ENDOTHELIN SIGNALING AND REGULATION OF PROTEIN KINASES

我们将验证这样一种假设，即细胞适配器蛋白和激酶的酪氨酸磷酸化是内皮素诱导的细胞内信号通路中的关键组成部分，从而导致内皮素的长期效应。我们还假设蛋白酪氨酸磷酸化是心血管和肾小球疾病演变的主要因素。我们认为，过度的蛋白酪氨酸磷酸化导致MAPKs激活介导了炎症性肾脏和心血管疾病，使用双特异性磷酸酶下调这些激酶将具有治疗益处。我们提出了研究非受体蛋白酪氨酸激酶的实验，它将G蛋白偶联的内皮素受体与肾小球系膜细胞和血管平滑肌细胞中的MAP激酶信号级联激活联系起来。我们将评估非受体酪氨酸激酶PYK2及其家族成员在ET-1长期效应中的作用。我们将重点研究ERK、SAP激酶和p38激酶在与理解肾脏和血管疾病相关的系统中的调节和作用。这些在培养的系膜和血管平滑肌细胞中进行的研究将有助于了解MAPK的生物学功能，并为开发新的血管和肾脏疾病治疗策略奠定基础。为此，我们将使用腺病毒介导的不同双特异性磷酸酶基因转移到培养细胞中，研究不同的MAPK信号通路在血管平滑肌和肾小球系膜细胞增殖和/或肥大中的作用。我们将评估一种假说，即过度的蛋白酪氨酸磷酸化导致MAPKs激活，并结合MAPKs信号级联成员的转录调控部分介导炎症性肾脏疾病。为了评估蛋白酪氨酸磷酸化的调节在肾脏疾病进展中的作用，我们将使用两种实验性肾损伤的动物模型。肾小球接头蛋白和蛋白激酶的磷酸化状态将被分析，并与疾病的进展相关。

We will test the hypothesis that the tyrosine phosphorylation of cellular adaptor proteins and kinases are crucial components in the endothelin-induced intracellular signaling pathways leading to long-term effects of endothelin. We also hypothesize that protein tyrosine phosphorylation is a principal factor in evolution of cardiovascular and glomerular diseases. We believe that excessive protein tyrosine phosphorylation leading to MAPKs activation mediates inflammatory renal and cardiovascular diseases and that down-regulation of these kinases, using dual-specificity phosphatases, will have therapeutic benefit. We propose experiments to study non-receptor protein tyrosine kinases which link G-protein coupled endothelin receptors to activation of MAP kinase signaling cascades in glomerular mesangial cells and vascular smooth muscle cells. We will evaluate the roles of non-receptor tyrosine kinase Pyk2 and members of Src family of tyrosine kinases in long-term effects of ET-1. We will focus our research on regulation and role of ERK, SAP kinase and p38 kinase cascades in systems relevant for understanding the renal and vascular diseases. These studies performed in cultured mesangial and vascular smooth muscle cells will add to understanding the biological functions of MAPKs and set the stage for the development of new therapeutic strategies for vascular and renal diseases. To this end we will use the adenoviral-mediated gene transfer of different dual-specificity phosphatases into cultured cells to investigate the role of distinct MAPK signaling cascades in proliferation and/or hypertrophy of vascular smooth muscle and glomerular mesangial cells. We will evaluate the hypothesis that excessive protein tyrosine phosphorylation leading to MAPKs activation in combination with transcriptional regulation of members of MAPKs signaling cascade partially mediates inflammatory renal diseases. To evaluate the role of regulation of protein tyrosine phosphorylation in the progression of renal disease we will use two animal models of experimental renal injury. The phosphorylation status of glomerular adaptor proteins and protein kinases will be analyzed and correlated with the progression of the disease.