Basic calcium phosphate crystals stimulate cell proliferation and collagenase message accumulation in cultured adult articular chondrocytes

Abstract

Objective. To investigate BCP (basic calcium phosphate) crystal-stimulated mitogenesis and collagenase gene transcription in primary cultures of porcine chondrocytes.Methods. The role of protein kinases in BCP crystal4imulated DNA synthesis was investigated using thymidine incorporation, kinase inhibitors, and protein kinase C (PKC) assays. Northern blot analysis was used to determine the levels of collagenase c-fos and c-jun message.Results. BCP crystals stimulated chondrocyte proliferation in a PKC-dependent manner. Increased levels of collagenase message were preceded by an increased accumulation of c-fos, but not c-jun.Conclusion. BCP crystals could contribute to the abnormal chondrocyte proliferation and collagenase secretion observed in some rheumatic diseases.Intraarticular deposition of basic calcium phosphate (BCP) crystals (hydroxyapatite, octacalcium phosphate, and tricalcium phosphate) and/or calcium pyrophosphate dihydrate (CPPD) crystals is associated with a number of destructive arthropathies involving cartilage degeneration (1,2). Collagenase activity has been demonstrated in the synovial fluid of patients with BCP crystal deposition disease (3,4), although Dieppe et a1 (5) were unable to show an increase in collagenase activity in synovial fluids from a series of patients with crystal deposition disease of the shoulder ("idiopathic destructive disease of the shoulder"). Both CPPD crystals (6,7) and BCP crystals (6)(7)(8)(9) are commonly found in osteoarthritic cartilage and have occasionally been observed within chondrocytes (6,9).It has previously been demonstrated that cultured human rheumatoid synovial cells or normal canine synovial cells will endocytose BCP or CPPD crystals (10). Crystal endocytosis was followed by the release of prostaglandins and, in addition, collagenase and neutral protease activity (11,12). It has also been demonstrated that BCP crystals are mitogenic in cultures of human foreskin fibroblasts, canine synovial fibroblasts (13), and murine fibroblasts (14). Crystalstimulated mitogenesis has been postulated to be a contributing factor in the synovial hyperplasia associated with BCP or CPPD crystal deposition diseases (1 3,15).Earlier studies demonstrated that the time to peak BCP crystal-stimulated thymidine uptake in fibroblasts was delayed by 2-3 hours compared with the peak uptake time using 10% serum (13). In addition, solubilization of the crystals was necessary in order for mitogenesis to proceed (16). In fibroblasts, BCP crystals acted similarly to a competence growth factor (17), since insulin-like growth factor 1 (IGF-1) was required in order to elicit a full mitogenic response (18). BCP crystals also induced accumulation of the competence genes c-fos and c-myc, in a protein kinase C (PKCwependent manner, in BALBk-3T3 mouse fibroblasts (14).The proliferative effects of BCP crystals on

Molecular Mechanism of Basic Calcium Phosphate Crystal-induced Activation of Human Fibroblasts

“…This postulate is supported in a study by Mitchell et al (6) who found that BCP crystal-induced mitogenesis in Balb/c/3T3 fibroblasts was inhibited when PKC was down-regulated using the tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), an analogue of diacylglycerol. Another index of PKC activation is translocation of the enzyme from the cytosolic compartment to the membrane compartment of the cell, and we have shown increased membrane-associated PKC activity in porcine chondrocytes treated with BCP crystals (10). These data suggest that the mitogenic response to BCP crystals involves activation of PKC.…”

Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes

“…Chondrocytes were enzymatically released from cartilage tissue as previously described (18), and plated at high density (4 ϫ 10 5 cells/cm 2 ) in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal calf serum and 1% penicillinstreptomycin-fungizone (PSF). These conditions maintain the differentiated chondrocyte phenotype (19). Experiments were completed within 3 days of plating.…”

Molecular Mechanism of the Induction of Metalloproteinases 1 and 3 in Human Fibroblasts by Basic Calcium Phosphate Crystals

“…The BCP crystal-induced PKC activity is blocked by co-treatment of crystal-stimulated cells with the PKC inhibitors, staurosporine and bisindolylmaleimide I (25). Furthermore, an increase in PKC activity associated with the membrane fraction is seen following BCP crystal stimulation of chondrocytes (26). Down-regulation of PKC activity by chronic treatment with the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate, an analog of DAG, blocks crystal-induced c-fos and c-myc expressions and mitogenesis in Balb/c 3T3 cells (7) whereas co-treatment with the PKC inhibitor, staurosporine, blocks BCP-induced c-fos expression and mitogenesis in HF (25), indicating that PKC activity is essential for these crystalinduced effects to occur.…”

“…Because PKC is known to be physiologically active only in the membrane-associated state and translocation of the PKC enzyme from the cytosol to the membrane of the cell is used to monitor its intracellular activation (25,26), we determined PKC activity in the membrane fractions of the cells in calcium-and magnesium-free HBSS and compared it with the activity in HBSS containing calcium and magnesium. Using Gö6976 as the specific inhibitor of the calcium-dependent PKC and PMA as the positive control for the PKC activity, we have shown that there was no PKC activity in the absence of calcium and magnesium as seen in Fig.…”