Protein kinase C and protein tyrosine kinase activity contribute to mitogenic signaling by endothelin-1. Cross-talk between G protein-coupled receptors and pp60c-src.

Endothelin (ET) peptides are potent growth factors that bind to G protein-coupled receptors. Although short-term signals activated by ET receptors have been extensively characterized, relatively little is known about mitogenic signal transduction. We investigated the ET receptor subtype involved in mitogenic signaling in glomerular mesangial cells and the role of protein kinase C (PKC) and protein tyrosine kinase (PTK) activity. Pertussis toxin attenuates increases in [Ca2+]i by ET-1 but not [3H]thymidine uptake. An ETA-selective receptor antagonist, BQ 123, blocks increments in [Ca2+]i by ET-1 and inhibits [3H]thymidine uptake. A nonselective ETA-ETB receptor antagonist (PD 142893) blocked [3H]thymidine uptake, but ETB receptor-selective agonists (S6c and [Ala1,Ala3,Ala11,Ala15]ET-1(6-21)) were unable to increase [Ca2+]i or [3H]thymidine uptake. Collectively, these data suggest that mitogenic signaling occurs through an ETA receptor subtype in mesangial cells. Experiments with both PKC inhibition and depletion demonstrate that PKC was necessary but not sufficient for mitogenic signaling. ET-1 increased tyrosine phosphorylation of cellular proteins in quiescent mesangial cells that was blocked by preincubation with herbimycin A. Two chemically and mechanistically dissimilar PTK inhibitors (herbimycin A and genistein) blocked [3H]thymidine uptake by ET-1. In addition, herbimycin A attenuated c-fos induction, AP-1 DNA binding, and transcription directed by an AP-1 cis-element in response to ET-1. Taken together, these data suggest that mitogenic signaling by ET-1 also involves a PTK-based mechanism. We further demonstrated that ET-1 stimulated autophosphorylation of pp60c-src and pp60c-src-catalyzed phosphorylation of a peptide substrate specific for PTK activity. That the dose-response relationship for ET-1-induced pp60c-src activation and [3H]thymidine uptake were similar suggests that these events might be functionally linked. Thus, cross-talk between G protein-coupled receptors and nonreceptor PTK such as pp60c-src might be involved in transcriptional regulation and mitogenic signaling by ET-1.