Thrombin mediated regulation of CCN1 regulates cell proliferation in an integrin dependent manner

Abstract

We previously reported that PAR1 (activated by thrombin) and the M3 muscarinic (activated by carbachol) receptors differentially effect AP‐1 mediated gene expression and DNA synthesis in 1321N1 astroglial cells. Microarray experiments were used to explore genes that were differently regulated by thrombin verses carbachol and revealed that the secreted protein cysteine‐rich 61 (Cyr61/CCN1) was robustly and selectively induced by thrombin. Thrombin but not carbachol activate RhoA therefore we postulated that Rho signaling mediates CCN1 expression. This was supported by experiments demonstrating that the C3 exoenzyme attenuates thrombin‐induced CCN1 expression and that other GPCR agonists that activate Rho also induce CCN1 expression while Gq coupled receptor agonists do not. Inhibition of CCN1 expression with siRNA decreased thrombin‐stimulated DNA synthesis by ~50% implicating CCN1 signaling in thrombin mediated proliferation. Currently integrins are the only known receptor for CCN1. Data shows that CCN1 is present on the cell surface of 1321N1 cells, that 1321N1 cells bind to CCN1 in an α6β1, α5β1, and β1 integrin dependent manner, and that functional inhibitory antibodies to these integrins attenuate thrombin mediated DNA synthesis. We suggest that CCN1 represents an intermediate by which G12/13 and Rho coupled GPCRs utilize integrins to elicit additional signals involved in cell proliferation.NIH 5R01‐GM36927