Nicotine Facilitates VSMC Dysfunction Through A MiR-200b/RhoGDIA/cytoskeleton Module

Cigarette smoking is considered to be a major risk factor for the development of cardiovascular diseases such as atherosclerosis and hypertension.It is reported that nicotine,an addictive agent in cigarette smoke,has a wide variety of effects on vascular dysfunction.For example,nicotine can induce the migration and proliferation of vascular smooth muscle cells?VSMCs?.However,the molecular mechanisms underlying this pathological process have not been elucidated.Our previous study demonstrated that nicotine could affect the expression of four cytoskeleton proteins in VSMCs,including?-actin,?-actin,tropomyosin 4 and F-actin,and as well as RhoGDIA?Rho-specific guanine nucleotide dissociation inhibitor A?,a negative regulator of the Rho GTPase pathway.So in the present study,we investigated the function of the Rho GTPase pathway underlying nicotine-induced abnormal behaviors in VSMCs and we sought to determine the mechanism underlying nicotine-induced deregulation of RhoGDIA.In the study,mouse primary VSMCs were obtained from the thoracic arteries of mice using the cell explant method and cells between passages2 and 4 were used for subsequent experiments and the experiments were performed on cells coming from different batches.In vitro,we used an experimental concentration of 10?M,which is consistent with the nicotine concentration(10^–5 to 10^–88 M)that was determined from plasma isolated from smokers.Meanwhile,mice were treated with 0.2 mg/100 g free-base nicotine in vivo according to the definition of?heavy smoking??a consumption?20 cigarettes daily?by the World Health Organization?WHO?.Since the mechanism of vascular senescence in aging mice is complex,mice were treated with nicotine for 20 weeks after sexually maturation to mimic nicotine-exposure at middle age.The control group received a daily equivalent of normal saline.Firstly,eight cytoskeletal markers??-actin,?-actin,F-actin,myosin light chain?MLC?,phosphorylated myosin light chain?p-MLC?,desmin,calponin and caldesmon?were detected.Meanwhile,to determine whether the Rho GTPase pathway was associated with nicotine-induced deregulation of proteins,?-BtX??7-nicotinic acetylcholine receptor,?7-nAchR specific antagonist?and Y27632?an inhibitor of Rho-Kinase activation?were added to the culture media 30 min before nicotine treatment.We found that treatment with nicotine significantly increased expression of cytoskeleton-related proteins and addition of either?-BtX or Y27632 inhibited the considerable increase in cytoskeletal protein expression induced by nicotine.To further understand the influence of nicotine on the Rho GTPase pathway,we examined the activation of the main Rho GTPases,namely RhoA-GTP,RAC1-GTP and CDC42-GTP as well as the downstream intracellular signaling proteins,including myosin phosphatase target subunit-1?MYPT1?,p21-activated kinase-1?PAK1?and phosphatidylinositde-3 kinase/protein kinase B?PI3K/AKT?and we found that nicotine can activate the Rho GTPase pathway and induce the synthesis of the cytoskeletal proteins in VSMCs through the activation of intracellular downstream signaling pathways,including targets such as MYPT1,PAK1 and PI3K/AKT pathway.To explore the molecular mechanisms underlying nicotine-induced Rho-GTPase-dependent migration and proliferation of VSMCs,the expression levels of Rho GTPase-related genes were detected.By qRT-PCR and Western blotting analysis,we found that upon nicotine treatment,the mRNA level of RhoGDIA is increased but protein level is decreased both in vitro and in vivo,which suggested a mechanism of post-translational regulation.In addition to the changes in RhoGDIA,there are also changes in mRNA and protein expression of total RhoA,RAC1 and CDC42,which hinted at more than one mechanism involved in the nicotine-induced Rho GTPase changes.Considering the abnormal co-activation of Rho GTPases and the function of RhoGDIA as a negative regulator of the Rho GTPase pathway,we thought that RhoGDIA may play an important role in the dysfunctions of VSMCs,so we focused on RhoGDIA and the mechanism underpinning its deregulation in the study.Subsequently,by the dual luciferase reporter assay and Western Blotting verification,we identified the microRNA-200b?miR-200b?as a modulator of the behavioural changes of VSMCs in response to nicotine through targeting RhoGDIA directly.Transfection with miR-200b mimics induced migration and proliferation in VSMCs.Furthermore,miR-200b inhibitors depressed migration and proliferation of VSMCs that were treated with either nicotine or miR-200b mimics.Given that miR-200b can promote dysfunction in VSMCs,we examined the activation of the main Rho GTPases after transfecting VSMCs with miR-200b mimics.Cells transfected with miR-200b had a sharp decrease in RhoGDIA expression and this effect in turn activated RhoA,RAC1 and CDC42.These findings suggest that miR-200b promotes the dysfunction VSMCs mainly through the targeting of RhoGDIA,which is a well-known negative regulator of Rho GTPases.Additionally,we found that hypomethylation in the CpG island shore region of miR-200b was responsible for the nicotine-induced miR-200b up-regulation in VSMCs.The methylation level?the percentage of methylated-CpGs compared to the total number of CpGs?was 88.0%in the control group and this level was decreased to 56.7%in the nicotine-treated group.Moreover,methylation occurred less frequently at sites 1,3,5,6 and 9.To confirm the epigenetic regulation of miR-200b,5-aza-2?-deoxycytidine?5-Aza-2?-DC?was used in VSMCs.Following methylation-specific and non-methylation-specific-PCR,low doses of 5-Aza-2?-DC were able to change the methylation status of the miR-200b promoter.This change was accompanied by an obvious increase in the level of miR-200b.In summary,our results demonstrated that nicotine treatment significantly increases miR-200b expression through promoter hypomethylation.The miR-200b-mediated down-regulation of RhoGDIA in turn facilitates migration and proliferation of VSMCs in a Rho GTPase-dependent manner.This newly identified mechanism involving miR-200b and RhoGDIA provides a new avenue in understanding the process of VSMC migration and proliferation.