Transcription Factor SOX5 Attenuates Warfarin Induced Aortic Valve Calcification

Aims:The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells(AVICs),but it lacks early detection and effective treatment methods,while the mechanism is not fully understood.Previously,transcription factor SOX5 was mainly involved in the regulation of embryonic development,cell cycle and cartilage formation.Recently,SOX5 has been found to be involved in osteogenic differentiation and calcification of AVICs,but its role in warfarin-induced valvular calcification has not been reported.Our study was designed to investigate the role of transcription factor SOX5 and anti-calcification factor SOX9 in aortic valve calcification induced by warfarin.Methods:1.Expression of SOX5 in calcified human aortic valves and warfarin-induced calcified murine aortic valvesExpression of SOX5 was determined by immunofluorescence staining and real-time polymerase chain reaction(RT-PCR)in human non-calcified aortic valves,calcific aortic valves tissue of rheumatic valvular heart disease treated with long-term warfarin,non-calcified aortic valves of control mice and calcific aortic valves of warfarin-induced calcified mice.2.Overexpression and knockdown of SOX5 on warfarin-induced calcification of aortic valve interstitial cells(AVICs)Porcine aortic valve interstitial cells(AVICs)were isolated and cultured in vitro,then infected with recombinant adenovirus Ad-SOX5 and Ad-shSOX5.After induction of 10 μM warfarin and 1.6 mM Pi,calcium content assay,alkaline phosphatase activity(ALP)assay,and Alizarin Red staining were performed to determine the effect of SOX5 on calcification.The mRNA expressions of osteogenic markers RUNX2,SOX9,BMP2 were determined by RT-PCR,the protein level of SOX5,RUNX2 and SOX9 were determined by Western blot.3.Changes of nuclear expression of SOX5 and anti-calcification factor SOX9 in calcified AVICsThe calcified nuclear proteins were collected and tested by Western blot to detect the expression of SOX5,SOX9 and ATF4.Immunofluorescence staining was used to detect the expression of SOX5 and SOX9 in calcified AVICs.4.Interaction between SOX5 and SOX9The SOX5 transcription complex was explored by reciprocal immunocoprecipitation(Co-IP)and Duo-link PLA in situ protein interaction technology.Chromatographic immunoprecipitation(Ch-IP)was used to investigate the effect of SOX5 on the promoter region of SOX9 gene.Results:1.Expression of SOX5 in calcified human aortic valves and warfarin-induced calcified murine aortic valvesRT-PCR and immunofluorescence staining confirmed that SOX5 was specifically expressed in human AVICs and was down-regulated in aortic valve calcification by more than 60%(p<0.05).Immunofluorescence staining confirmed that SOX5 was decreased significantly in mouse calcification model induced by warfarin injection.2.Overexpression and knockdown of SOX5 on warfarin-induced calcification of aortic valve interstitial cells(AVICs)Calcium phenotypes such as intracellular calcium content and alizarin red staining showed that overexpression of SOX5 significantly reduced warfarin-induced calcium deposition in AVICs,and ALP activity was significantly reduced when overexpressing SOX5(p<0.001).Western Blot showed that overexpression of SOX5 in warfarin-induced AVICs led to decrease of RUNX2 by 25%(p<0.001),and increase of SOX9 by 4 times compared to the control group(p<0.001).RT-PCR showed that the BMP2 and RUNX2 were significantly reduced(p<0.001)and SOX9 was significantly higher(p<0.001).After interfering with the expression of SOX5,intracellular calcium content and alizarin red staining showed that warfarin-induced calcium deposition in AVICs increased,and the ALP activity was 2 times higher than that of the control group after interference with SOX5(p<0.01).Western Blot showed that the interference of SOX5 caused RUNX2 1.2 times higher compared to the control group(p<0.05),SOX9 was reduced by 50%compared to the control group(p<0.01).3.Changes of nuclear expression of SOX5 and anti-calcification factor SOX9 in calcified AVICsAt nuclear protein level,SOX9 expression was reduced by 28%in warfarin-induced calcified AVICs(p<0.05),but SOX9 was increased to the initial level after overexpression of SOX5(p<0.05).Immunofluorescence staining showed that SOX9 expression was significantly increased in the nucleus after overexpression of SOX5.4.Interaction between SOX5 and SOX9Co-IP indicated that SOX5 combined directly to SOX9 and the binding is increased upon calcification.Duo-link PLA confirmed that SOX5 binded directly to SOX9,and the binding is increased during calcification and more pronounced in nuclear.Ch-IP confirmed that SOX5 increased the activity of promoter of SOX9 gene(p<0.001).Conclusions:1.The transcription factor SOX5 significantly inhibits warfarin-induced aortic valve calcification by inhibiting osteogenic differentiation of AVICs.2.SOX5 can directly bind with SOX9 and inhibit warfarin-induced calcification of AVICs by promoting SOX9 nuclear localization.Overexpression of SOX5 also increases the activity of SOX9 promoter,thereby increasing SOX9 expression and inhibiting calcification of AVICs.