Abstract 1174: Ser188 Phosphorylation of RhoA Induces Vascular Smooth Muscle Cells Proliferation and Migration by Activation of the PI3K/Akt Pathway
Although over-activation of RhoA is recognized as a common component for the pathogenesis of hypertension and vascular proliferative disorders, the molecular mechanisms regulating RhoA activity in vascular smooth muscle cells (VSMC) are still unknown. We have previously shown that in VSMC, RhoA is phosphorylated on Ser188 by NO-stimulated cGMP-dependent kinase (PKG). This Ser188 phosphorylation of RhoA leads to inhibition of RhoA-Rho kinase pathway. Here we hypothesize that in addition to this inhibition, cytosolic Ser188-phosphorylated RhoA has its own functions. To address this hypothesis we analyzed VSMC functions in cell expressing wild-type RhoA (WT), constitutively active (Q63L), phosphoresistant (S188A), phosphomimetic (S188E) and double-mutants Q63L-S188A and Q63L-188E. Using a scratch-wound repair assay in VSMC monolayers, we first have measured the ability of these mutants to influence the healing process at 24h-post wounding. Expression of phosphoresistant mutants reduced the wound-closure (37.5% ± 1.4 for Q63L-S188A compared to 49.0% ± 3.1 for Q63L; p < 0.01; n = 5). In contrast, phosphomimetic mutants greatly accelerated the wound-closure (91.5% ± 3.8 Q63L-S188E; p< 0.01, n = 5). The NO donor sodium nitroprusside (100 μM) or the PKG activator 8pCPTcGMP (100 μM) accelerated the wound-closure in VSMC expressing the WT RhoA (77.5% ± 4.3 and 90.0% ± 8.6 respectively vs 57.5 ± 2.6; p < 0.01, n = 3). This effect of PKG-stimulating agents was inhibited in VSMC expressing the phosphoresistant mutant S188A. The positive effect of phosphomimetic RhoA mutants resulted on stimulation of both VSMC migration and proliferation. Phosphomimetic Q63-S188E mutant increased VSMC proliferation assessed by BrDU staining to 182 ± 14% of control (48h post-transfection). Analysis of signaling pathways involved revealed that Q63L-S188E enhanced Akt phosphorylation (298% ± 52 of the Akt phosphorylation in VSMC expressing the Q63L, n = 3). Pharmacological blockers of the PI3K-Akt signaling pathway (LY294002, 25 μM; Akt inhibitor IV, 10 μM) inhibited the accelerated wound-healing induced by Q63L-S188E. Our work thus demonstrates that PKG-induced phosphorylation of RhoA facilitates VSMC proliferation and migration by activation of the PI3K/Akt pathway.