Progesterone induces RhoA inactivation in male rat aortic smooth muscle cells through up-regulation of p27^kip1

Previously, we showed that progesterone (P4) at physiologic concentrations (5nM-500nM) inhibits proliferation and migration of rat aortic smooth muscle cells (RASMCs). The P4-induced migration inhibition in RASMC was resulted from Rat sacroma homolog gene family, member A (RhoA) inactivation induced by activating the cSrc/AKT/ERK 2/p38 mitogen-activated protein kinase-mediated signaling pathway. We also demonstrated that up-regulation of cyclin-dependent kinase inhibitor 1B (p27^kip1) is involved in the P4-induced migration inhibition in RASMC. Because P4 can increase formation of the p27^kip1-RhoA complex in RASMC, this finding led us to hypothesize that the P4-induced inactivation in RhoA might be caused by up-regulation of p27^kip1. Here, we showed that P4 increased phosphorylation of p27^kip1 at Ser10 in the nucleus, which in turn caused p27^kip1 translocation from the nucleus to the cytosol, subsequently increasing formation of the p27^kip1-RhoA complex. These effects were blocked by knocking-down kinase-interacting stathmin (KIS) using KIS small interfering RNA. Knock-down of p27^kip1 abolished the P4-induced decreases in the level of RhoA protein in RASMC. However, pretreatment of RASMC with the proteasome inhibitor, N-(benzyloxycarbonyl)leucinylleucinylleucinal (MG132), prevented the P4-induced degradation of p27^kip1 and RhoA. Taken together, our investigation of P4-induced migration inhibition in RASMC showed a sequence of associated intracellular events that included 1) increase in formation of the KIS-p27^kip1 complex in the nucleus; 2) phosphorylated nuclear p27^kip1 at Ser10; 3) increased cytosolic translocation of p27^kip1 and formation of the p27^kip1-RhoA complex in the cytosol; and 4) degradation of p27^kip1 and RhoA through the ubiquitin-proteasome pathway. These findings highlight the molecular mechanisms underlying P4-induced migration inhibition in RASMC.