Rho- and GPCR-mediated signaling through novel cellular effectors

通过新型细胞效应器 Rho 和 GPCR 介导的信号传导

• 批准号: 7655625
• 负责人: JOAN HELLER BROWN
• 金额: $ 33.6万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-08-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7655625
• 关键词: 1,2-diacylglycerol; Agonist; Astrocytes; Back; Binding; Biological Assay; Brain; Brain Injuries; Cell Line; Cell Proliferation; Cell surface; Cells; Characteristics; Comparative Study; Controlled Study; Cues; Cytokine Gene; DNA biosynthesis; Development; Diglycerides; Enzymes; F2R gene; Fluorescence Resonance Energy Transfer; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Generations; Glioblastoma; Glioma; Goals; Growth Factor; Guanine Nucleotide Exchange Factors; Human; Image; Immediate-Early Genes; In Vitro; Inflammation; Inflammation Mediators; Inflammatory Response; Integrin Signaling Pathway; Integrins; Knock-out; Knockout Mice; Ligands; Link; Lysophosphatidic Acid Receptors; Malignant Neoplasms; Mediating; Mitogen-Activated Protein Kinases; Modeling; Monomeric GTP-Binding Proteins; Mus; Mutation; Nude Mice; Pain; Pathway interactions; Phospholipase; Phospholipase C; Phosphotransferases; Play; Process; Proliferating; Protein Kinase; Proteins; Receptor Activation; Regulation; Role; Signal Pathway; Signal Transduction; Spinal Injuries; Spinal cord injury; Spinal nerve structure; Stimulation of Cell Proliferation; Stimulus; Testing; Thrombin; Up-Regulation; Work; astrogliosis; base; cancer cell; cell growth; cell growth regulation; cell injury; cell type; chorioallantoic membrane; extracellular; human disease; in vivo; neoplastic cell; nerve injury; novel; overexpression; phospholipase C epsilon; public health relevance; receptor; receptor-mediated signaling; research study; response; response to injury; rho; rho GTP-Binding Proteins; small hairpin RNA; therapeutic target; tumor

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.

描述（由申请人提供）：这项工作的长期目标是检查两个新的信号通路，似乎参与细胞生长的调节。一个涉及特定磷脂酶C（PLC 5）的激活，其将来自G蛋白偶联受体（GPCR）的信号整合到MAP激酶途径。另一个涉及Cyr61（CCN 1）的转录上调，CCN 1是一种表达为立即早期基因的蛋白质，通过整联蛋白受体分泌和发出信号。调节这些过程的细胞外刺激（凝血酶、LPA、S1P）和细胞内途径（小GTdR RhoA的活化）通常与细胞损伤、炎症和癌症相关。该提案测试了PLC 5和Cyr 61在这些病理生理条件下发挥关键信号作用的假设，并利用体外和体内研究，对小鼠星形胶质细胞和人胶质母细胞瘤细胞系发现可以靶向阻断这些途径的调控机制。第一个具体的目的是检查PLC 5作为GPCR激活的靶点，以及作为导致细胞增殖和基因表达的下游反应的效应子的参与。待测试的假设是PLC 5通过激活Rap1/ERK信号级联和通过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的信号整合到对DNA合成至关重要的信号中。拟议的实验使用原代星形胶质细胞PLC5基因敲除小鼠描绘的凝血酶，S1P和LPA受体的PLC5激活的途径，以确定是否PLC5作为一个鸟嘌呤核苷酸交换因子Rap1的激活和随后的ERK的激活，并检查PLC5在介导星形胶质细胞基因表达和细胞增殖体外发挥的作用。第二个具体目的是阐明增加CCN 1/Cyr61表达在Rho介导的对GPCR激动剂的反应中所起的作用。提出的实验将使用1321N1胶质母细胞瘤细胞和其他细胞系来确定CCN 1基因表达是否由于GPCR激活G 12/13和Rho介导的途径而受到转录调节，它是否通过整联蛋白信号传导途径对细胞起反作用以诱导持续的应答，并证明响应于GPCR激动剂的持续信号传导和DNA合成依赖于CCN 1上调。第三个具体目标是研究PLC 5在脑和脊髓损伤后星形胶质细胞增生中的体内病理生理作用以及CCN 1在神经胶质肿瘤发展中的作用。待测试的假设是Rho信号传导途径和激活它们的GPCR配体通过它们对PLC 5和CCN 1的作用促进这些反应。拟议的实验使用PLC5基因敲除小鼠来检查这种酶在响应于体内脑或脊髓损伤而产生的星形胶质细胞增生中的作用。在1321N1和其他胶质母细胞瘤细胞中用shRNA敲低CCN 1用于在鸡绒毛尿囊膜（CAM）测定和裸鼠中检查CCN 1在肿瘤细胞生长中的作用。 公共卫生相关性：细胞接受环境信号，通过一个称为信号转导的过程，引导它们增殖、迁移或死亡。一种名为RhoA的蛋白质转导源自细胞表面G蛋白偶联受体的信号，并且似乎在癌症和细胞损伤中受到异常调节。我们已经发现了RhoA在细胞内与之对话的两个“目标”（磷脂酶C5和CCN 1），并建议研究它们是如何被控制的，它们做什么以及抑制它们的功能是否会使体外和脑损伤和癌症模型中的细胞信号正常化。