Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein. Each of its five subunits is approximately 100,000 Da in molecular weight. COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon. To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture. METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent. During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium. The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies. The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels. For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined. RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts. COMP could be easily detected by immunoprecipitation in all cell types. Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used. COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells. The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively). CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts. These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.

Expression of the basement membrane heparan sulfate proteoglycan (perlecan) in human synovium and in cultured human synovial cells.

BACKGROUND: Perlecan is a heparan sulfate proteoglycan that has a core protein of 466 kDa and is composed of five modules, four of which share a high degree of homology with a variety of biologically important molecules including the low-density lipoprotein receptor, laminin, and the neural cell adhesion molecule. Previously, this specialized proteoglycan, characteristic of the basement membrane, was not identified as a constitutive extracellular component of human synovium. EXPERIMENTAL DESIGN: Using human synovium and cultured human synovial cells, we examined perlecan gene expression in human synovium, a tissue lacking a classic basement membrane. The modulation of gene expression of the perlecan core protein by transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (FGF) was examined in cultures of normal human synovial cells, and by metabolic labeling using radioactive sodium sulfate, the question of whether this specialized protein is produced by synovial cells as a proteoglycan was addressed. RESULTS: Cultures of normal human synovial cells were shown to contain the large 14.5 kb perlecan mRNA and produced substantial amounts of perlecan core protein as shown by immunohistochemistry employing specific human perlecan Ab. Immunohistochemical detection showed intense staining in the intimal and subintimal layers of human synovial membrane. Moreover, the perlecan core protein was shown to be up-regulated by TGF-beta and down-regulated by basic FGF. In addition, a sizable portion (approximately 25%) of the synovial cell-produced proteoglycan was shown to contain heparan sulfate, providing evidence that synovial cell perlecan is produced as a proteoglycan. CONCLUSIONS: The presence of perlecan in human synovium and the modulation of the biosynthesis of its core protein by TGF-beta and FGF suggest that, in addition to its structural role, this unique heparan sulfate proteoglycan may be involved in normal synovial membrane function and in the pathogenesis of arthritis.