Endothelin Signaling and Regulation of Protein Kinases

描述(申请人提供)：内皮素-1(ET-1)对肾血管和肾小球系膜细胞(GMC)起血管收缩、肥大和促有丝分裂作用。ET-1依赖的肾小球系膜细胞收缩可调节肾小球超滤系数，ET-1诱导的肾小球肥大和增殖可能参与多种类型的增生性和硬化性肾小球疾病。ET-1刺激GMC可引起多种信号事件；然而，ET-1诱导的细胞增殖和肥大主要通过激活细胞外信号调节激酶(ERK)来实现。GMC的收缩反应依赖于p38MAPK的激活。我们假设，蛋白质-蛋白质相互作用是内皮素诱导的细胞内信号通路中的关键组成部分，导致ET-1的短期和长期效应，并且经常调节这些相互作用的蛋白质酪氨酸磷酸化可能是肾脏疾病演变的主要因素。特异性目标1将评估酪氨酸激酶PYK2在ET-1介导的GMC收缩中的可能参与。我们将通过腺病毒将编码显性干扰突变体和野生型PYK2的基因转移到大鼠和人的GMC中，并分析通过PYK2抑制信号对ET-1介导的系膜细胞收缩、调节p38MAPK的小GTP酶的激活、MAPK激活的蛋白激酶2/3(p38MAPK的下游底物)、HSP25的磷酸化和肌动蛋白细丝动力学的调节的影响。特定目的2将评估鸟核苷酸交换因子Pix介导内皮素刺激GMC中小GTP酶CDc42的假说。我们将研究a)PIX与参与ET-1信号转导的异源三聚体G蛋白的直接相互作用；b)PIX显性干扰突变体抑制ET-L依赖的CDC42激活的能力；c)PIX显性干扰突变体抑制ET-1依赖的MAPK激活的能力。特异性目标3将评估这一假说，即双特异性磷酸酶对细胞内MAPK水平的负性调节是ET-1正常信号转导的重要组成部分。我们推测，MKP-3抑制ET-1介导的ERK激活，而MKP-1减弱p38MAPK，MKP-1和MKP-3对MAPK信号的严格调控控制着GMC肥大关键基因的表达。

DESCRIPTION (provided by applicant): Endothelin 1 (ET-1) exerts vasoconstrictor, hypertrophic and mitogenic actions on the renal vasculature and glomerular mesangial cells (GMC). ET-1-dependent contraction of GMC can regulate the glomerular ultrafiltration coefficient and ET-1 induced glomerular hypertrophy and proliferation may contribute to diverse types of proliferative and sclerotic glomerular diseases. Stimulation of GMC with ET-1 evokes a wide variety of signaling events; however, ET-1-induced cell proliferation and hypertrophy occurs primarily via activation of the extracellular signal-regulated kinase (ERK). Contractile responsiveness of GMC was shown to depend on activation of the p38 MAPK. We hypothesize that protein-protein interactions are among crucial components in the endothelin-induced intracellular signaling pathways leading to short- and long-term effects of ET-1 and that protein tyrosine phosphorylation, which often regulates these interactions, may be a principal factor in evolution of renal diseases. Specific Aim 1 will evaluate the putative involvement of tyrosine kinase Pyk2 in ET-1-mediated GMC contractility. We will carry out adenoviral mediated transfer of genes encoding dominant interfering mutant and wild type Pyk2 into rat and human GMC and analyze the effect of inhibition of signaling via Pyk2 upon ET-1 mediated mesangial cell contraction, activation of small GTPases regulating p38 MAPK, activation of MAP kinase-activated protein kinase 2/3 (downstream substrate of p38 MAPK), phosphorylation of HSP 25 and regulation of actin filament dynamics. Specific Aim 2 will evaluate the hypothesis that guanine nucleotide exchange factor Pix mediates stimulation of small GTPase cdc42 by endothelin in GMC. We will study a) the direct interaction of Pix with heterotrimeric G proteins, involved in ET-1 signaling in mesangial cells; b) ability of dominant interfering mutants of Pix to inhibit ET-l-dependent activation of cdc42; c) ability of dominant interfering mutants of Pix to inhibit ET-1- dependent activation of MAPKs. Specific Aim 3 will evaluate the hypothesis that negative regulation of endothelin signaling on the level of intracellular MAPK cascades by dual specificity phosphatases is an essential component of the normal signal transduction by ET-1. We hypothesize that MKP-3 inhibits ET-1 mediated ERK activation, whereas MKP-1 attenuates p38 MAPK and tight regulation of MAPK signaling by MKP-1 and MKP-3 controls expression of genes critical for GMC hypertrophy.