Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

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Abstract
The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by PH thymidine incorporation in a dose-dependent manner with an ED10 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 ± 15% of TPA alone to 393 ± 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of M1 80000, pI 4.5 (termed 80K) and M1 28000, pI 5.7–5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED10 of 8 ng/ml TPA. Exposure or mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4x-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role of protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates