Rabbit synoviocyte inositol phospholipid metabolism is stimulated by hydroxyapatite crystals

Calcium-containing crystals such as basic calcium phosphate (BCP) 1 and calcium pyrophosphate dihydrate (CPPD) are two of the most common forms of pathologic articular materials that are associated with destructive arthropathies involving cartilage degeneration (1, 2). At concentrations found in pathologic human joint fluids, these crystals exert biological effects on cultured cells in a manner similar to growth factors like platelet-derived growth factor, epidermal growth factor, and serum. It has been demonstrated that BCP crystals stimulate fibroblast, synoviocyte, and chondrocyte mitogenesis in vitro (3); stimulate the production of prostaglandin via the phospholipase A 2 /cyclo-oxygenase pathway (4); activate phospholipase C and inositol phospholipid hydrolysis (5); induce the expression of the proto-oncogenes, c-fos and c-myc (6, 7); and induce the synthesis and secretion of metalloproteinases (MMPs) 1, 3, 8, and 13 (8 -12).In contrast to other mitogenic and growth factors, BCP crystal-elicited signal transduction pathways have not been completely studied. However, we have identified some of the component molecules involved in calcium-containing crystal signal transduction mechanisms. One pathway activated upon crystal stimulation of human fibroblasts (HF) is the p44 and p42 mitogen-activated protein kinase (p44/42 MAPK) pathway, also known as extracellular signal-related mitogen protein kinases 1 and 2 (ERK1 and ERK2), respectively. The MAPK cascade can be blocked by the selective inhibitors, PD98059 (13) and U0126 (14), which hinder the activation and phosphorylation of MEK (MAPK/ERK kinase). Co-treatment of HF with BCP crystals and PD98059 blocks crystal-induced p44/42 MAPK activation and mitogenesis (15) in addition to crystalinduced up-regulation of MMP-1 and MMP-3 mRNA and protein expressions (16). Moreover, phosphocitrate (PC), a specific inhibitor of the biological effects of BCP and CPPD crystals (17), also blocks crystal-induced activation of p44/42 MAPK, further supporting the role of this signal pathway in crystalinduced responses in HF (15).Another messenger with an apparent role in crystal-activated signal transduction is calcium. We have previously shown that treatment of HF with BCP crystals induces a rapid transient rise of intracellular calcium levels in seconds due to calcium influx from outside the cell, followed by a slow and sustained increase of intracellular calcium within 60 min after stimulation, due to crystal dissolution (18). Removal of calcium from the cell culture medium attenuates the BCP crystal in-

mentioning

confidence: 99%

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

Reuben

Brogley

Sun

et al. 2002

“…BCP crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (8). Current data suggest that BCP crystals enhance phospholipase C activity in synovial fibroblasts (9). If BCP crystals increase phospholipase C activity, resultant diacylglycerol accumulation followed by increased protein kinase C (PKC) activity should ensue.…”

mentioning

confidence: 99%

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

McCarthy

Augustine

Baldwin

et al. 1998

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. BCP crystals cause mitogenesis of articular cells and stimulate matrix metalloprotease production, thus promoting degradation of articular tissues. Previous work suggested that BCP crystal-induced cell activation required intracellular crystal dissolution, induction of proto-oncogene expression, and activation of signal transduction pathways involving protein kinase C and mitogen-activated protein kinases. Here we further elucidate the mechanisms of BCP crystal-induced cell activation as BCP crystals activate transcription factors nuclear factor B and activator protein 1 in human fibroblasts. We confirm the role of protein kinase C in BCP crystal-induced mitogenesis in human fibroblasts.In contrast, we demonstrate that BCP crystals do not activate signal transduction pathways involving protein tyrosine kinases or phosphatidylinositol 3-kinase. These data further define the mechanism of cell activation by BCP crystals and confirm its selectivity, an observation that may have therapeutic implications. Synovial fluid basic calcium phosphate (BCP)1 (hydroxyapatite, octacalcium phosphate, and tricalcium phosphate) crystals are common in osteoarthritis (OA) and are associated with severe degenerative arthropathies (1). The prevalence of BCP crystals in synovial fluid from patients with knee OA is between 30 and 60%, and their presence correlates strongly with radiographic evidence of cartilage degeneration (2). Larger joint effusions are seen in affected joints when compared with joint fluid from OA knees without BCP crystals (3).Clinical and pathological studies have demonstrated that synovial lining proliferation of varying degrees is associated with BCP crystals in osteoarthritis (4). BCP crystals themselves are at least partly responsible for such proliferation since they stimulate cell replication in vitro (5). Increased cellularity in the synovial lining enhances the capacity for secretion of cytokines, which may promote chondrolysis. Noninflammatory destruction of matrix-rich articular structures including cartilage, ligament, and tendon is also characteristic of BCP crystal deposition (1). BCP crystals promote tissue damage by induction of matrix metalloprotease (MMP) synthesis and secretion. Since there are no available drugs to inhibit deposition or affect reabsorption of these crystals, prevention of the biological consequences of the destructive processes initiated by BCP crystals is an attractive therapeutic strategy.The in vitro effects of BCP crystals emphasize their pathogenic potential. BCP crystals induce mitogenesis in cultured fibroblasts (5, 6). However, mechanisms by which BCP crystals induce mitogenesis have been incompletely studied. Endocytosis and intracellular dissolution of the crystals, producing an elevated cytoplasmic calcium concentration, are important (7). BCP crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MA...

“…Moreover, BCP crystals and PDGF exert similar biologic effects on cultured cells, such as stimulation of PGE 2 production via the phospholipase A 2 /cyclo-oxygenase pathway (4), activation of phospholipase C and inositol phospholipid hydrolysis (5), induction of collagenase and neutral protease synthesis (6 -8), and induction of proto-oncogenes (c-fos and c-myc) (9,10).…”

mentioning

confidence: 99%

Phosphocitrate Inhibits a Basic Calcium Phosphate and Calcium Pyrophosphate Dihydrate Crystal-induced Mitogen-activated Protein Kinase Cascade Signal Transduction Pathway

Nair

Misra

Sallis

et al. 1997

Calcium deposition diseases caused by calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals are a significant source of morbidity in the elderly. We have shown previously that both types of crystals can induce mitogenesis, as well as metalloproteinase synthesis and secretion by fibroblasts and chondrocytes. These responses may promote degradation of articular tissues. We have also shown previously that both CPPD and BCP crystals activate expression of the c-fos and c-jun proto-oncogenes. Phosphocitrate (PC) can specifically block mitogenesis and proto-oncogene expression induced by either BCP or CPPD crystals in 3T3 cells and human fibroblasts, suggesting that PC may be an effective therapy for calcium deposition diseases. To understand how PC inhibits BCP and CPPD-mediated cellular effects, we have investigated the mechanism by which BCP and CPPD transduce signals to the nucleus. Here we demonstrate that BCP and CPPD crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (ERK 2 and ERK 1). BCP and CPPD also cause phosphorylation of a nuclear transcription factor, cyclic AMP response element-binding protein (CREB), on serine 133, a residue essential for CREB's ability to transactivate. Treatment of cells with PC at concentrations of 10 ؊3 to 10 ؊5 M blocked both the activation of p42/p44 MAP kinases, and CREB serine 133 phosphorylation, in a dose-dependent fashion. At 10 ؊3 M, a PC analogue, n-sulfo-2-aminotricarballylate and citrate also modulate this signal transduction pathway. Inhibition by PC is specific for BCP-and CPPD-mediated signaling, since all three compounds had no effect on serum-induced p42/P44 or interleukin-1␤ induced p38 MAP kinase activities. Treatment of cells with an inhibitor of MEK1, an upstream activator of MAPKs, significantly inhibited crystal-induced cell proliferation, suggesting that the MAPK pathway is a significant mediator of crystal-induced signals.