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-β1 (TGF-β1) 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-β1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [³⁵S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-β1 on matrix production. However, there was little, if any, effect on TGF-β1-dependent increases in [³H]thymidine incorporation, and TGF-β1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-β1 caused a dose-dependent increase in prostaglandin E₂ (PGE₂) production which was further increased by PKC inhibition. The increase was regulated by TGF-β1-dependent effects on phospholipase A₂ (PLA₂). Activation of PLA₂ inhibited TGF-β1 effects on PKC, and inhibition of PLA₂ activated TGF-β1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-β1-dependent increases in PKC. The effects of TGF-β1 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-β1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE₂ and protein kinase A (PKA). Inhibition of PKA also decreases TGF-β1-dependent proliferation. We have previously shown that PGE₂ stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways. Copyright (C) 2000 Elsevier Science B.V.