The Mechanism Of SM22α Inhibiting Ras Activation And Targeting Interference Of Baicalin

Objective: Vascular smooth muscle cell (VSMC) phenotypic remodelingand migration are the key events of vascular diseases, including hypertension,atherosclerosis and restenosis. Platelet-derived growth factor (PDGF)-BB isthe most potent mitogenic factor and chemoattractant. Baicalin, anherb-derived flavonoid compound, plays strong protective roles incardiovascular system. As a differentiation marker of VSMC, smooth muscle22alpha (SM22α) inhibits VSMC proliferation, inflammation and oxidantstress. Ras protein, a member of small G protein family, is involved regulationof cell growth, differentiation, secretion and transportation of proteins. Ourprevious studies showed that baicalin inhibited PDGF-BB-induced VSMCproliferation, but the mechanism was not well characterized.Methods and results:1Baicalin inhibits PDGF-BB-induced VSMC phenotypic remodelingVSMC was treated with PDGF-BB (20ng/ml) for24h, Western blotshowed that PDGF-BB significantly promoted the expression of VSMCproliferation marker PCNA, and inhibited the expression of differentiationmarker SM22α, caldesmon, calponin,and SM α-actin.VSMC was pretreated with baicalin for24h followed by stimulation withPDGF-BB for24h. Western blot showed that baicalin inhibitedPDGF-BB-induced upregulation of PCNA and downregulation ofdifferentiation gene SM22α, caldesmon, calponin, and SM α-action. Theseresults suggested that baicalin inhibited PDGF-BB-induced VSMC phenotypicswitching.2SM22α inhibits PDGF-BB-induced the formation of SOS1-Rassignaling complex via blockade of the interaction of SOS1and RasThe interaction between SOS1and Ras plays an important role in Ras activation. VSMC was treated with PDGF-BB for2min. Thenimmunoprecipitation analysis found that PDGF-BB promoted the interactionbetween SOS1and Ras, whereas overexpression of SM22α inhibited it. Theseresults suggested that SM22α inhibited Ras activation via preventing theinteraction of SOS1with Ras.3The SM22α phosphorylation mutant promotes the interaction betweenSOS1and RasImmunoprecipitation analysis showed that overexpression of SM22αnon-phosphorylated mutant S181A inhibited the interaction of Ras with SOS1,consistent with the role of wild-type SM22α while overexpression of SM22αphosphorylated mutant S181D promoted the interaction of Ras and SOS1.These results verified the above findings.4Baicalin inhibits the expression of ICAM-1and VCAM-1and intimalhyperplasia in carotid arteries after balloon injuryImmunohistochemical staining showed that: Baicalin significantlyinhibited balloon injury-induced ICAM-1and VCAM-1expression andintimal hyperplasia in balloon-injured carotid arteries, compared with injurygroup.Conclusions：1Baicalin inhibits PDGF-BB-induced VSMC phenotypic switching fromcontractile to synthetic state.2SM22α inhibits PDGF-BB-induced the formation of Ras-SOS1signalingcomplex.3The SM22α phosphorylation mutant promotes the formation of SOS1-Rassignaling complex.4Baicalin inhibits the expression of ICAM-1and VCAM-1and intimalhyperplasia induced by balloon injury.