SOX2 Hinders Osteogenic Differentiation of Valvular Interstitial Cells through Attenuation of NOTCH1 Promoter Methylation via a NOTCH1-SOX2 Positive-Feedback Loop

Abstract

Background: Calcific aortic stenosis’s pathology is associated with calcification of valvular interstitial cells (VICs). SOX2 (SRY (sex determining region Y)-box 2) has been reported to regulate osteogenic differentiation that leads to calcification and as a mediator of the methylation of NOTCH1 (The Notch homolog 1 translocation-associated). This study characterized the specific role of SOX2 in osteogenic differentiation of VICs. Methods: VICs isolated from porcine hearts, identified by immunofluorescence assay, were induced into osteogenesis. SOX2 overexpression/knockdown plasmids and notch intracellular domain (NICD) overexpression plasmids were used to investigate the role of SOX2 and NOTCH1 on the osteogenic differentiation of VICs. Alkaline phosphatase activity, mineralized nodules, and calcium concentration were assessed by alkaline phosphatase staining, Alizarin Red S staining, and Arsenazo III dye assay, respectively. In these osteogenically differentiated VICs cells, the expressions of SOX2, NOTCH1, and osteogenesis-related markers (RUNX2 (Runt-related transcriptionfactor2), BMP2 (Bone morphogenetic protein 2) and OPN (Osteopontin)) were analyzed by western blot and/or quantitative reverse transcription-PCR (polymerase chain reaction). NOTCH1 promoter methylation was analyzed by bisulfite sequencing-PCR and methylated DNA (deoxyribonucleic acid) immunoprecipitation. Results: In osteogenically differentiated isolated VICs, SOX2, and NOTCH1 expressions were decreased (p < 0.01;p < 0.001). SOX2 overexpression or NICD overexpression suppressed alkaline phosphatase activity, inhibited mineralized nodule formation, decreased calcium concentration (p < 0.001), osteogenesis-related markers (p < 0.05;p < 0.001), and NOTCH1 methylation (p < 0.001), and increased the expression of NOTCH1 (p < 0.05;p < 0.001). By contrast, SOX2 knockdown produced the opposite effect (p < 0.05). NICD overexpression reversed the SOX2 knockdown-induced effect (p < 0.001), which in turn abolished the NICD-induced effect. Conclusions: SOX2 hinders VIC osteogenic differentiation by attenuating NOTCH1 promoter methylation via a NOTCH1-SOX2 positive-feedback loop.