Molecular Mechanisms Of Increased Proliferation And Migration Of Aortic Smooth Muscle Cell Induced By The Inactivation Of Cysteine 674 In The Sarco(endo)plasmic Reticulum Calcium ATPases 2 (SERCA2)

BACKGROUND:Vascular remodeling,a common pathological feature of cardiovascular diseases such as hypertension,atherosclerosis,and restenosis after angioplasty,mainly includes four processes:cell growth,cell death,cell migration,as well as production and degradation of extracellular matrix.The aortic wall is composed of different types of cells,including endothelial cells,smooth muscle cells（SMC）and fibroblasts,of which SMC is the main cell type.Normal SMCs are mainly contractile and undergo a phenotypic transition to a synthetic type in response to external stimulation.Synthetic SMC always exhibits reduced expression of contractile marker proteins and reduced contractile capacity,increased synthesis and secretion of extracellular matrix,and re-entering the cell cycle with increased cell proliferation and migration.SMC phenotypic conversion is the key process of vascular remodeling,and it is important to investigate the factors affecting SMC proliferation and migration for the prevention and treatment of vascular remodeling diseases.Sarcoplasmic/endoplasmic reticulum calcium ATPase（SERCA）is a key membrane transporter that maintains Ca²⁺homeostasis in the cytoplasm and the only active protein that transports Ca²⁺from the cytosol to sarcoplasmic/endoplasmic reticulum.The expression of SERCA is tissue-specific,with SERCA2 being the major isoform in cardiovascular.The cysteine 674（C674）of SERCA2is an important redox site,which is important for maintaining the normal physiological function of SERCA2.The pathological condition of increased reactive oxygen species（ROS）is highly susceptible to inactivation of C674.Our previous study found that in aortic smooth muscle cells（ASMC）,C674 inactivation in SERCA2 caused increased cytoplasmic Ca²⁺,and activated calcineurin（Ca N）/NFAT4/p65NFκB pathway,and downregulated PPARγ,leading to the phenotypic transition of ASMC and increased cell proliferation and migration.Inhibition of the Ca N/NFAT4/p65NFκB pathway or activation of PPARγcould inhibit ASMC proliferation and migration induced by C674inactivation.Function changes of SERCA2,a key Ca²⁺regulatory protein,may have broad implications on cellular signaling pathways.We hypothesize that other molecular mechanisms may be involved in the regulation of ASMC proliferation and migration by C674 inactivation.SERCA2 C674 inactivation promotes the development of vascular remodeling diseases such as hypertension and aortic aneurysms,therefore,it is important to investigate the mechanisms by which C674 inactivation in SERCA2 promotes ASMC proliferation and migration.METHODS and RESULTS:To explore the molecular mechanisms by which C674inactivation in SERCA2 promotes ASMC proliferation and migration,we constructed SERCA2 C674S mutant knock-in（SKI）mice by mutating cysteine to serine at site 674（serine 674,S674）in SERCA2 to mimic C674 inactivation under pathological conditions.ASMC proliferation and migration were increased in SKI mice compared to ASMC from littermate wild type（WT）mice.There was no difference in SERCA2 protein expression between WT mice and SKI mice ASMC,indicating that C674 inactivation affected the function of SERCA2,but had no effect on the expression of SERCA2 protein.Ca²⁺chelator BAPTA-AM was used to reduce ASMC cytoplasmic Ca²⁺levels in SKI mice ASMC and we found that cell proliferation and migration were reduced,suggesting that C674 inactivation promotes ASMC proliferation and migration by increasing cytoplasmic Ca²⁺.Further exploration revealed that compared to ASMC of WT mice,ASMC of SKI mice:1)The proporation of G0/G1 phase cells decreased and the percentage of S phase cells increased.We found increased expression of cyclin D1,that regulates the G0/G1phase of cells,increased expression of cyclin A1 and cyclin A2,which promote the transition from G1 to S phase,and decreased expression of p21 and p27,which inhibit the cyclin D-cyclin-dependent kinase 4/6（CDK4/6）complex and cyclin A-CDK2complex,indicating that C674 inactivation accelerates the cell cycle and promotes cell proliferation and migration through the regulation of these cell cycle protein;2)We found activation of the renin-angiotensin system（RAS）associated with proliferation,decreased expression of the proliferation-suppressing p53 protein,and activation of the extracellular signal-regulated kinase 1/2（ERK1/2）pathway associated with proliferation.The expression of angiotensin-converting enzyme（ACE）and angiotensin type 1 receptor（AT1R）increased,expression of ACE2 decreased,and expression of G-coupled protein receptor of angiotensin-（1-7）（Mas）did not change in RAS;3)ROS production was increased.The expression of protein that inhibit ROS production were decreased including glutathione peroxidase-1（GPx-1）,NAD[P]H quinone oxidoreductase1（NQO1）,heme oxygenase-1（HO-1）and superoxide dismutase1（SOD1）.In SKI mice ASMC,the AT1R blocker olmesartan:1)Inhibited the expression of cyclin A1 and increased the expression of p53;2)Inhibited the production of ROS and increased the protein expression of GPx-1 and SOD2;3)Inhibited cell proliferation and migration,suggesting that RAS activation at least partially mediated the regulation of C674 inactivation on ASMC proliferation and migration,which may be related to the regulation of cell cycle protein and ROS production.Then we explored the upstream regulatory protein of RAS.In ASMC of SKI mice,endoplasmic reticulum stress inhibitor 4-PBA and AMPK agonist AICAR inhibited the protein expression of ACE;PPARγactivator pioglitazone increased the protein expression of ACE2;The Ca N inhibitor Cs A had no significant effect on RAS,suggesting that RAS activation induced by C674 inactivation was regulated by endoplasmic reticulum stress,AMPK and PPARγpathways.In SKI mice ASMC,the ERK1/2 pathway inhibitor U0126:1)Inhibited the protein expression of cyclin A2;2)Inhibited ROS production and increased the protein expression of NQO1 and HO-1;3)Inhibited the proliferation and migration of ASMC.These results suggest that activation of the ERK1/2 pathway at least partially mediates the regulation of ASMC proliferation and migration by C674 inactivation,which may be related to the regulation of cell cycle protein and ROS production.In SKI mice ASMC,the ROS scavenger Tempol:1)Inhibited the ROS production and increased the protein expression of GPx-1,HO-1 and SOD1;2)Inhibited the protein expression of cyclin A1,cyclin A2 and cyclin D1 and increased the expression of proliferation-inhibiting protein p27;3)Inhibited the activation of RAS（ACE and AT1R）and ERK1/2 pathway;4)Inhibited the proliferation and migration of ASMC.These results suggest that ROS mediates the regulation of ASMC proliferation and migration by C674 inactivation,which may be related to its regulation of RAS and ERK1/2 pathways activation.Finally,we explored whether activation of SERCA2 improves the regulation of cell proliferation and migration by C674 inactivation using CDN1163,an agonist of SERCA2.In SKI mice ASMC,CDN1163:1)Increased the protein expression of SERCA2,inhibited the protein expression of cyclin A2 and cyclin D1,and increased the protein expression of p27;2)Inhibited ASMC proliferation and migration.These results suggest that activation of SERCA2 in the case of SERCA2dysfunction inhibits ASMC proliferation and migration.CONCLUSION:Inactivation of C674 in SERCA2 causes oxidative stress,activates RAS and ERK1/2 pathway,and promotes ASMC proliferation and migration.The activation of RAS and ERK1/2 pathway in ASMC is also involved in the regulation of ROS generation,cell proliferation and migration by C674 inactivation.