Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cellDNA synthesis and migration

Aberrant regulation of smooth muscle cell proliferation and migration is as sociated with the pathophysiology of vascular disorders such as hypertensio n, atherosclerosis, restenosis, and graft rejection. To elucidate molecular mechanisms that regulate proliferation and migration of vascular smooth mu scle cells, we determined whether signaling through the small G protein Rho is involved in thrombin- and phenylephrine-stimulated proliferation and mi gration of rat aortic smooth muscle cells (RASMCs). Thrombin and the thromb in peptide SFLLRNP stimulated DNA synthesis of RASMCs as measured by [H-3]t hymidine incorporation, Both ligands also increased cell migration as measu red by the Boyden chamber method. L-Phenylephrine failed to induce either o f these responses but increased inositol phosphate accumulation and mitogen -activated protein kinase activation in these cells, which indicated that t he cells were responsive to alpha(1)-adrenergic stimulation. The C3 exoenzy me, which ADP-ribosylates and inactivates Rho, fully inhibited both thrombi n-stimulated proliferation and migration but had no effect on inositol phos phate accumulation. III addition, Y-27632, an inhibitor of the Rho effector p160ROCK/Rho kinase, decreased thrombin-stimulated DNA synthesis and migra tion. To directly examine Rho activation, Rho-[S-35]GTP gamma S binding was measured. The addition of the thrombin peptide SFLLRNP, but not phenylephr ine, to RASMC lysates resulted in a significant increase in Rho-[S-35]GTP g amma S binding. Thrombin and SFLLRNP, but not phenylephrine, also increased membrane-associated Rho in intact RASMCs, consistent with selective activa tion of Rho by thrombin. These results indicate that thrombin activates Rho in RASMCs and establish Rho as a critical mediator of thrombin receptor af fects on DNA synthesis and cell migration in these cells.