Transforming growth factor-β1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways

Previous studies have shown that transforming growth factor-beta 1 (TGF-beta 1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-beta 1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7, [S-23]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-beta 1 on matrix production. However, there was little, if any, effect on TGF-beta 1-dependent increases in [H-3]thymidine incorporation, and TGF-beta 1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC, TGF-beta 1 caused a dose-dependent increase in prostaglandin E-2 (PGE(2)) production which was further increased by PKC inhibition. The increase was regulated by TGF-beta 1-dependent effects on phospholipase A(2) (PLA(2)). Activation of PLA(2) inhibited TGF-beta 1 effects on PKC, and inhibition of PLA2 activated TGF-pl-dependent PKC, Exogenous arachidonic acid also inhibited TGF-beta 1-dependent increases in PKC, The effects of TGF-beta 1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed. These results support the hypothesis that TGF-beta 1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE(2) and protein kinase A (PKA). Inhibition of PKA also decreases TGF-beta 1-dependent proliferation. We have previously shown that PGE(2) stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways, (C) 2000 Elsevier Science B.V. All rights reserved.