PDGF induces tyrosine phosphorylation in osteoblast-like cells: relevance to mitogenesis

We have studied the association between protein tyrosine phosphorylation and the mitogenic effect induced by platelet-derived growth factor (PDGF) in the osteoblast-like cell line MC3T3-E1. PDGF caused a dose-dependent increase in [3H]thymidine incorporation in MC3T3-E1 cells, reaching a plateau at 10 ng/ml. Vanadate, a potent phosphatase inhibitor, induced a twofold increase in thymidine incorporation. The combination of vanadate and PDGF resulted in a dose-dependent synergistic effect on thymidine incorporation. Genistein, a tyrosine kinase inhibitor, inhibited in a dose-related manner (2-20 microM) the mitogenic effect induced by either PDGF or the combination of vanadate and PDGF. These observations suggest that tyrosine kinases are involved in mediating the mitogenic effect of PDGF in these cells. PDGF treatment of MC3T3-E1 cells and subsequent immunoblotting with antiphosphotyrosine antibodies resulted in a marked phosphorylation of the PDGF receptor. Vanadate had a lesser effect on PDGF receptor phosphorylation, but given together with PDGF it induced a significant increase in the intensity of receptor phosphorylation. Preincubation with genistein abrogated these effects. Taken together, these findings indicate a direct correlation between thymidine incorporation and tyrosine phosphorylation in MC3T3-E1 cells and suggest that tyrosine phosphorylation plays a role in PDGF-induced mitogenic activity in osteoblast-like cells.