Involvement of PKC-α in PDGF-mediated mitogenic signaling in human mesangial cells

Platelet-derived growth factor (PDGF) is a potent mitogen for a variety of cells. The calcium/ phospholipid-dependent protein kinase C (PKC) represents a major signal transduction pathway for many growth stimuli including PDGF. Various isoforms of PKC are differentially expressed in the same or in different cells and tissues, and diverse stimuli may selectively activate one or more PKC isoforms. We studied the effect of PDGF on DNA synthesis and on the activity of PKC in human mesangial cells and vascular pericytes in the glomerular microvascular bed. PKC activity was measured as the amount of phosphorylated myelin basic protein-derived peptide substrate in the absence and presence of an inhibitor, a peptide spanning the pseudosubstrate region of PKC. PDGF (15 ng/ml) stimulated PKC activity within 5 min, and the effect was sustained for 60 min. Pretreatment of mesangial cells with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, abolished the stimulation of PKC and DNA synthesis in response to PDGF. This effect of H-7 was specific, because H-7 did not inhibit the tyrosine phosphorylation of the PDGF receptor in vivo when added to the cells or the in vitro kinase activity in the PDGF β-receptor immunoprecipitates. Utilizing isotype-specific antibodies against PKC-α, -β, or -γ for immunoprecipitation of PDGF-treated mesangial cell extracts, followed by assay of PKC activity, we demonstrated the activation of PKC-α only. Northern blot analysis of mRNA prepared from mesangial cells also revealed two transcripts, 3.7 kb and 1.8 kb, that hybridized with cDNA specific for PKC-α. Moreover, specific ribonuclease (RNase) protection assay using cRNAs specific for PKC-α, -β, and -γ as probes revealed predominantly the presence of PKC-α in mesangial cells. These studies demonstrate that in mesangial cells stimulation of DNA synthesis in response to PDGF is dependent on activation of PKC and that the α-isoform of this kinase may mediate the mitogenic effect of PDGF.