Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar,growth plate,and articular cartilage in rats

Summary Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. Therefore, we concluded that TIMP-1 and TIMP-2 were general inhibitors of MMP-2 and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix.     

17β-estradiol reduces expression of MMP-1, -3, and -13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system

Clinical observations have suggested a relationship between osteoarthritis and a changed sex-hormone metabolism, especially in menopausal women. This study analyzes the effect of 17β-estradiol on expression of matrix metalloproteinases-1, -3, -13 (MMP-1, -3, -13) and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) in articular chondrocytes. An imbalance of matrix metalloproteinases (MMPs) specialized on degradation of articular cartilage matrix over the respective inhibitors of these enzymes (TIMPs) that leads to matrix destruction was postulated in the pathogenesis of osteoarthritis. Primary human articular chondrocytes from patients of both genders were cultured in alginate beads at 5% O(2) to which 10(-11)M-10(-5)M 17β-estradiol had been added and analyzed by means of immunohistochemistry, immunocytochemistry and real-time RT-PCR. Since articular chondrocytes in vivo are adapted to a low oxygen tension, culture was performed at 5% O(2). Immunohistochemical staining in articular cartilage tissue from patients and immunocytochemical staining in articular chondrocytes cultured in alginate beads was positive for type II collagen, estrogen receptor α, MMP-1, and -13. It was negative for type I collagen, MMP-3, TIMP-1 and -2. Using real-time RT-PCR, it was demonstrated that physiological and supraphysiological doses of 17β-estradiol suppress mRNA levels of MMP-3 and -13 significantly in articular chondrocytes of female patients. A significant suppressing effect was also seen in MMP-1 mRNA after a high dose of 10(-5)M 17β-estradiol. Furthermore, high doses of this hormone led to tendentially lower TIMP-1 levels whereas the TIMP-2 mRNA level was not influenced. In male patients, only incubations with high doses (10(-5)M) of 17β-estradiol were followed by a tendency to suppressed MMP-1 and TIMP-1 levels while TIMP-2 mRNA level was decreased significantly. There was no effect on MMP-13 expression of cells from male patients. Taken together, application of 17β-estradiol in physiological doses will improve the imbalance between the amounts of MMPs and TIMPs in articular chondrocytes from female patients. Downregulation of TIMP-2 by 17β-estradiol in male patients would not be articular cartilage protective.     

Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes

Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the underlying molecular events that become activated. In this study, hyaluronan oligosaccharides induced the loss of extracellular matrix proteoglycan and collagen from cultured slices of normal adult human articular cartilage. This loss was coincident with an increased expression of matrix metalloproteinase (MMP)-13. MMP-13 expression was also induced in articular chondrocytes by hyaluronan (HA) hexasaccharides but not by HA tetrasaccharides nor high molecular weight hyaluronan. MMP-13 promoter-reporter constructs in CD44-null COS-7 cells revealed that both CD44-dependent and CD44-independent events mediate the induction of MMP-13 by hyaluronan oligosaccharides. Electromobility gel shift assays demonstrated the activation of chondrocyte NFkappaB by hyaluronan oligosaccharides. NFkappaB activation was also documented in C-28/I2 immortalized human chondrocytes by luciferase promoter assays and phosphorylation of IKK-alpha/beta. The link between activation of NFkappaB and MMP-13 induction by HA oligosaccharides was further confirmed through the use of the NFkappaB inhibitor helenalin. Inhibition of MAP kinases also demonstrated the involvement of p38 MAP kinase in the hyaluronan oligosaccharide induction of MMP-13. Our findings suggest that hyaluronan-CD44 interactions affect matrix metabolism via activation of NFkappaB and p38 MAP kinase.     

Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway

Oncostatin M (OSM), a member of the IL-6 superfamily of cytokines, is elevated in patients with rheumatoid arthritis and, in synergy with IL-1, promotes cartilage degeneration by matrix metalloproteinases (MMPs). We have previously shown that OSM induces MMP and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression in chondrocytes by protein tyrosine kinase-dependent mechanisms. In the present study, we investigated signaling pathways regulating the induction of MMP and TIMP-3 genes by OSM. We demonstrate that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, JAK2, JAK3, and STAT1 as well as extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase 1/2 mitogen-activated protein kinases in primary bovine and human chondrocytes. A JAK3-specific inhibitor blocked OSM-stimulated STAT1 tyrosine phosphorylation, DNA-binding activity of STAT1 as well as collagenase-1 (MMP-1), stromelysin-1 (MMP-3), collagenase-3 (MMP-13), and TIMP-3 RNA expression. In contrast, a JAK2-specific inhibitor, AG490, had no impact on these events. OSM-induced ERK1/2 activation was also not affected by these inhibitors. Similarly, curcumin (diferuloylmethane), an anti-inflammatory agent, suppressed OSM-stimulated STAT1 phosphorylation, DNA-binding activity of STAT1, and c-Jun N-terminal kinase activation without affecting JAK1, JAK2, JAK3, ERK1/2, and p38 phosphorylation. Curcumin also inhibited OSM-induced MMP-1, MMP-3, MMP-13, and TIMP-3 gene expression. Thus, OSM induces MMP and TIMP-3 genes in chondrocytes by activating JAK/STAT and mitogen-activated protein kinase signaling cascades, and interference with these pathways may be a useful approach to block the catabolic actions of OSM.     

Interleukin-17F affects cartilage matrix turnover by increasing the expression of collagenases and stromelysin-1 and by decreasing the expression of their inhibitors and extracellular matrix components in chondrocytes

Interleukin (IL)-17, a proinflammatory cytokine, is produced primarily by activated Th17 cells. IL-17 consists of six ligands that signal through five receptors (IL-17Rs); IL-17A and IL-17F share the highest homology in the family. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling whereas tissue inhibitor of metalloproteinases (TIMPs) inhibit the action of MMPs. In the present study, we examined the effect of IL-17F on the degradation and synthesis of the extracellular matrix in cartilage using human articular chondrocytes. We examined the effect of IL-17F on the expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and cyclooxygenases (COXs), as well as on prostaglandin E₂ (PGE₂) production. We also examined the indirect effect of PGE₂ on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2. Cells were cultured with or without IL-17F in the presence or absence of either an IL-17R antibody or NS-398 for up to 28 days. Expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and COXs at mRNA and protein levels was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. PGE₂ production was determined by ELISA. The expression of all types of IL-17Rs was detected in chondrocytes. However, IL-17RE expression was extremely low, compared with other IL-17Rs. The expression of MMP-1, MMP-3, MMP-13, and COX-2 as well as PGE₂ production were increased by addition of IL-17F, whereas the expression of IL-17RD, TIMP-2, TIMP-4, type II collagen, aggrecan, link protein, and COX-1 was decreased. The expression of IL-17RA, IL-17RB, IL-17RC, MMP-2, MMP-14, TIMP-1, and TIMP-3 was unaffected by addition of IL-17F. The IL-17R antibody blocked the stimulating/reducing effect of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, aggrecan, and link protein. NS-398 blocked the reducing effect of IL-17F on aggrecan expression, whereas it did not completely block the stimulating/reducing effects of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, and link protein. Our results suggest that IL-17F stimulates cartilage degradation by increasing the expression of collagenases (MMP-1 and -13) and stromelysin-1 (MMP-3) and by decreasing expression of their inhibitors (TIMP-2 and -4), type II collagen, aggrecan, and link protein in chondrocytes. Furthermore, our results suggest that the expression of aggrecan, link protein, and TIMP-4 decrease through the autocrine action of PGE₂ in chondrocytes.     

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton

Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.     

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.     

Mithramycin downregulates proinflammatory cytokine-induced matrix metalloproteinase gene expression in articular chondrocytes

Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-α) are the main proinflammatory cytokines implicated in cartilage breakdown by matrix metalloproteinase (MMPs) in arthritic joints. We studied the impact of an anti-neoplastic antibiotic, mithramycin, on the induction of MMPs in chondrocytes. MMP-3 and MMP-13 gene expression induced by IL-1β, TNF-α and IL-17 was downregulated by mithramycin in human chondrosarcoma SW1353 cells and in primary human and bovine femoral head chondrocytes. Constitutive and IL-1-stimulated MMP-13 levels in bovine and human cartilage explants were also suppressed. Mithramycin did not significantly affect the phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase. Despite effective inhibition of MMP expression by mithramycin and its potential to reduce cartilage degeneration, the agent might work through multiple unidentified mechanisms.     

The effect of IL-1beta on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes

Interleukin-1 (IL-1) plays key roles in altering cartilage matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). In the present study, we examined the effect of IL-1beta on cell proliferation, alkaline phosphatase (ALPase) activity, and the expression of MMPs, and TIMPs in chondrocytes derived from normal human femoral cartilage. The cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and 0, 1, 10, or 100 U/ml of IL-1beta for up to 28 days. The level of expression of MMPs and TIMPs was estimated by determining mRNA levels using real-time PCR and by determining protein levels using an enzyme-linked immunosorbent assay. Cell proliferation decreased in the presence of IL-1beta after day 21 of culture. ALPase activity decreased significantly in the presence of IL-1beta after day 10 of culture. The expression of MMP-1, -2, and -3 increased markedly in the presence of IL-1beta after day 21 of culture. MMP-13 expression increased markedly in the presence of IL-1beta on day 1 of culture, but decreased markedly after day 7. The expression of TIMP-1 increased significantly after day 14 of culture. The expression of TIMP-2 decreased significantly on day 1, but increased significantly from day 3 to day 14 of culture. These results suggest that IL-1beta may stimulate cartilage matrix turnover by increasing mainly MMP-13 production by the cells.     

miR146a up-regulation is involved in small HA oligosaccharides-induced pro-inflammatory response in human chondrocytes

BackgroundSmall HA fragments are produced during cartilage degradation and their role seems to be preponderant during pathologies in which cartilage injury contribute to trigger and perpetuate the inflammatory mechanism.Several reports have increasingly shown that MicroRNAs (miRs), a small non-coding mRNAs are involved in the regulation of multiple biological processes, including cell proliferation and inflammation response in different pathologies, among them miR146a seems to be involved in inflammatory processes.MethodsStarting by these evidences we investigated the levels of miR146a and its correlation with inflammatory mediators in an experimental model of 6-mer HA-induced inflammatory response in human cultured chondrocytes.ResultsTreatment of chondrocytes with 6-mer HA showed up-regulation in inflammation parameters such as TLR-4, and CD44 receptors activation, IL-6, IL-1β and MMP-13 mRNA expression and proteins production, as well as NF-kB activation. We also revealed an up-regulation of miR146a. Transfection with a miR146a mimic or miR146a inhibitor produced the following effects: chondrocytes receiving miR146a mimic and then 6-mer HA significantly reduced inflammatory cytokines and MMP-13, while exposition of chondrocytes with miR146a inhibitor and then the 6-mer HA incremented the activity of damaging cytokines and MMP13. Expression of CD44 receptor was not affected by miR-146a treatments, while TLR-4 expression and NF-kB activation were modified.ConclusionsWe concluded that up-regulation of miR146a occurred in 6-mer HA-induced inflammation response may reduce the inflammatory cascade by modulating TLR-4 and NF-kB activation.General significanceThese results could be useful in develop new therapeutic strategies with the aim to reduce OA and RA incidence.     

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT-PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related "a disintegrin and metalloproteinase" (ADAM) family proteinases in chick corneal development. While MMP-13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase-1 (TIMP-1), was expressed from ED 18 to 2 days post-hatching (P2). Early MMP-13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane-bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad-spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell-derived mesenchymal cell migration. Finally, we identified high levels of active membrane-type 3-MMP (MT3-MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3-MMP takes part in MMP-2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development.     

Localization of CD44 (hyaluronan receptor) and hyaluronan in rat mandibular condyle.

CD44 is a multifunctional adhesion molecule that binds to hyaluronan (HA), type I collagen, and fibronectin. We investigated localization of CD44 and HA in mandibular condylar cartilage compared with the growth plate and the articular cartilage, to clarify the characteristics of chondrocytes. We also performed Western blotting using a lysate of mandibular condyle. In mandibular condyle, CD44-positive cells were seen in the surface region of the fibrous cell layer and in the proliferative cell layer. Western blotting revealed that the molecular weight of CD44 in condyle was 78 to 86 kD. Intense reactivity for HA was detected on the surface of the condyle and the lacunae of the hypertrophic cell layer. Moderate labeling was seen in cartilage matrix of the proliferative and maturative layer. Weak labeling was also seen in the fibrous cell layer. In growth plate and articular cartilage, HA was detected in all cell layers. However, chondrocytes of these cartilages did not exhibit reactivity for CD44. These results suggest that chondrocytes in the mandibular condylar cartilage differ in expression of CD44 from those in tibial growth plate and articular cartilage. Cell-matrix interaction between CD44 and HA may play an important role in the proliferation of chondrocytes in the mandibular condyle.     

The effects of high magnitude cyclic tensile load on cartilage matrix metabolism in cultured chondrocytes

Excessive mechanical load is thought to be responsible for the onset of osteoarthrosis (OA), but the mechanisms of cartilage destruction caused by mechanical loads remain unknown. In this study we applied a high magnitude cyclic tensile load to cultured chondrocytes using a Flexercell strain unit, which produces a change in cell morphology from a polygonal to spindle-like shape, and examined the protein level of cartilage matrixes and the gene expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and proinflammatory cytokines such as IL-1beta and TNF-alpha. Toluidine blue staining, type II collagen immunostaining, and an assay of the incorporation of [35S]sulfate into proteoglycans revealed a decrease in the level of cartilage-specific matrixes in chondrocyte cultures subjected to high magnitude cyclic tensile load. PCR-Southern blot analysis showed that the high magnitude cyclic tensile load increased the mRNA level of MMP-1, MMP-3, MMP-9, IL-1beta, TNF-alpha and TIMP-1 in the cultured chondrocytes, while the mRNA level of MMP-2 and TIMP-2 was unchanged. Moreover, the induction of MMP-1, MMP-3 and MMP-9 mRNA expression was observed in the presence of cycloheximide, an inhibitor of protein synthesis. These findings suggest that excessive mechanical load directly changes the metabolism of cartilage by reducing the matrix components and causing a quantitative imbalance between MMPs and TIMPs.     

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.     

Tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA is constitutively expressed in bovine,human normal,and osteoarthritic articular chondrocytes

Tissue inhibitors of metalloproteinases (TIMPs) inhibit the extracellular matrix (ECM) metalloproteinases (MMPs). To determine the source of TIMPs in synovial fluids of patients with osteoarthritis (OA), the ability of chondrocytes to express TIMP-2 and its regulation by agents found in inflammed joints was investigated. The constitutive TIMP-2 mRNA expression was demonstrated in chondrocytes from normal bovine, human OA and normal cartilage. The cross-hybridization of human and bovine TIMP-2 suggested its evolutionary conservation. Serum, IL-1, IL-6 and TGF-β were unable to augment considerably the basal expression of TIMP-2 mRNA. TIMP-1 RNA expression in chondrocytes from human OA cartilage was elevated compared to non-OA chondrocytes, while TIMP-2 mRNA levels were similar in both. IL-1β, IL-6 and TGF-β did not affect TIMP-2 expression but TGF-β induced TIMP-1 mRNA in human OA chondrocytes. TIMP-2 and TIMP-1 are therefore differentially regulated in chondrocytes and the basal TIMP-2 levels may be needed for the cartilage ECM integrity. © 1996 Wiley-Liss, Inc.