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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036927-25
Application #
8323539
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Dunsmore, Sarah
Project Start
1982-08-01
Project End
2013-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
25
Fiscal Year
2012
Total Cost
$329,351
Indirect Cost
$116,179
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha et al. (2014) The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth. J Biol Chem 289:17689-98
Zhao, Xia; Ding, Eric Y; Yu, Olivia M et al. (2014) Induction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection. J Mol Cell Cardiol 75:152-61
Smrcka, Alan V; Brown, Joan Heller; Holz, George G (2012) Role of phospholipase Cýý in physiological phosphoinositide signaling networks. Cell Signal 24:1333-43
Carretero-Ortega, Jorge; Walsh, Colin T; Hernandez-Garcia, Ricardo et al. (2010) Phosphatidylinositol 3,4,5-triphosphate-dependent Rac exchanger 1 (P-Rex-1), a guanine nucleotide exchange factor for Rac, mediates angiogenic responses to stromal cell-derived factor-1/chemokine stromal cell derived factor-1 (SDF-1/CXCL-12) linked to Rac Mol Pharmacol 77:435-42
Walsh, Colin T; Radeff-Huang, Julie; Matteo, Rosalia et al. (2008) Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61. FASEB J 22:4011-21
Walsh, Colin T; Stupack, Dwayne; Brown, Joan Heller (2008) G protein-coupled receptors go extracellular: RhoA integrates the integrins. Mol Interv 8:165-73
Haga, Jason H; Kaunas, Roland; Radeff-Huang, Julie et al. (2008) Pulsatile equibiaxial stretch inhibits thrombin-induced RhoA and NF-kappaB activation. Biochem Biophys Res Commun 372:216-20
Radeff-Huang, Julie; Seasholtz, Tammy M; Chang, Jenny W et al. (2007) Tumor necrosis factor-alpha-stimulated cell proliferation is mediated through sphingosine kinase-dependent Akt activation and cyclin D expression. J Biol Chem 282:863-70
Citro, Simona; Malik, Sundeep; Oestreich, Emily A et al. (2007) Phospholipase Cepsilon is a nexus for Rho and Rap-mediated G protein-coupled receptor-induced astrocyte proliferation. Proc Natl Acad Sci U S A 104:15543-8
Radeff-Huang, Julie; Seasholtz, Tammy M; Matteo, Rosalia G et al. (2004) G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival. J Cell Biochem 92:949-66

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