Abstract
Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are often associated with destructive arthropathies involving cartilage degeneration. These crystals are mitogenic and induce oncogene expression and matrix metalloproteinase (MMP) synthesis and secretion in human fibroblasts. To date, BCP crystal-elicited signal transduction pathways have not been completely studied. Because protein kinase C (PKC) is known to play an important role in signal transduction, we investigated the participation of this pathway in the BCP crystal induction of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts. Using reverse transcription/polymerase chain reaction (RT-PCR) and Northern and Western blotting techniques, we show here that BCP crystal stimulation of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts is dependent upon the calcium-dependent PKC signal transduction pathway and that the PKC alpha isozyme is specifically involved in the pathway. We have previously shown that BCP crystal induction of MMP-1 and MMP-3 is also dependent on the p44/42 mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We now show that these two pathways operate independently and seem to complement each other. This leads to our hypothesis that the two pathways initially function independently, ultimately leading to an increase in mitogenesis and MMP synthesis, and may converge downstream of PKC and p44/42 MAPK to mediate BCP crystal-induced cellular responses.
Highlights
Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are often associated with destructive arthropathies involving cartilage degeneration
Using reverse transcription/polymerase chain reaction (RT-PCR) and Northern and Western blotting techniques, we show here that BCP crystal stimulation of matrix metalloproteinase (MMP)-1 and MMP-3 mRNA and protein expressions in human fibroblasts is dependent upon the calcium-dependent protein kinase C (PKC) signal transduction pathway and that the PKC␣ isozyme is involved in the pathway
To determine whether a protein kinase signaling is necessary for the BCP crystal-induced expression of these MMPs, we examined the effect of staurosporine, a potent, cell-permeable and broad spectrum inhibitor of protein kinases on the BCP crystal-induced MMP mRNA and protein levels by reverse transcription (RT)-PCR and Northern and Western blots
Summary
Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are often associated with destructive arthropathies involving cartilage degeneration. Phosphocitrate (PC), a specific inhibitor of the biological effects of BCP and CPPD crystals [17], blocks crystal-induced activation of p44/42 MAPK, further supporting the role of this signal pathway in crystalinduced responses in HF [15]. Removal of calcium from the cell culture medium attenuates the BCP crystal innase, MEK, MAPK/ERK kinase; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PC, phosphocitrate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PVDF, polyvinylidene fluoride; RT, reverse transcription; TBS, Tris-buffered saline; TMB-8, 8-(N,Ndiethylamino)-octyl-3,4,5-trimethoxy benzoate, HCl; PI-PLC, phosphatidylinositol-specific phospholipase C. duction of c-fos mRNA [18], suggesting that an influx of extracellular calcium is required for maximal induction of c-fos expression. The atypical isozymes are made up of zeta (), iota (), and mu (), and their activities are neither calcium- nor phospholipid-dependent [23, 24]
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