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Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61

Colin T. Walsh^*, Julie Radeff-Huang^*, Rosalia Matteo^*, Albert Hsiao, Shankar Subramaniam, Dwayne Stupack and Joan Heller Brown^*,1

^* Department of Pharmacology,

Department of Bioengineering, and

Department of Pathology and The John and Rebecca Moores Cancer Center, University of California San Diego, La Jolla, California, USA

¹Correspondence: Joan Heller Brown, Department of Pharmacology, University of California San Diego, 9500 Gilman Dr.-0636, La Jolla, CA 92093, USA. E-mail: jhbrown{at}ucsd.edu

A subset of G-protein coupled receptors (GPCRs), including the thrombin receptor (PAR1), elicits mitogenic responses. Thrombin also activates Ras homolog gene family member A (RhoA) and activating protein (AP-1) -mediated gene expression in 1321N1 astrocytoma cells, whereas the nonmitogenic agonist carbachol does not. Transcriptomic analysis was used to explore differential gene induction by these agonists and revealed that the matricellular protein cysteine-rich 61 (Cyr61/CCN1) is selectively induced by thrombin. The ability of GPCR agonists to induce Cyr61 parallels their ability to activate RhoA; agonist-stimulated Cyr61 expression is inhibited by C3 toxin. When Cyr61 is down-regulated using short interfering RNA (siRNA) or short-hairpin RNA (shRNA), thrombin-induced DNA synthesis is significantly attenuated. When Cyr61 expression is induced, it appears in the extracellular compartment and on the cell surface. Extracellular Cyr61 interacts with ₅, ₆, and β₁ integrins on these cells, and monoclonal antibodies directed against ₅ and β₁ integrins inhibit thrombin-induced DNA synthesis. Functional blockade of Cyr61 with soluble heparin or anti-Cyr61 antibodies also inhibits thrombin-induced DNA synthesis. Thus Cyr61 is a highly inducible, secreted extracellular factor through which GPCR and RhoA signaling pathways engage integrins that contribute to GPCR-mediated proliferation.—Walsh, C. T., Radeff-Huang, J., Matteo, R., Hsiao, A., Subramaniam, S., Stupack, D., and Brown, J. H. Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61.

Key Words: G-protein • S1P receptor • PAR1 • matricellular protein • glioblastoma