Simonson M S, Herman W H
Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
J Biol Chem. 1993 May 5;268(13):9347-57.
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.
内皮素（ET）肽是与G蛋白偶联受体结合的强效生长因子。尽管ET受体激活的短期信号已得到广泛表征，但关于促有丝分裂信号转导的了解相对较少。我们研究了肾小球系膜细胞中参与促有丝分裂信号传导的ET受体亚型以及蛋白激酶C（PKC）和蛋白酪氨酸激酶（PTK）活性的作用。百日咳毒素可减弱ET-1引起的[Ca2+]i升高，但不影响[3H]胸苷摄取。一种ETA选择性受体拮抗剂BQ 123可阻断ET-1引起的[Ca2+]i升高，并抑制[3H]胸苷摄取。一种非选择性ETA-ETB受体拮抗剂（PD 142893）可阻断[3H]胸苷摄取，但ETB受体选择性激动剂（S6c和[Ala1,Ala3,Ala11,Ala15]ET-1(6-21)）无法增加[Ca2+]i或[3H]胸苷摄取。总体而言，这些数据表明促有丝分裂信号传导通过系膜细胞中的ETA受体亚型发生。PKC抑制和耗竭实验均表明，PKC对于促有丝分裂信号传导是必要的，但并不充分。ET-1可增加静止系膜细胞中细胞蛋白的酪氨酸磷酸化，而预先用赫曲霉素A孵育可阻断这种磷酸化。两种化学和机制不同的PTK抑制剂（赫曲霉素A和染料木黄酮）可阻断ET-1引起的[3H]胸苷摄取。此外，赫曲霉素A可减弱ET-1诱导的c-fos诱导、AP-1 DNA结合以及由AP-1顺式元件介导的转录。综上所述，这些数据表明ET-1的促有丝分裂信号传导还涉及基于PTK的机制。我们进一步证明，ET-1可刺激pp60c-src的自磷酸化以及pp60c-src催化的对PTK活性特异的肽底物的磷酸化。ET-1诱导的pp60c-src激活与[3H]胸苷摄取的剂量反应关系相似，这表明这些事件可能在功能上相关联。因此，G蛋白偶联受体与非受体PTK（如pp60c-src）之间的相互作用可能参与了ET-1的转录调控和促有丝分裂信号传导。
