Basic calcium phosphate crystals activate p44/42 MAPK signal transduction pathway via protein kinase Cμ in human fibroblasts

Abstract

Although basic calcium phosphate (BCP) crystals are common in osteoarthritis, the crystal-induced signal transduction pathways in human fibroblasts have not been fully comprehended. We have previously demonstrated that the induction of matrix metalloproteinases (MMP) 1 and 3 by BCP crystals follows both the calcium-dependent protein kinase C (PKC) pathway and the calcium-independent p44/42 mitogen-activated protein kinase (p44/42 MAPK) pathway. Although we showed that the calcium-dependent PKC pathway was characterized by calcium-dependent PKCα, here we show that the calcium-independent p44/42 MAPK pathway is mediated by calcium-independent PKCμ. Inhibition of PKCμ synthesis and activity by antisense oligodeoxynucleotides and H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5- isoquinolinesulfonamide), respectively, results in the inhibition of p44/42 MAPK activation, thus demonstrating that p44/42 MAPK activity is dependent upon PKCμ. Reverse transcription-polymerase chain reaction and Western blotting also show that inhibition of PKCμ results in the inhibition of MMP-1 and MMP-3 mRNA and protein expression as a result of p44/42 MAPK inhibition. These results now lead us to the conclusion that BCP crystal activation of human fibroblasts follows two pathways: 1) the calcium-dependent PKC pathway characterized by PKCα and 2) the calcium-independent p44/42 MAPK pathway mediated by PKCμ, which operate independently leading to an increase in mitogenesis and MMP synthesis and ultimately complementing each other for the efficient regulation of cellular responses to BCP crystal stimulation of human fibroblasts.