PROTEIN-KINASE-C SIGNALS THROMBOXANE INDUCED INCREASES IN FIBRONECTIN SYNTHESIS AND TGF-BETA BIOACTIVITY IN MESANGIAL CELLS

Previous studies have demonstrated that thromboxane (TX) stimulates matrix protein synthesis in mesangial cells (MC), and that this action is signalled by receptor mediated activation of protein kinase C (PKC). In the present study, we examined the hypothesis that activation of PKC by TX signals increases in transforming growth factor beta (TGF-beta) bioactivity, which in turn induces enhanced matrix protein synthesis. In cultured rat MC, the TXA(2)/prostaglandin endoperoxide analogue U-46619, but not exogenous human platelet TGF-beta(1), activated PKC as reflected by enhanced in situ phosphorylation of MARCKS protein, an endogenous substrate of PKC. U-46619 and TGF-beta(1) stimulated fibronectin (Fn) synthesis in MC, as shown by [S-35]methionine incorporation into immunoprecipitable Fn. Pan-specific rabbit anti-TGF-beta antibody blocked the increases in Fn synthesis induced by exogenous TGF-beta and those induced by U-46619 at 24 lo 72 hours after addition. Anti-TGF-beta antibody did not block the small increases in Fn synthesis observed six hours after addition of U-46619, suggesting that this acute response was not dependent on TGF-beta. Anti-TGF-beta antibody also failed to block activation of PKC by U-46619. U-46619 and 50 nM of the PKC agonist phorbol dibutyrate (PDBu) significantly increased both the active fraction and total (Latent plus active) TGF-beta in MC culture media, as assayed with the mink lung epithelial cell bioassay system. PKC inhibition with bisindolylmaleimide GF 109203X (GFX) or down-regulation of PKC in MC by prior exposure to a high concentration (0.5 mu M) of phorbol myristate acetate (PMA) blocked increases in TGF-beta bioactivity induced by either U-46619 or PDBu. PKC down-regulation in MC also blocked increases in Fn synthesis induced by U-46619. By contrast, exogenous TGF-beta stimulated Fn synthesis in both intact MC and in MC with down-regulated PKC. The findings indicate that activation of PKC by U-46619 signals an increase in TGF-beta bioactivity, which in turn stimulates Fn synthesis in MC by processes not dependent on PKC.