Basic calcium phosphate crystal induction of collagenase 1 and stromelysin expression is dependent on a p42/44 mitogen-activated protein kinase signal transduction pathway.
Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. These crystals are mitogenic and induce protooncogene expression and matrix metalloproteinase (MMP) synthesis and secretion in human fibroblasts, effects that are specifically blocked by phosphocitrate (PC). We have recently determined that crystals transduce signals to the nucleus via the activation of the p42 and p44 mitogen-activated protein (MAP) kinases (Nair et al., 1997, J Biol Chem 272:18920-18925). Treatment of human fibroblasts (HF) with BCP induces phosphorylation of p42/44 MAPK, which is inhibited by PC in a dose-dependent manner. Blocking of p42/44 MAPK signal transduction with an inhibitor (PD98059) of MEK1, an upstream activator of MAPKs, reduces crystal-induced p42/44 MAPK activation and significantly inhibits crystal-induced cell proliferation. Based on these findings, we sought to determine the role of the p42/44 MAPK signal transduction pathway in crystal-induced expression of matrix MMPs. We demonstrate suppression of crystal-induced MMPs via the utilization of two different MEK inhibitors: PD98059 and the recently described U0126, a novel inhibitor of MEK1 and MEK2. Treatment of HF with PD98059 blocks the induction of crystal-stimulated collagenase 1 (MMP-1) and stromelysin (MMP-3) expression. PD98059 and PC reduced the level of crystal-induced MMP-1 and MMP-3 mRNA expression to that observed in nonstimulated cells. Likewise, PD98059 treatment of HF blocked the epidermal growth factor (EGF)- and crystal-induced increases in MMP-1 and MMP-3 protein expression and secretion as demonstrated by Western blotting and zymography. Treatment of HF with U0126 inhibits EGF-induced phosphorylation of p42/44 MAPK as well as crystal- and EGF-induced upregulation of MMP-1 mRNA. Additionally, we demonstrate that treatment of HF with BCP, EGF, or PD98059 does not significantly alter levels of gelatinase A (MMP-2) mRNA and protein expression.
Research Science and Geriatric Research, Education, and Clinical Center, VA Medical Center, and Department of Medicine (Arthritis), University of Miami School of Medicine, Florida 33135, USA.
M A
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Brogley
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Cruz
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Cheung
D015152:Blotting, Northern; D002070:Butadienes / Q000494:pharmacology; D002114:Calcinosis / Q000378:metabolism*; D002130:Calcium Phosphates / Q000737:chemistry* / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics*; D003460:Crystallization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D018093:Gelatinases / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D020781:Matrix Metalloproteinase 1; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3 / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009570:Nitriles / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000235:genetics; D012867:Skin / Q000166:cytology / Q000201:enzymology
D002070:Butadienes; D002130:Calcium Phosphates; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009570:Nitriles; D012333:RNA, Messenger; C113580:U 0126; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1; C485829:calcium phosphate, dibasic, anhydrous; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one