Rho- and GPCR-mediated signaling through novel cellular effectors

描述（由申请人提供）：这项工作的长期目标是研究似乎参与细胞生长调节的两种新的信号通路。一种涉及特定磷脂酶C （PLC5）的激活，该酶将来自g蛋白偶联受体（gpcr）的信号整合到MAP激酶途径。另一种涉及Cyr61 （CCN1）的转录上调，Cyr61是一种作为即时早期基因表达的蛋白质，通过整合素受体分泌并发出信号。调节这些过程的细胞外刺激（凝血酶、LPA、S1P）和细胞内途径（小GTPase RhoA的激活）通常与细胞损伤、炎症和癌症有关。该提案验证了PLC5和Cyr61在这些病理生理条件中起关键信号作用的假设，并利用小鼠星形胶质细胞和人类胶质母细胞瘤细胞系的体外和体内研究来发现可能靶向阻断这些途径的调节机制。第一个具体目的是研究PLC5作为gpcr激活的靶标，以及作为导致细胞增殖和基因表达的下游反应的效应者的参与。待验证的假设是，PLC5通过激活Rap1/ERK信号级联、局部生成二酰基甘油、激活其下游蛋白激酶靶点和改变基因表达，将激活Rho的信号整合为DNA合成的关键信号。拟建的实验利用PLC5敲除小鼠的原代星形胶质细胞来描绘PLC5被凝血酶、S1P和LPA受体激活的途径，以确定PLC5是否作为鸟嘌呤核苷酸交换因子激活Rap1和随后激活ERK，并在体外研究PLC5在介导星形胶质基因表达和细胞增殖中的作用。第二个具体目的是阐明CCN1/Cyr61表达增加在rho介导的对GPCR激动剂的反应中所起的作用。拟议的实验将使用1321N1胶质母细胞瘤细胞和其他细胞系来确定CCN1基因表达是否因GPCR激活g12 /13和Rho介导途径而受到转录调节，是否通过整合素信号通路作用于细胞诱导持续反应，并证明响应GPCR激动剂的持续信号传导和DNA合成依赖于CCN1上调。第三个具体目的是研究PLC5在脑和脊髓损伤后星形胶质细胞形成中的体内病理生理作用，以及CCN1在胶质肿瘤发展中的作用。待验证的假设是Rho信号通路和激活它们的GPCR配体通过对PLC5和CCN1的作用促进这些反应。拟议的实验使用PLC5敲除小鼠来检测该酶在体内脑或脊髓损伤后产生的星形胶质细胞形成中的作用。利用shRNA在1321N1和其他胶质母细胞瘤细胞中敲低CCN1，在鸡绒毛膜尿囊膜（CAM）实验和裸鼠实验中检测CCN1在肿瘤细胞生长中的作用。

DESCRIPTION (provided by applicant): The long term objective of this work is to examine two new signaling pathways that appear to be involved in regulation of cell growth. One involves activation of a specific phospholipase C (PLC5) that integrates signals from G-protein coupled receptors (GPCRs) to the MAP kinase pathway. The other involves transcriptional upregulation of Cyr61 (CCN1), a protein that is expressed as an immediate early gene, secreted and signals through integrin receptors. The extracellular stimuli (thrombin, LPA, S1P) and the intracellular pathway (activation of the small GTPase RhoA) that regulate these processes are commonly associated with cell injury, inflammation and cancer. The proposal tests the hypothesis that PLC5 and Cyr61 subserve critical signaling roles in these pathophysiological conditions and utilizes both in vitro and in vivo studies, on mouse astrocytes and a human glioblastoma cell line to discover regulatory mechanisms that could be targeted to block these pathways. The first specific aim is to examine the involvement of PLC5 as a target for activation by GPCRs, and as an effector of downstream responses leading to cell proliferation and gene expression. The hypothesis to be tested is that PLC5 integrates signals that activate Rho into signals critical for DNA synthesis by activating a Rap1/ERK signal cascade and by localized generation of diacylglycerol, activation of its downstream protein kinase targets and altered gene expression. Proposed experiments use primary astrocytes from PLC5 knockout mice to delineate pathways for PLC5 activation by thrombin, S1P, and LPA receptors, to determine whether PLC5 serves as a guanine nucleotide exchange factor for activation of Rap1 and subsequent activation of ERK and to examine the role played by PLC5 in mediating astroglial gene expression and cell proliferation in vitro. The second specific aim is to elucidate the role played by increased CCN1/Cyr61 expression in Rho-mediated responses to GPCR agonists. Proposed experiments will use 1321N1 glioblastoma cells and other cell lines to determine whether CCN1 gene expression is transcriptionally regulated as a consequence of GPCR activation of G 12/13 and Rho mediated pathways, whether it acts back on the cell through integrin signaling pathways to induce sustained responses, and to demonstrate that sustained signaling and DNA synthesis in response to GPCR agonists depends on CCN1 upregulation. The third specific aim examines the in vivo pathophysiological roles of PLC5 in astrogliosis following brain and spinal injury and of CCN1 in glial tumor development. The hypothesis to be tested is that Rho signaling pathways and the GPCR ligands that activate them promote these responses through their effects on PLC5 and CCN1. Proposed experiments use PLC5 knockout mice to examine the role of this enzyme in astrogliosis produced in response to in vivo brain or spinal cord injury. Knockdown of CCN1 with shRNA in 1321N1 and other glioblastoma cells is used to examine the role of CCN1 in tumor cell growth in the chick chorioallantoic membrane (CAM) assay and in nude mice. PUBLIC HEALTH RELEVANCE: Cells receive environmental cues which direct them to proliferate, migrate or die, using a process called signal transduction. A protein called RhoA transduces signals that originate from G-protein coupled receptors on the cell surface and appears to be abnormally regulated in cancer and cell injury. We have discovered two "targets" that RhoA talks to inside the cell (phospholipase C5 and CCN1) and propose to study how they are controlled, what they do and whether inhibiting their function normalizes cell signaling in vitro and in models of brain injury and cancer.