The Effects Of Deubiquitin Enzymes USP22 On Vascular Calcification Via Participating In ER? Mediated Gene Transactivation

Objective:With the more and more aging people,the morbidity of vascular calcification(VC)become increasing.VC,as a complication of cardiovascular diseases(CVD),increases its morbidity and mortality.Clinical study shows that hormone therapy(HT)obviously reduces the morbidity of coronary artery calcification.Some experimental study suggests that estrogen(E2)/estrogen receptor(ER?)play inhibited roles in VC via affecting COX2,RANKL,Gas6.So,as a member of the nuclear receptor subfamily,ER?may exert protective effects on VC.Deubiquitin enzymes USP22 is a member of a ubiquitin protease system.USP22 exerts important function in cardiovascular diseases.USP22,as a cofactor of nuclear receptor,participate in androgen receptor(AR)-dependent transcactivation in prostate cancer.Whether USP22 participates in ER?-dependent transcactivation is unclear.The objective of this study is to build human artery smooth muscle cells(hASMCs)calcification model and verifies the effects and mechanism of USP22 on VC.Methods:Human aortic smooth muscle cells(hASMCs)vaccinated respectively in the normal DMEM,1.25 Mmol/L?-glycerol phosphate and 0.625Mmol/L calcium chloride(1.25 Mm CAL)DMEM,2.5 Mmol/L?-glycerol phosphate and 1.25 Mmol/L calcium chloride(2.5 Mm CAL)DMEM,5 Mmol/L?-glycerol phosphate and 2.5Mmol/L calcium chloride(5 Mm CAL)DMEM culture 0,3,7,9 days,with a 1%/2%Alizarin red S-dyeing,and the inverted phase contrast microscope imaging;Under the condition of 5 Mm CAL cultivating 0,3,7,9 days,western blot experiment tests the expression of calcification associated protein BMP2,RANKL,COX2,Gas6,ER?.Luciferase double gene report experimental detectes in hASMCs deubiquitin enzyme USP22 whether to participate in the ER?mediated gene transcription;With overexpression of USP22,western blot test was used to detect the expression of calcification associated protein BMP2,RANKL,COX2,Gas6,ER?.Result:Human aortic smooth muscle cells(hASMCs)have significant calcium deposition in the 9~thh days of 1.25Mm CAL condition culturing.In the condition of2.5Mm CAL and 5Mm CAL culture condition,hASMCs have small amount of calcium deposition in the 7~thh days,but in the 9~thh days,hASMCs have obvious calcium deposition.In 5Mm CAL conditions to developing,the increased expression of BMP2 suggests smooth muscle cell phenotype transform into ostegenesis phenotypic;The increasing of USP22 expression demonstrates that USP22 may promot hASMCs calcification.Other experiments results demonstrated that deubiquitin enzyme USP22 inhibit ER?mediated target gene Gas6 transcription regulation,and then promots hASMCs calcification.Conclusion:with the condition of 5Mm CAL concentration culture time,the phenotype of smooth muscle cells is transformed into osteogenic phenotype;The expression of deubiquitin enzyme USP22 protein also increase,which suggest USP22 may exert promoting effect.Deubiquitin enzyme USP22 inhibit ER?mediated target genes Gas6gene transcription,and then facilitates human aortic smooth muscle cells(hASMCs)calcification.