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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

We have previously reported the up-regulation of matrix metalloproteinase 10 (MMP-10) following treatment with the procatabolic stimulus of interleukin-1 (IL-1) and oncostatin M (OSM) in chondrocytes. Although MMP-10 is closely related to MMP-3, little is known about the role of MMP-10 in cartilage catabolism. The purpose of this study was to determine whether MMP-10 is expressed in connective tissue cells and to assess how it may contribute to cartilage collagenolysis.

Methods

MMP gene expression was assessed by real-time polymerase chain reaction using RNA from human articular chondrocytes and synovial fibroblasts stimulated with IL-1 plus OSM or tumor necrosis factor α (TNFα) plus OSM. Synovial fluid levels of MMP-10 were determined by specific immunoassay. Recombinant procollagenases were used in activation studies. Immunohistochemistry assessed MMP-10 expression in diseased joint tissues.

Results

MMP-10 expression was confirmed in both chondrocytes and synovial fibroblasts following stimulation with either IL-1 plus OSM or TNFα plus OSM, and MMP-10 was detected in synovial fluid samples from patients with various arthropathies. Exogenous MMP-10 significantly enhanced collagenolysis from IL-1 plus OSM–stimulated cartilage, and MMP-10 activated proMMP-1, proMMP-8, and proMMP-13. Immunohistochemistry revealed the presence of MMP-10 in the synovium and cartilage of an IL-1 plus OSM–induced model of arthritis as well as in samples of diseased human tissues.

Conclusion

We confirm that both synovial fibroblasts and articular chondrocytes express MMP-10 following treatment with procatabolic stimuli. Furthermore, the detectable levels of synovial fluid MMP-10 and the histologic detection of this proteinase in diseased joint tissues strongly implicate MMP-10 in the cartilage degradome during arthritis. The ability of MMP-10 to superactivate procollagenases that are relevant to cartilage degradation suggests that this activation represents an important mechanism by which this MMP contributes to tissue destruction in arthritis.

The arthritides are debilitating diseases in which degradation of articular cartilage is a major feature. The cartilage extracellular matrix (ECM) is composed primarily of type II collagen and proteoglycan (aggrecan), structural components that provide tensile strength and resistance to compressive forces. The matrix metalloproteinases (MMPs) are a key family of potent enzymes in tissue degradomics (1), including the cartilage degradome, since collectively they can degrade all the ECM components and have been strongly implicated in arthritic disease (2, 3). Aggrecanolysis is thought to be predominantly mediated by ADAMTS proteinases (4), although this ECM component can be replaced relatively rapidly once the stimulus, such as interleukin-1 (IL-1), has been removed (5). In contrast, collagenolysis is much less readily achieved, but when degradation does occur, the structural integrity of the tissue is irreversibly lost (6).

The collagenolytic MMPs (MMPs 1, 8, and 13) have all been implicated in pathologic collagenolysis. These proteinases require activation of their latent pro forms via the proteolytic removal of a propeptide, which can be mediated by serine proteinases (7–9) or by MMPs. For example, MMP-3 (also called stromelysin 1) activates the pro forms of MMPs 1, 7, 8, and 13 (10–14), and such activation represents a key step in cartilage collagenolysis (8, 9). MMP-3 expression is a marker of disease activity and joint damage progression in early rheumatoid arthritis (RA) (15) and may play a role in other degenerative arthritides, including osteoarthritis (OA) (16) and juvenile idiopathic arthritis (JIA) (17).

MMP-10 (stromelysin 2) has a structure and substrate specificity similar to that of MMP-3, also activates MMPs 1, 7, 8, and 9 pro forms (18), and its expression correlates with the invasive properties of RA fibroblast-like synoviocytes (19). However, there are relatively few data concerning the potential role of MMP-10 in the context of the arthritides. In the present study, we demonstrate that MMP-10 expression in human articular chondrocytes is induced by proinflammatory stimuli known to promote cartilage catabolism (20–24), that MMP-10 is detectable in synovial fluids and joint tissues of arthritis patients, and that MMP-10 potentiates cartilage collagenolysis via the activation of procollagenase(s).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Materials and reagents.

Chemicals were obtained from the following suppliers. Human IL-1 was a generous gift from Dr. Keith Ray (GlaxoSmithKline, Stevenage, UK), and recombinant human oncostatin M (OSM) was kindly provided by Prof. John Heath (University of Birmingham, Birmingham, UK). Superscript II reverse transcriptase was obtained from Invitrogen (Paisley, UK), and real-time polymerase chain reaction (PCR) Master Mix reagents were obtained from Roche (Lewes, UK). All fast-protein liquid chromatography resins and columns were purchased from Amersham Biosciences (Chesham, UK). MMP-10 polyclonal antibody was a gift from Dr. Ros Hembry (University of East Anglia, Norwich, UK). Vectastain kit PK-6105 was purchased from Vector (Burlingame, CA). Oligonucleotides and analytical-grade chemicals were obtained from Sigma (Poole, UK) or were obtained as described elsewhere (25).

Synovial fluid samples.

The experiments were performed on archived RA, OA, and JIA synovial fluid samples from the Musculoskeletal Unit at the Freeman Hospital (Newcastle-upon-Tyne, UK). JIA was defined according to the ILAR criteria (26). Permission was obtained from the local research ethics committee and consent from the patient or parent was obtained at the time of sampling. Synovial fluids were obtained during routine aspiration for active joint diseases and were centrifuged at 1,300g for 30 minutes. The supernatants were aliquoted and stored at –80°C prior to assay.

Enzyme-linked immunosorbent assay (ELISA) for MMP-10.

MMP-10 Quantikine ELISA was purchased from R&D Systems (Minneapolis, MN) and was performed according to the manufacturer's instructions.

Cell culture and RNA extraction.

Human articular cartilage and synovium were obtained with consent from OA patients undergoing joint replacement surgery at a local hospital. Chondrocytes were isolated from the tissue by sequential proteolysis, as described previously (27). For synovial fibroblast isolation, all steps were performed in the presence of 100 μg/ml of streptomycin, 100 units/ml of penicillin, 40 units/ml of nystatin, and 2 mM glutamine. Synovial tissue was finely minced with scissors and washed twice in phosphate buffered saline (PBS). The tissue was then digested with bacterial collagenase (2 mg/ml) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS; Invitrogen) for 4 hours at 37°C, with shaking (110 revolutions per minute). The cell suspension was then filtered through a 100-μm cell strainer (Biotrace International, Bridgend, UK), centrifuged at 1,200g for 5 minutes, and the pellet was resuspended in DMEM containing 10% FCS.

Chondrocytes or fibroblasts were seeded into 75-cm2 tissue culture flasks (Corning/Costar, High Wycombe, UK) at 5 × 106 cells per flask and grown to 85% confluence in DMEM containing 25 mM HEPES supplemented with 10% FCS. Chondrocytes were always used as primary cultures, whereas the fibroblasts were used from passage 2 onward so as to ensure a homogeneous population of type II synoviocytes. Confluent cells, used at passage 4 or 5, were washed with Dulbecco's PBS, and the medium was replaced with serum-free DMEM and maintained overnight prior to stimulation with serum-free medium containing IL-1 (0.02 ng/ml) and/or OSM (10 ng/ml). Serum was excluded from stimulated cells because it can markedly alter cell metabolism in the absence of exogenous cytokines (28). Since these experiments represent models of tissue destruction, we avoided using chondroprotective agents such as insulin-like growth factor 1 (29), which are known to be present in serum and are able to replace it (30). The absence of serum has also been shown not to affect cell viability (data not shown), and previous studies have shown that tissue in serum-free culture for up to 8–9 days can respond to serum and other growth factors (31).

Following stimulation, cells were lysed with RNeasy lysis buffer (Qiagen, Crawley, UK), and total cellular RNA was isolated according to the manufacturer's instructions. DNA was removed using the DNase I set (Qiagen) during the isolation, and RNA was quantified using RiboGreen reagent (Molecular Probes, Eugene, OR). RNA quality was assessed spectrophotometrically, and only samples with a 260 nm:280 nm ratio >1.8 were used in subsequent experiments. Isolated RNA was stored at –80°C until required.

Real-time PCR.

Oligonucleotide primers for human MMP-10 were designed using Primer Express 2.0 software (Applied Biosystems, Warrington, UK). Primers were placed within different exons close to, or spanning, an intron/exon boundary so as to prevent amplification of any residual genomic DNA. The sequences were as follows: forward, 5′-AATGAGGTACAAGCAGGTTATCCAAAGTCTTCCAAT-3′ and reverse, 5′-ACAGCTGCATCAATTTTCCTTATGCCTACTGTTGCTGTG-3′. Since MMP-3 and MMP-10 share marked sequence similarity (86% at the DNA level), BLAST searches (online at www.ncbi.nlm.nih.gov/BLAST/) were performed to select primers that annealed only to MMP-10. To confirm this empirically, the primers were tested using real-time PCR and plasmids containing the complementary DNA (cDNA) sequences for MMP-3 or MMP-10.

Total RNA (1 μg) was reverse transcribed in a 20-μl reaction using 2 μg of random hexamers and Superscript II reverse transcriptase according to the manufacturer's instructions. Relative quantification of gene expression was performed using a 7900HT PCR system (Applied Biosystems). PCR reactions were performed in triplicate using 2 μl of HotStart SYBR Green Master Mix, 5 mM MgCl2, and 1 μM of each primer in a 20-μl reaction. Thermocycler conditions comprised an initial activation step at 95°C for 10 minutes, followed by a 3-step program consisting of 95°C for 20 seconds, 60°C for 5 seconds, and 72°C for 10 seconds for 45 cycles. A 1-step melt curve analysis was also performed at the end of each run to ensure that the crossover values obtained were due to the amplification of a specific product. GAPDH was used as an endogenous control to normalize for differences in the amount of total RNA in each sample, as described previously (25).

Arthritis model and immunohistochemical analysis.

Joint sections were prepared from an established model of inflammatory arthritis that uses replication-deficient adenovirus engineered to express murine IL-1 or tumor necrosis factor α (TNFα) in combination with OSM at 1 × 106 plaque-forming units/joint/vector, as described previously (21, 23). Antigen retrieval of deparaffinized and rehydrated sections (5 μm) was performed by incubating sections in 10 mM sodium citrate, pH 6.0, for 2 hours at room temperature. Sections were then placed in 3% H2O2 in Tris buffered saline (TBS; 100 mM Tris, 154 mM NaCl, pH 7.6) for 15 minutes. Thereafter, serial sections were blocked with 1.5% normal rabbit serum in TBS for 30 minutes and then incubated for 90 minutes at room temperature with a polyclonal sheep anti-human MMP-10 antibody diluted 1:1,000 as supplied (32). Sections were subsequently washed twice in TBS for 5 minutes and then incubated with biotinylated rabbit anti-sheep IgG (diluted 50-fold in TBS) in 1.5% rabbit serum in TBS for 30 minutes, followed by incubation with avidin–biotin complex for 30 minutes according to instructions for the Vectastain kit.

After washing sections twice for 5 minutes in TBS, the signal was developed using diaminobenzidine tetrahydrochloride (DAB; Dako, Ely, UK) according to the manufacturer's protocol. Sections were counterstained with hematoxylin for 5 seconds and washed extensively in water. Images of stained sections were captured using a 3-CCD color video camera (JVC, Tokyo, Japan) and displayed on a computer monitor.

Human joint tissues and immunohistochemical analysis.

Cartilage and synovium obtained from patients undergoing joint replacement surgery and were frozen in ice-cold isopentane. Serial cryostat sections (10 μm) on 3-aminopropyltriethoxysilane–coated (2%) slides were air-dried for 1 hour after cutting and stored at –80°C until required. Prior to immunohistochemical analysis, slides were equilibrated at room temperature for 30 minutes and fixed in 4% paraformaldehyde for 10 minutes. Sections were subsequently washed twice in PBS for 5 minutes and then placed in 3% H2O2 in methanol for 30 minutes. Normal rabbit serum (1.5%) was used for blocking (10 minutes), and sections were incubated with the anti–MMP-10 antibody (1:1,000 dilution) for 90 minutes at room temperature. Sections were subsequently washed twice in PBS for 5 minutes and then incubated with biotinylated secondary antibody (rabbit anti-sheep IgG, diluted 50-fold in PBS according to the Vectastain kit instructions) in 1.5% rabbit serum in PBS for 30 minutes, followed by incubation with avidin–biotin complex for 30 minutes using Vectastain kit PK-6105 according to the manufacturer's instructions. Sections were washed twice for 5 minutes in PBS, and signal development and image capture were performed as described above, except that nickel–DAB was used for the synovium sections.

Expression and purification of recombinant human proMMPs.

ProMMP sequences, except MMP-1, were amplified from cDNA derived from primary human articular chondrocytes using oligonucleotides designed to generate full-length proMMP sequences, each incorporating an initiating ATG in the forward primer and a TAG stop codon in the reverse primer. For human proMMP-1, cDNA clones were generously provided by Dr. Alan Galloway (British Biotech, Oxford, UK) as 0.3-kb and 1.7-kb Eco RI fragments in pUC19. Plasmid DNA containing the 0.3-kb fragment was partially digested with Eco RI, dephosphorylated, and purified. This linearized plasmid was ligated to the 1.7-kb Eco RI fragment to generate pUC19/proMMP-1. Correct orientation was verified by Xmn I restriction digestion. PCR of pGEX2T/MMP-1 (33) generated a DNA fragment representing the stabilized form of mature MMP-1 (33). This PCR fragment was Xmn I–digested and gel purified, and the larger 3′ fragment was ligated to Aat II/Xmn I–digested pUC19/proMMP-1. The ligation product was purified, and PCR was performed with Pfu DNA polymerase and T4 polynucleotide kinase–treated primers to generate a fragment representing full-length, stabilized proMMP-1. The resulting PCR product was purified and ligated to dephosphorylated Sma I–digested pUC18 (Amersham Biosciences) to generate pUC18/proMMP-1. Correct orientation and the presence of the mutation to improve proteinase stability were assessed by Nde I and Bgl II digestion, respectively, prior to transfer to the pIB/V5-His TOPO TA vector (Invitrogen) by PCR.

The DNA sequence of all plasmids was verified by dideoxy sequencing, and 1 μg of each plasmid used to transfect High Five insect cells (1.8–2.3 × 106 cells/ml >95% viability), according to the instructions with the pIB/V5-His TOPO TA expression kit. Express Five SFM serum-free medium (Invitrogen) was used for transfection and subsequent cell culture. Stable cell lines from each of the individual transfections were selected using blasticidin, and clones that produced high levels of each proMMP (as assessed by Western blotting and collagenolytic or gelatinolytic activity assays) were chosen for long-term storage and pro-protein production. Conditioned medium (100 ml) containing proMMP was incubated with 100 ml of SP-Sepharose slurry (prepared according to the manufacturer's instructions) and 100 ml of 20 mM sodium cacodylate, 150 mM NaCl, 0.05% Brij 35, and 0.02% azide (pH 6.5) for 16 hours at 4°C, with agitation. After incubation, the binding mixture was poured into a column, washed with the same buffer, and bound proteins were batch-eluted with 1M NaCl. Aliquots were stored at –20°C after dialysis against the same buffer containing no NaCl. ProMMP proteins were further purified using Mono S and an NaCl gradient (0–1M; 40 ml at 1 ml/minute), followed by Superdex 75 gel filtration of 0.5 ml samples (0.25 ml/minute). Fractions containing protein were retained, and proMMP was identified by Western blotting with specific antibody and either collagenolytic (34) or caseinolytic activity (35).

Cartilage degradation assay.

An established in vitro degradation assay (20) was used. Briefly, discs (∼2 mm3) were punched from bovine nasal septum cartilage. Three discs per well in a 24-well plate were incubated overnight at 37°C in DMEM containing 25 mM HEPES supplemented with 2 mM glutamine, 100 μg/ml of streptomycin, 100 units/ml of penicillin, 2.5 μg/ml of gentamicin, and 40 units/ml of nystatin. Fresh medium, with or without IL-1 (1 ng/ml) and/or OSM (10 ng/ml), with or without MMP-10 (5 μg/ml) (4 wells for each condition), was added (day 0), and the plate was incubated at 37°C for 7 days. Media were removed and retained for analysis. Fresh media containing IL-1 and/or OSM, with or without MMP-10, were added to the cartilage and incubated at 37°C for a further 7 days, and again, the media were retained. The remaining cartilage was papain digested (20), and the amount of hydroxyproline in each sample was determined (as a measure of collagen) using a microtiter modification of a previously described method (36, 37).

Enzyme activity assays.

Collagenolytic activity for proMMP-1, proMMP-8, and proMMP-13 was determined using a 96-well plate modification of the 3H-acetylated collagen diffuse fibril assay (34). All assays were performed in duplicate. APMA (0.67 mM) or MMP-10 was added to activate the procollagenase. One unit of collagenase activity degrades 1 μg of collagen per minute at 37°C.

Gelatinase activity was assessed using the 3H-acetylated type I collagen from above, following denaturation as described previously (35). For caseinase activity, 3H-acetylated casein replaced the gelatin in the assay.

Statistical analysis.

Statistical analysis was performed using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test. SPSS software (SPSS, Chicago, IL) was used to perform the analyses.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Induction of MMP-10 gene expression in human articular chondrocytes and synovial fibroblasts by IL-1 plus OSM.

We have previously shown by gene array and real-time PCR analyses that MMP-10 is up-regulated in chondrocytes treated with IL-1 plus OSM (25). In the present study, we confirm that MMP-10 expression is also induced in human articular chondrocyte populations following stimulation with IL-1 plus OSM as well as stimulation with TNFα plus OSM. For both stimuli, the cytokine combination resulted in higher mean levels of MMP-10 expression than either cytokine alone in all 4 chondrocyte populations examined (Figure 1A), although some heterogeneity in terms of the magnitude of response was seen, as reported previously for primary chondrocytes (25). Similar observations were also found with synovial fibroblasts; the magnitude of the responses was less than that for chondrocytes, with TNFα-containing treatments showing the most marked response (Figure 1B).

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Figure 1. Induction of matrix metalloproteinase 10 (MMP-10) gene expression in chondrocytes and synovial fibroblasts by proinflammatory cytokines. A, Primary human articular chondrocytes and B, synovial fibroblasts (passage 4 or 5) were stimulated for 24 hours with medium alone (control), oncostatin M (OSM; 10 ng/ml), interleukin-1 (IL-1; 0.02 ng/ml), tumor necrosis factor α (TNFα; 10 ng/ml), IL-1 plus OSM, or TNFα plus OSM. Total RNA from 4 separate cell preparations was isolated and analyzed in triplicate by real-time reverse transcription–polymerase chain reaction. The data are presented relative to GAPDH and are expressed as the fold induction compared with control. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines inside the boxes represent the mean. Whiskers represent the maximum and minimum range. P values are versus controls, as determined by one-way analysis of variance; ns = not significant.

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Presence of MMP-10 in synovium and cartilage in a murine model of arthritis.

In order to confirm the in vivo presence of MMP-10, we analyzed joint sections from mice with an arthritis specifically induced by overexpression of IL-1 plus OSM or by TNFα plus OSM (21, 23). Immunohistochemistry revealed low levels of MMP-10 in control cartilage, which correlated with the real-time PCR data. However, significantly higher levels of MMP-10 were present in cartilage from the cytokine-treated joints (Figure 2A). MMP-10 was absent in control synovium but was present in synovium from IL-1 plus OSM–treated and TNFα plus OSM–treated mice (Figure 2B). No staining with the isotype control antibody was observed in any sections (data not shown).

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Figure 2. Elevated expression of matrix metalloproteinase 10 (MMP-10) in proinflammatory cytokine–induced arthritis in vivo. Murine joints were treated with vector alone (control), vectors encoding murine tumor necrosis factor α (TNFα) plus murine oncostatin M (OSM), or vectors encoding murine interleukin-1 (IL-1) plus murine OSM, as described in Materials and Methods. Joints were embedded in paraffin, and 5-μm serial sections were prepared. MMP-10 was detected using a sheep anti-human MMP-10 polyclonal antibody for A, cartilage and B, synovium. Bars = 20 μm.

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Acceleration of IL-1 plus OSM–induced cartilage collagenolysis by active MMP-10.

Since MMP-10 was clearly detected in the articular cartilage of IL-1 plus OSM–treated arthritic mice, we assessed the effect of the exogenous addition of MMP-10 to IL-1 plus OSM–treated cartilage. In this in vitro model of degradation, collagen release is rarely seen by day 7 of culture (20), findings which were confirmed in the present study, but the addition of active MMP-10 led to significant collagenolysis by day 7 (Figure 3). No significant collagen release was observed for any other treatment, including MMP-10 alone, confirming an indirect role of this MMP in the collagen degradation observed.

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Figure 3. Acceleration of interleukin-1 (IL-1) plus oncostatin M (OSM)–induced cartilage collagenolysis by matrix metalloproteinase 10 (MMP-10). Bovine nasal cartilage discs were cultured in the presence of medium alone, IL-1 (1 ng/ml), OSM (10 ng/ml), or IL-1 plus OSM, with (solid bars) or without (open bars) MMP-10 (5 μg/ml). Media were removed on day 7, and fresh reagents were added until day 14, when the media were removed and the remaining cartilage was digested with papain. The hydroxyproline assay was used to measure collagen content, and the release of collagen into the medium on day 7 was expressed as a percentage of the total. Values are the mean and SD of 4 experiments. The Bonferroni multiple comparison test was used to compare IL-1 plus OSM plus MMP-10 with all other treatments. ∗∗∗ = P ≤ 0.001.

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MMP-10 mediation of proMMP-1, proMMP-8, and proMMP-13 activation.

Since MMP-10 promoted collagenolysis by day 7 under conditions that have been shown to induce significant levels of procollagenases but not active collagenase(s) (20), we assessed which of the collagenolytic MMPs could be processed to the mature (active) form by MMP-10 (Figure 4). Inclusion of MMP-10, which is not collagenolytic (38), to preparations of recombinant proMMP-1, proMMP-8, or proMMP-13 (lane P in Figures 4A–C) led to markedly increased levels of collagenolytic activity (lanes 3 and 4) compared with the procollagenase preparations alone, all of which underwent a limited amount of autoactivation during the prolonged bioassay incubation (lane 1). Indeed, these levels were similar to those produced following APMA activation (lane 2 in Figures 4A–C). Western blot analyses also confirmed the conversion of all 3 procollagenase enzymes to a mature form following addition of MMP-10.

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Figure 4. Mediation of pro–matrix metalloproteinase 1 (proMMP-1), proMMP-8, and proMMP-13 activation by MMP-10. MMP-10 was incubated with 150 ng of A, proMMP-1, B, proMMP-8, or C, proMMP-13 for 16 hours at 37°C and then assessed for activation by activity assay (n = 2) or Western blotting with specific antibodies, as described in Materials and Methods. For the bioassay, procollagenase was incubated alone (open bars) or in the presence of 0.67 mM APMA (solid bars), 15 ng of MMP-10 (horizontal striped bars), or 7.5 ng of MMP-10 (diagonal striped bars), where 1 unit of collagenase activity degrades 1 μg of collagen per minute at 37°C. For Western blot analyses, recombinant procollagenase was incubated alone (lane 1) or in the presence of 0.67 mM APMA (lane 2), 15 ng of MMP-10 (lane 3), or 7.5 ng of MMP-10 (lane 4), with a procollagenase standard (lane P). The sizes of the active MMP-1, MMP-8, and MMP-13 enzymes are 44 kd, 58 kd, and 48 kd, respectively. Results are representative of 2 separate experiments.

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Presence of MMP-10 in synovial fluid samples from arthritis patients.

Since both chondrocytes and synovial fibroblasts express MMP-10 following treatment with catabolic stimuli known to promote arthritic disease (21, 23, 24), we assessed by ELISA the MMP-10 levels in synovial fluid samples from patients with different arthropathies. MMP-10 levels ranged from 44.6 to 763.5 pg/ml for OA samples, 4.13 to 1,339 pg/ml for RA, and 4.13 to 2,256 pg/ml for JIA, with mean ± SEM values of 359.2 ± 54.1 pg/ml, 268.2 ± 64.7 pg/ml, and 267.3 ± 206.9 pg/ml, respectively. No significant differences in the MMP-10 concentrations were observed between the different arthropathies (Figure 5).

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Figure 5. Presence of matrix metalloproteinase 10 (MMP-10) in synovial fluid samples from patients with different arthropathies. Samples of synovial fluid from patients with osteoarthritis (OA; n = 16), rheumatoid arthritis (RA; n = 20), and juvenile idiopathic arthritis (JIA; n = 10) were assayed for MMP-10 by specific enzyme-linked immunosorbent assay. Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines inside the boxes represent the median. Whiskers represent the maximum and minimum range. Mean MMP-10 levels were 359.2 pg/ml, 268.2 pg/ml, and 267.3 pg/ml in the OA, RA, and JIA patients, respectively. There were no significant differences in MMP-10 levels between any of the groups.

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Presence of MMP-10 in RA and OA joint tissues.

All of the above data implicate MMP-10 in arthritic disease and specifically as an activator of procollagenases. Since synovium invades cartilage in disease states (19) and since cartilage collagenolysis is a pericellular event (39, 40), we hypothesized that MMP-10 expression in diseased tissues from human joints would be localized to synovial fibroblasts and would be associated with the articular chondrocytes. Immunohistochemistry confirmed this (Figure 6). In cartilage samples, MMP-10 staining was localized to the chondrocytes predominantly in the superficial and middle zones (Figures 6A–D), being less evident distal to obvious lesions (results not shown). In synovium samples, MMP-10 staining was localized to the most superficial layers of the tissue (insets in Figures 6E–H), with evidence of MMP-10 staining at sites of neovascularization in the RA samples (Figure 6F).

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Figure 6. Presence of matrix metalloproteinase 10 (MMP-10) in joint tissues from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Cartilage and synovium from OA patients (n = 5) and RA patients (n = 3) were embedded in OCT, and 10-μm sections were obtained with a cryostat. MMP-10 was detected using a sheep anti-human MMP-10 polyclonal antibody for A–D, cartilage and E–H, synovium. Insets, Lower-magnification views of the same tissue sections. Shown are representative sections from each patient group. Bars = 50 μm in the cartilage sections; 100 μm in the synovium sections.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

During previous gene array analyses of IL-1 plus OSM–stimulated chondrocytes, we observed a synergistic induction of MMP-10 (25). In the present study, we confirmed MMP-10 induction in primary human articular chondrocytes as well as synovial fibroblasts following stimuli known to promote cartilage collagenolysis (20, 22) and arthritis in vivo (21, 23). Relatively few data exist on MMP-10 expression in arthritis, and it was reported by Hembry and coworkers (32) to be absent in RA and OA synovial tissues, although in vitro, synovial fibroblasts do express MMP-10, which correlates with an invasive phenotype (19). Herein we confirm synovial MMP-10 expression in both an animal model of inflammatory arthritis and in synovial fluids and tissues from RA and OA patients. Like many MMPs, MMP-10 is induced by proinflammatory cytokines such as IL-1 and TNFα, and the increasing data that support a role for such mediators of inflammation in OA as well as RA (41, 42) explain the presence of MMP-10 in OA synovial tissues with evidence of synovitis. It is likely, therefore, that MMP-10 will be less abundant in noninflamed OA tissues (32).

Data on MMP-10 expression in cartilage are limited, although both normal and OA cartilage have low levels of mRNA expression (43). The present findings clearly demonstrate the ability of chondrocytes to also produce this proteinase following a proinflammatory stimulus. Thus, the synovial fluid levels found in 3 different arthropathies examined in the present study are probably a combined result of both synovium- and cartilage-derived production, although it is probable that synovial production will predominate during the early stages in any inflamed joint, since this is the major site of proinflammatory cytokine production. There is clear evidence in both RA and OA that chondrocytes contribute significantly to cartilage degradation (25, 39, 44), which may be partly due to the release of factors that are stimulatory for synoviocytes (44). The present study confirms that both RA and OA tissues express MMP-10, such that procollagenase activation is possible, and hence collagenolysis is likely to occur, both within the cartilage and at pannus–cartilage interfaces.

MMP-10 expression has also been reported in osteoblasts and osteophytes and in growth plate chondrocytes during development (45, 46); these observations provide further evidence of a role of MMP-10 in ECM remodeling events. Of significance is the induction of MMP-10 by 2 potent cytokine combinations already known to induce a highly degradative arthritis with both aggrecanolysis and collagenolysis (20–23). Moreover, inclusion of MMP-10 in an in vitro model of cartilage degradation significantly (up to 50%) exacerbated the extent of collagen release at a time point when collagenolysis is rarely observed (8, 20). This extent of collagen release is very similar to the findings of our previous study using equivalent amounts of MMP-3 (8), which indicated that these stromelysins have comparable potencies in terms of procollagenase activation. Indeed, MMP-10 has previously been shown to “superactivate” MMP-1 as does MMP-3 (38).

This “superactivation” results in a collagenase form that has ≥10-fold higher specific activity than that of procollagenases activated by APMA, trypsin, or plasmin (10, 38, 47), and we observed this phenomenon with MMP-10 for proMMP-1, proMMP-8, and proMMP-13. We therefore conclude that once active, MMP-10 could help to superactivate the procollagenase reservoir known to be present by day 7 of culture in this model (8, 20). Such superactivation may well have significant implications for tissue integrity once the endogenous pool of tissue inhibitors of metalloproteinases has been exceeded, resulting in rapid collagenolysis (8, 9, 20–22).

Furthermore, the present study is the first to confirm that MMP-10 activates proMMP-13, which is highly significant, since we have previously shown that this collagenase is a major collagenolytic MMP in this degradation assay (9), as well as MMP-1 (20, 21). While stimulation with IL-1 plus OSM only weakly induces MMP-8 expression in bovine chondrocytes (21), the ability of MMP-10 to activate this collagenase may still be pathophysiologically relevant, especially in reactive arthritis (48). Indeed, we have proposed that procollagenase activation is a key and rate-limiting step in cartilage collagenolysis (8, 9), and the present study clearly implicates MMP-10 as such an activator. Thus, the activation cascades for the procollagenases are numerous and complex and include the closely related MMP-3 (stromelysin 1) as well as MMP-14 (2) and possibly 2 different serine proteinase–dependent mechanisms (8, 9).

For either MMP-10 or MMP-3 to be a pathophysiologic activator of procollagenases, the pro forms of these proteinases would also require proteolytic activation. The serine proteinase plasmin is known to activate proMMP-3, and it may be that the proposed serine proteinase activation cascades for the procollagenases (8, 9) involve intermediates such as MMP-3 and/or MMP-10. However, the identity of specific serine proteinases involved in the proteolytic events that lead to cartilage collagenolysis will need to be elucidated. These observations could imply a level of redundancy in terms of procollagenase activation, and could help to explain in part the lack of efficacy of MMP inhibitors that have been used therapeutically to date (49).

It is becoming increasingly apparent that the degradome that drives cartilage destruction in arthritis is complex and involves multiple proteinases. For many of these proteinases, we are only just beginning to understand their substrate specificities and preferences in relation to the cartilage ECM. Thus, our data clearly demonstrate the presence of MMP-10 in arthritic tissues and its ability to activate procollagenases. This suggests that it will be prudent to include MMP-10 in the ever more diverse list of proteinases that are likely to be active players in the proteolytic disassembly of the cartilage ECM during degenerative joint diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Dr. Keith Ray, Dr. Ros Hembry, and Prof. John Heath for providing reagents, and we are indebted to Dr. Chris Morris for the use of his immunohistochemistry facilities.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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