotein 130 (gp130) [13]. OSM primarily mediates effects via Janus kinase/signal transducers and activators of transcription (JAK/STAT), and mitogen-activated protein kinase (MAPK) pathways [13]. All-trans-retinoic acid (RetA) is a naturally occurring, biologically active derivative of retinol, a vitamin A family member. Its effects are mediated by RetA receptors (RARs) and retinoid X receptors (RXRs), both nuclear receptors of the steroid/thyroid nuclear receptor superfamily [14], which when activated bind RetA response elements in gene promoters [15]. RetA is important in cartilage development [16], profoundly affecting cartilage and bone metabolism, and we have previously demonstrated that RetA and IL-1 synergize to promote cartilage collagenolysis via MMP-1 and -13 [17].

RetA resembles OSM in several respects since both: (i) induce TIMP-1 production from connective tissue cells [18, 19]; (ii) induce cartilage aggrecanolysis [5, 20]; (iii) increase plasminogen activator activity [21, 22], which could contribute to procollagenase activation [5]; and (iv) synergize with IL-1 to increase MMP expression and cartilage collagenolysis [7, 17]. Moreover, both RetA and OSM have been shown to synergize with other mediators, modulating MMP/TIMP levels in favour of MMPs, thus promoting collagenolysis [7, 17, 23]. However, the effects of these two putative chondroprotective agents together have not been previously reported and are the focus of the present study.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Recombinant human OSM was a generous gift from Prof. J. Heath (University of Birmingham, UK), and recombinant human IL-1{alpha} was from GlaxoSmithKline (Stevenage, UK). All-trans-RetA was from Sigma (Poole, UK). Fetal calf serum (FCS) was from Invitrogen (Paisley, UK). BB-94 was a gift from British Biotech Pharmaceuticals (Oxford, UK). Recombinant human TIMP-1 was produced in-house [24]. cDNAs for human MMPs, TIMPs, gp130, OSM-specific receptor (OSMRß) and rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were as described [6]. The anti-human MMP-1 and -13 antibodies were raised in-house [5]. All other reagents were commercially available analytical grade obtained from Fisher (Loughborough, UK) or Sigma, or have been described previously [5–7].

Cartilage degradation assay
Samples of bovine nasal septum cartilage and porcine articular cartilage (metacarpophalangeal joint) were prepared from material provided by a local abattoir as described [17]. Briefly, three discs or cartilage pieces per well of a 24-well plate were incubated at 37°C in 600 µl of control medium [Dulbecco's Modified Eagle Medium (DMEM) containing 25 mM HEPES (Invitrogen) supplemented with 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin and 40 U/ml nystatin] for 24 h. Fresh control medium (600 µl), with or without test reagents (four wells for each condition), was added and incubated at 37°C for 3 days. Supernatants were harvested and replaced with fresh medium containing test reagents. This was repeated at day 7 and the experiment was continued to day 14. In some experiments the medium was only changed at day 7. All culture supernatants were stored at –20°C until assayed. The remaining cartilage was papain-digested until completion [17]. For histology, cartilage samples were incubated for 4 h in 4% paraformaldehyde at room temperature (RT), rinsed with PBS and then incubated in 30% sucrose/PBS (48 h, 4°C) before snap-freezing in OCT.

Viability of cartilage explants was assessed by screening for the production of lactate dehydrogenase (LDH) using the Cytotox 96 assay (Promega, Southampton, UK). This is always performed when new combinations are used with cartilage explants. No increase in LDH levels with any of the cytokine or inhibitor combinations was found (data not shown). Since this model is one of cartilage breakdown, we excluded serum, which does not affect tissue viability (see [6] and references therein).

Proteoglycan and collagen assays
Media samples and papain digests were assayed for sulphated glycosaminoglycan (GAG; a measure of proteoglycan release) using a microtitre plate modification of the 1,9-dimethylmethylene blue dye binding assay [25]. Hydroxyproline (OHPro) release (a measure of collagen degradation) was assayed using a microtitre plate assay [17, 26]. The extent of release was then calculated as a percentage of the total.

Enzyme and inhibitor assays
Collagenolytic activity was measured using a modified diffuse fibril assay with 3H-acetylated calf skin collagen [27]. Trypsin was used to confirm that the collagen was native. Aminophenylmercuric acetate (APMA) was included (0.7 mM final concentration) to allow measurement of total (pro + active) collagenolytic activity since it activates procollagenases. Inhibitory activity of samples was determined by the addition of a known amount of active collagenase to each sample in the diffuse fibril assay, and the percentage inhibition calculated. One unit of collagenase activity degrades 1 µg of collagen per minute at 37°C and one unit of inhibitory activity inhibits two units of collagenase by 50%.

Chondrocyte culture
The human chondrocyte cell line T/C28a4 was cultured as described [6]. Samples of bovine nasal cartilage or human articular cartilage (full-depth, macroscopically normal articular cartilage obtained with consent under local ethical committee approval from osteoarthritis patients undergoing joint replacement surgery) were cut into pieces (~2 mm3), washed three times in PBS containing penicillin (100 U/ml), streptomycin (100 µg/ml) and nystatin (40 U/ml). Primary chondrocytes were isolated using sequential enzymatic digestion [28], and 1 x 106 cells were seeded per 25 cm2 culture flask (Corning/Costar, High Wycombe, UK) in control medium supplemented with 10% FCS. Once 80% confluent, cells were washed with PBS and serum-starved for 24 h. Fresh control medium with or without RetA or OSM or combinations thereof (IL-1 was also included in some experiments at 1 ng/ml) was then added for various time points. Media were stored at –20°C until assayed, and cells harvested into RNeasy lysis buffer (Qiagen, Crawley, UK) for total RNA isolation.

Northern blotting
Northern blot analyses were performed on total RNA (20 µg/lane) as described [6]. Probe-specific mRNA was visualized using autoradiographic film or by exposure to a phosphor screen (Molecular Dynamics, Chesham, UK). The signal for GAPDH was used to normalize for RNA loading.

Histology
Frozen sections (5 µm) were prepared using a CM1900 cryostat (Leica Microsystems, Milton Keynes, UK) set at –20°C. Sections were placed in 100% acetone (10 min, RT), air-dried, rehydrated in 70% acetone and then in Tris-buffered saline (TBS) (pH 7.6) (2 min, RT). Endogenous peroxidase was then blocked using 20 mM NaN3, 0.3% H2O2 in PBS for 10 min (RT). Thereafter, serial sections were blocked with 20% normal pig serum in TBS for 1 h at 4°C, and then incubated overnight at 4°C with the following polyclonal primary antibodies: sheep anti-pig MMP-1 at 2 µg/ml or rabbit anti-human MMP-13 at 10 µg/ml; negative controls were performed by replacing the primary antibody with an appropriate sheep or rabbit control IgG at the same concentration. Sections were subsequently washed twice (5 min) in TBS, and then incubated with biotinylated secondary antibody: rabbit anti-sheep IgG (Vectastain Kit PK-6106) for MMP-1 or goat anti-rabbit IgG (Vectastain Kit PK-6101) for MMP-13, both in 20% normal swine serum in TBS (1 h, RT). Sections were washed twice (5 min) in TBS and streptavidin–biotin–peroxidase complex solution (Dako) added (30 min, RT) according to the manufacturer's instructions. Sections were then washed twice (5 min) in TBS, and signals (brown colour) developed using 3,3'-diaminobenzidine tetrahydrochloride following the manufacturer's protocol (Sigma). Sections were counterstained with Mayer's haematoxylin (10 s) and washed extensively in tap water before being dehydrated through ascending grades of alcohol. Both anti-MMP antibodies used recognize proenzyme, active and TIMP-complexed enzyme.

Some sections (5 µm) were stained with safranin O (for proteoglycans) with Weigert's haematoxylin and fast green counterstaining. Images of stained sections were captured using a JVC 3-CCD colour video camera (Victor Company of Japan, Tokyo, Japan) with Leica QWin software and displayed on a computer monitor.

Statistics
The data shown are the means ± S.D. Statistical significance was assessed using ANOVA with post hoc Bonferroni's multiple comparison test using commercially available software (SPSS, version 11.0).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Degradation of animal cartilages
The combination of RetA + OSM rapidly and reproducibly induced degradation of bovine cartilage. RetA alone (10–7 M) induced proteoglycan release by day 3 of culture (Fig. 1A), which was more marked by day 7 or at 10–6 M (data not shown). When in combination with OSM, a dose-dependent increase in aggrecanolysis was observed at day 3 (Fig. 1A). At 10–6 M, RetA induced significant proteoglycan release, as reported elsewhere [20], and a small but statistically significant amount of collagen release (13.3 ± 6.1%, P<0.01; six separate experiments) by day 14 (not shown). Indeed, RetA-induced collagenolysis was variable, as previously reported [17]. Collagenolysis was not always seen at 10–7 M, never at 10–8 M and not by day 7 irrespective of treatment (not shown). OSM alone had little effect on collagen release, as previously reported [5, 6]. The RetA + OSM combination promoted synergistic and dose-dependent collagenolysis, which was most marked with OSM at 10 ng/ml (Fig. 1B).


Figure 1
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  		FIG. 1. Effect of RetA + OSM on the degradation of bovine cartilage. Bovine nasal cartilage was stimulated with RetA (10–7 M) or OSM (1 or 10 ng/ml) or combinations thereof. Media were harvested and replaced with identical treatments at days 3 and 7, and the final harvest was at day 14. (A) Proteoglycan release (as determined by glycosaminoglycan) was expressed as a percentage of the total, and the data presented are for the release by day 3. (B) Cumulative collagen release (as determined by OHPro) at day 14 expressed as a percentage of the total. Bonferroni's multiple comparison test was used to assess statistical significance. ***P ≤ 0.001; **P ≤ 0.01. The data are representative of six separate experiments.

 
RetA also induced modest collagenolysis from porcine cartilage to an extent similar to that seen in bovine tissue, whilst RetA + OSM also promoted synergistic release of collagen from porcine cartilage (Fig. 2), although a higher concentration of OSM was necessary, as previously reported [5]. However, like other potent combinations for animal cartilages [5, 6], RetA + OSM failed to induce collagen release from human articular cartilage (not shown).


Figure 2
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  		FIG. 2. Effect of RetA + OSM on the degradation of porcine cartilage. Porcine articular cartilage was stimulated with RetA (10–6 M) and/or OSM (50 ng/ml), as detailed in Fig. 1. Proteoglycan and collagen releases were expressed as a percentage of the total. Bonferroni's multiple comparison test was used to assess statistical significance. ***P ≤ 0.001. The data are representative of three separate experiments.

 
Bioassay data from the media samples revealed a rapid but transient induction of TIMP inhibitory activity for both RetA and OSM alone, being most evident for RetA + OSM. However, only OSM alone had significantly detectable TIMP levels by day 14 (Fig. 3A). Conversely, collagenolytic activity was only measurable in day-14 supernatants, being most marked for RetA + OSM, although RetA alone did have detectable levels of collagenolytic activity in excess of TIMP (Fig. 3B). Detection of collagenolytic activity mirrored the observed collagenolysis (compare Figs 3B and 1B).


Figure 3
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  		FIG. 3. TIMP and collagenolytic activity levels in RetA + OSM-treated bovine cartilage. Conditioned culture media from RetA (10–7 M)- and/or OSM (10 ng/ml)-stimulated bovine cartilage explants in Fig. 1 were assayed for (A) TIMP inhibitory activity or (B) total collagenolytic activity. Bonferroni's multiple comparison test was used to assess statistical significance. ***P ≤ 0.001; *P ≤ 0.05. The data are representative of six separate experiments.

 
Inclusion of the hydroxamate-based metalloproteinase inhibitor BB94 into the bovine cartilage assay partially abolished RetA + OSM-induced aggrecanolysis, whereas TIMP-1 did not (Fig. 4A). Addition of either BB-94 or TIMP-1 effectively abolished RetA + OSM-induced collagenolysis (Fig. 4B), further demonstrating that this is MMP-mediated.


Figure 4
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  		FIG. 4. Effect of metalloproteinase inhibitors on RetA + OSM-induced cartilage degradation. Bovine nasal cartilage was stimulated with RetA (10–7 M), OSM (10 ng/ml) or combinations thereof with or without BB-94 (10–5 M) or TIMP-1 (100 units/ml), as described in Fig. 1. Releases of (A) proteoglycan and (B) collagen were expressed as a percentage of the total. Bonferroni's multiple comparison test was used to assess statistical significance. ***P ≤ 0.001. The data are representative of three separate experiments.

 
Collagenase and TIMP expression in bovine chondrocytes and cartilage
To further identify the collagenase(s) responsible for the observed collagenolysis from bovine cartilage, total RNA isolated from stimulated bovine chondrocytes was analysed. MMP-13 mRNA was induced following RetA + OSM stimulation, with maximal induction at 48 h (Fig. 5), which persisted for 7 days (not shown). No MMP-1 mRNA was detected when blots were probed with a human MMP-1 cDNA probe known to cross-react with the bovine counterpart [17, 28]. Signals for MMP-2 and MMP-14 were also detected but neither enzyme was regulated in response to RetA and/or OSM, and no signal was detected for MMP-8 (not shown). Low TIMP-1 mRNA levels were detected throughout the culture period but were modestly up-regulated by OSM (Fig. 5). There was a marked induction of TIMP-1 with IL-1 + OSM at 48 h, confirming previous studies [29]. Thus, the only collagenase that RetA + OSM appeared to specifically induce was MMP-13. Significant levels of collagenolytic activity (proform only) were detected in the medium of RetA + OSM-treated chondrocytes; this induction was not seen until day 3 but again persisted through to day 7 (Fig. 6).


Figure 5
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  		FIG. 5. MMP expression in RetA + OSM-treated bovine chondrocytes. Bovine nasal chondrocytes were stimulated for 24 or 48 h, and total cellular RNA was harvested and subjected to northern blotting. GAPDH was used for normalization. C, control; I, IL-1 (1 ng/ml); O, OSM (10 ng/ml); R, RetA (10–6 M); I/O, IL-1 + OSM (1 and 10 ng/ml); R/I, RetA + IL-1 (10–6 M and 1 ng/ml); R/O, RetA + OSM (10–6 M and 10 ng/ml). The IL-1 + OSM and RetA + IL-1 combinations were included for comparative purposes [7, 17]. The data are representative of three separate experiments.

 

Figure 6
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  		FIG. 6. Effect of RetA + OSM on the production of collagenolytic activity from bovine chondrocytes. Bovine nasal chondrocytes were stimulated with RetA (10–6 M) and/or OSM (10 ng/ml). Media samples were harvested over a 7-day period and assayed for active and total collagenolytic activity. Bonferroni's multiple comparison test was used to compare RetA + OSM with each individual treatment at a given time point. ***P ≤ 0.001. The data are representative of three separate experiments.

 
Safranin O staining of stimulated bovine cartilage revealed that, compared with control (Fig. 7A), both RetA (Fig. 7B) and OSM (not shown) induced modest aggrecanolysis whilst RetA + OSM resulted in essentially complete proteoglycan release by day 7 (Fig. 7C); this is concordant with the day-3 data (compare Figs 7A–C and 1A). We failed to detect MMP-1 staining in RetA-, OSM- or RetA + OSM-treated cartilage (not shown), in line with mRNA expression data. Little or no MMP-13 staining was seen in control, OSM- or RetA-stimulated cartilage at any time point (Fig 7D, E and G). However, MMP-13 staining was evident in RetA + OSM-treated cartilage at day 10 (Fig. 7F), with evidence of matrix dissolution, which was more pronounced by day 14 (Fig. 7I). RetA + IL-1-treated cartilage had similar staining, with even more marked ECM perturbation (Fig. 7H), in line with the increased MMP-13 expression of this combination (see Fig. 5).


Figure 7
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  		FIG. 7. Histological analyses of RetA + OSM-stimulated bovine cartilage. Bovine nasal cartilage was stimulated with RetA (10–6 M), OSM (10 ng/ml), IL-1 (1 ng/ml) or combinations thereof, as described in Fig. 1. Cartilage was frozen at days 7, 10 and 14, and sections (5 µm) were stained for (A–C) safranin O (for proteoglycan) with haematoxylin and fast green counterstaining or (D-I) MMP-13 with haematoxylin counterstaining. The treatments were: A and D, control; B and G, RetA; C, F and I, RetA + OSM; E, OSM; H, RetA + IL-1. The bars represent 50 µm.

 
Collagenase and TIMP expression in human chondrocytes
RetA and OSM failed to induce collagenolysis from human articular cartilage, even when in combination (not shown). In primary human chondrocytes, OSM alone modestly induced MMP-1 and MMP-13 gene expression, but markedly induced TIMP-1 (Fig. 8A), as found previously [6]. RetA alone reduced constitutively expressed MMP-1 whilst modestly inducing TIMP-1. Notably, RetA + OSM had little effect on MMP-1 and MMP-13 expression levels at early time points, which reduced at later time points. Sustained and marked synergistic induction of TIMP-1 occurred with RetA + OSM (Fig. 8A). MMP-2 and MMP-14 mRNAs were detected in human chondrocytes, but as in bovine cells there was no significant modulation in response to RetA or OSM (not shown).


Figure 8
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  		FIG. 8. MMP and receptor expression in RetA + OSM-treated human chondrocytes. Cells were stimulated as in Fig. 5 for the indicated time periods, and total cellular RNA was subjected to northern blotting. GAPDH was used for normalization. MMP-1 is shown for comparative purposes. The data presented are representative of at least two separate experiments. (A) Primary human articular chondrocytes. (B) T/C28a4 chondrocytes.

 
Assessment of the cell surface receptors used by OSM in a chondrocyte cell line shown previously to respond similarly to primary chondrocytes [5, 6] indicated no significant modulation of either gp130 or OSMRß. Furthermore, the RetA + OSM combination failed to induce MMP-1, unlike IL-1+OSM (Fig. 8B).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Here we report, for the first time, that RetA and OSM combine to promote collagenolysis from bovine and porcine cartilages. Although RetA alone does not always induce collagen release from bovine cartilage [17], collagenolysis is always seen when in combination with OSM. This is also true for IL-1 in combination with OSM [5, 6] or RetA [17]. However, the mechanisms by which RetA induces collagenolysis when in combination with IL-1 or OSM appear to be different. The present study has demonstrated that RetA + OSM-induced collagen degradation was independent of MMP-1 in bovine cartilage but was less marked than other combinations due to the modest induction of MMP-13. The combinations of IL-1 ± RetA and IL-1 ± OSM markedly up-regulate both MMP-1 and MMP-13 [5, 6, 17], resulting in a higher degree of collagenolysis (≥90% compared with 30–50%). This reduced collagen release, compared with other cytokine combinations [5, 6, 17], is also reflected in lower levels of detectable collagenolytic activity, although the bioassay used type I collagen as substrate [27], which underestimates the potency of MMP-13 as a collagenase. Indeed, MMP-13 is the most efficient collagenase against type II collagen [10], such that the modest MMP-13 mRNA induction still leads to significant RetA ± OSM-induced cartilage collagenolysis in the absence of TIMP expression. MMP-1 and MMP-13 are important in pathological cartilage proteolysis [1], and a role for both collagenases in this bovine cartilage model is further supported by the observation that an MMP-13-specific inhibitor fails to completely inhibit IL-1-induced degradation [30].

It has been suggested that RetA-induced collagen degradation may be MMP-independent [31, 32], although prevention of RetA + OSM-induced collagenolysis by TIMP-1 strongly supports MMP involvement. Data from northern blot analyses and immunolocalization also clearly suggest a role for MMP-13. There was no detectable MMP-8, and although both MMP-2 and MMP-14 are collagenolytic [33, 34], neither MMP was significantly modulated by RetA + OSM, and MMP-14 is poorly inhibited by TIMP-1, which effectively blocked collagenolysis.

No active collagenolytic activity was detected in supernatants from resorbing bovine cartilage even though TIMP-1 was able to prevent collagenolysis. Since MMP-13 appears to be responsible for this, the lack of active enzyme is probably explained by its instability in culture [17], its susceptibility to autolysis [9] and its ability to be internalized [35]. Indeed, proMMP-13 could only be detected in culture supernatants if a proteinase inhibitor cocktail was added at the time of harvest [17]. These factors also probably explain the absence of MMP-13 in diseased synovial fluids despite strong evidence for a role in pathogenesis [36]. This is further exacerbated since MMP-13 is a very effective collagenase against type II collagen [9], such that relatively low levels will still have a marked collagenolytic potential in cartilage.

RetA induces aggrecanolysis from bovine and porcine cartilages via aggrecanase-2 [20], and as predicted the inclusion of TIMP-1 did not block proteoglycan release since only TIMP-3 is inhibitory for aggrecanolysis [37]. Inclusion of BB-94, however, partially inhibited proteoglycan release at day 3, which was no longer apparent at day 7. It is therefore likely that more than one aggrecan-degrading proteinase is responsible for cartilage aggrecanolysis [38]. Recent evidence suggests that hyaluronidase(s) may be responsible [39], and such an activity would be insensitive to metalloproteinase inhibitors.

To date, the only combination of cytokines reported to promote collagenolysis from human articular cartilage is IL-1 + OSM [5]. This combination induces low but significant collagen release in ~50% of samples [40], although reproducible and synergistic induction of MMP-1 and MMP-13 is observed in human chondrocytes [6, 7]. In the present study, RetA + OSM failed to induce collagenolysis from human articular cartilage, with an absence of MMP-13 expression in human chondrocytes contrary to that seen in bovine cartilage or chondrocytes. RetA + OSM did, however, markedly induce TIMP-1, suggesting this combination may be chondroprotective in human cells if results from cell culture mimic the behaviour of ECM-embedded cells.

In some cells, RetA down-regulates gp130 [41], although we found no significant modulation of either gp130 or OSMRß. In human chondrocytes, OSM uses a gp130/OSMRß receptor complex [6], leading to STAT activation [6, 42]. OSM induction of MMP-1 has been suggested to be via STATs [43], and RetA interacts both positively and negatively with STAT proteins [44, 45]. If similar interactions occur in chondrocytes between the signalling components for RetA and OSM, this could explain the observed synergy in bovine cells as well as the collagenase repression and TIMP-1 induction seen in human cells. RetA repression of MMP-1 in human cells occurs through RAR/RXR heterodimers, reduced activator protein-1 (AP-1) binding and sequestration of Fos/Jun proteins [46, 47]. Direct interactions between RARs and AP-1 complexes occur [46], and the transcriptional activation of MMPs (including collagenases) is AP-1-dependent [47]. MMP-13 induction by RetA is dependent on core binding factor1 (cbfa1) [48], and it may be that species differences and/or cell-type specific phenomena account for the lack of MMP-13 induction in human chondrocytes by RetA. The presence of novel RetA response elements could also account for this, as has recently been reported for type XI collagen [49]. This may also help to explain the intriguing and marked TIMP-1 induction in human chondrocytes, which is worthy of further study. Indeed, retinoids have already been proposed as potential therapeutics for arthritis [50] and the present study highlights a possible mechanism by which they may act.
Figure 9


   Acknowledgments
 
The authors wish to thank British Biotech Pharmaceuticals Ltd, GlaxoSmithKline and John Heath for the provision of reagents. Funding was from the Arthritis Research Campaign and the Dunhill Medical Trust.

The authors have declared no conflicts of interest.


   Notes
 
*Present address: Unilever R&D Colworth, Sharnbrook, Bedford, UK. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 8 July 2005; revised version accepted 6 January 2006.
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