Activation of PLC and PI 3 kinase by PDGF receptor α is not sufficient for mitogenesis and migration in mesangial cells

Background. Platelet-derived growth factor (PDGF) isoforms act through two distinct cell surface α and β receptors. Glomerular mesangial cells express both receptors. PDGF BB and AB are potent mitogens for glomerular mesangial cells, and PDGF BB stimulates cell migration in a phosphatidylinositol 3 (PI 3) kinase-dependent manner. In this study, we investigated the effect of PDGF AA on cell migration, PI 3 kinase and phospholipase C (PLC) activation, and the role of these two enzymes in mediating biological responses in these cells in response to all three isoforms. Methods. ³H-thymidine incorporation and modified Boyden chamber assay were used to determine DNA synthesis and directed migration, respectively, in response to all three PDGF isoforms. Differential activation of α and β receptors was studied by immunecomplex tyrosine kinase assay of corresponding receptor immunoprecipitates. PLCγ1 activity was determined by measuring total inositol phosphates in response to different PDGF isoforms. PI 3 kinase activity was determined in antiphosphotyrosine or PDGF receptor immunoprecipitates. Results. Both PDGF BB and AB resulted in stimulation of DNA synthesis and directed migration of mesangial cells. AA was neither chemotactic nor mitogenic. However, all three isoforms increased tyrosine phosphorylation of a 180 kD protein in antiphosphotyrosine immunoprecipitates, suggesting activation of respective receptors. Direct immunecomplex tyrosine kinase assay of α and β receptors demonstrated significant activation of both of these receptors when cells are treated with PDGF BB or AB. PDGF AA increased tyrosine kinase activity of the α receptor but not the β receptor. All three isoforms significantly stimulated the production of inositol phosphates with order of potency being BB > AB > AA. PDGF AA also dose dependently stimulated PI 3 kinase activity measured in antiphosphotyrosine immunoprecipitates of treated cells. A comparison of PI 3 kinase activity in antiphosphotyrosine immunoprecipitates from mesangial cells stimulated with three different PDGF isoforms showed significant activation of this enzyme with a decreasing order of activity: BB > AB > AA. Conclusion. Taken together, these data demonstrate that all three isoforms of PDGF significantly stimulate PLCγ1 and PI 3 kinase, two enzymes necessary for both DNA synthesis and directed migration. However, activation of α receptor by PDGF AA with a subsequent increase in PLC and PI 3 kinase activities is not sufficient to induce these biological responses in mesangial cells. These data indicate that the extent of activation of signal transduction pathways may be a major determinant of the biological activity of different PDGF isoforms in mesangial cells.