The long term of objective of this work is to examine two new pathways that appear to be involved in regulation of cell growth. One involves activation of a specific phospholipase C (PLCe) that integrates signals from Gproteins coupled receptors (GPCRs) to the MAP kinase pathway. The other involves transcriptional upregulation of Cyr61 or 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 we demonstrate to regulate these processes are commonly associated with cell injury, inflammation and cancer. The proposal tests the hypothesis that PLCe and Cyr61 subserve critical signaling roles in these pathophysiological conditions and uses 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 PLCe as a target for activation by GPCRs, as an effector of downstream responses, and as a mediator of GPCR mediated cell proliferation and gene expression. The hypothesis to be tested is that PLCe integrates signals that activate Rho into signals critical for DNA synthesis and cell migration by activating a Rap1/ERK signal cascade and by localized generation of diacylglycerol and activation of its downstream targets. Proposed experiments use primary astrocytes from PLCe? knockout mice to delineate pathways for PLCe activation by thrombin, S1P, and LPA receptors, to determine whether PLCe serves as a guanine nucleotide exchange factor for activation of Rap1 and subsequent activation of ERK and to examine the role played by PLCe 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 reponses to GPCR agonists. Proposed experiments 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 PLCe in astrogliosis following brain 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 PLCe and CCN1. Proposed experiments use PLCe knockout mice to examine the role of this enzyme in astrogliosis produced in response to in vivo spiral 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.
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