Regulation of vascular smooth muscle proliferation and migration by β2-chimaerin, a non-protein kinase C phorbol ester receptor

Abstract

The proliferation and migration of vascular smooth muscle cells (SMC) are important aspects of atherogenesis. Activated growth factor signaling in injured vessels subsequently promotes a number of intracellular events resulting in the phenotypic modulation of SMC. Here, we investigated the role of β2-chimaerin, a non-protein kinase C phorbol ester receptor with Rac-GTPase-activating protein activity, in growth factor-stimulated SMC. The endogenous expression of β2-chimaerin was detected in cultured human SMC by reverse transcription-polymerase chain reaction and immunohistochemistry. Next, the overexpression of HA-tagged wild-type human β2-chimaerin was attempted using cultured rat SMC with a recombinant adenovirus (Adv-β2-Chim). Adv-LZ encoding β-galactosidase (LacZ) was used as the control. The proliferation of SMC stimulated by platelet-derived growth factor (PDGF-BB, 10 ng/ml), as measured by cell-counting and 5-bromo-2′deoxyuridine incorporation assay, was suppressed by infection with Adv-β2-Chim (50-200 MOI), but not with control viruses. PDGF-induced SMC migration was inhibited by approximately 25% after infection with Adv-β2-Chim (200 MOI) using a modified Boyden's chamber assay with a fibronectin-coated membrane. Confocal microscopy revealed that PDGF stimulation altered the sub-cellular localization of β2-chimaerin. The administration of 12-O-tetradecanoyl phorbol 13-acetate also induced changes in the sub-cellular localization of β2-chimaerin, which was not affected by a presence of the PKC inhibitor (GF109203X). Finally, PDGF-induced Rac1 activation was found to be inhibited in the Adv-β2-Chim-infected cells. Thus, we demonstrated that β2-chimaerin regulates the proliferation and migration of SMC downstream of growth factor signaling pathway via the regulation of Rac1 activity. The signaling mediated by β2-chimaerin may play a role in the regulation of SMC phenotypes, thereby implicating human atherogenesis.