Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells.
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 [3H]thymidine incorporation in a dose-dependent manner with an ED50 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% for 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 Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of 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 [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.
Issandou
M
M
INSERM U133, Faculté de Médecine, Toulouse, France.
Darbon
J M
JM
D000818:Animals; D002478:Cells, Cultured; D002999:Clone Cells; D004261:DNA Replication / Q000187:drug effects*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013936:Thymidine / Q000378:metabolism
D007328:Insulin; D010750:Phosphoproteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D013936:Thymidine