Calcific aortic valve disease (CAVD) is an important health burden due to its increasing prevalence and lack of available approaches. Osteogenic transdifferentiation of aortic valve interstitial cells (AVICs) contributes to valve calcification. SRY-related HMG-box transcription factor 5 (SOX5) is essential for cartilage development. Whether SOX5 is involved in AVIC calcification has not been determined. This study aimed to explore the role of SOX5 in warfarin-induced AVIC calcification. Immunostaining showed decreased SOX5 in human calcific AV and warfarin induced mouse calcific AV tissues compared with human noncalcific AV and control mouse AV tissues. In calcific human AVICs (hAVICs) and porcine AVICS (pAVICs), both knockdown and overexpression of SOX5 inhibited calcium deposition and osteogenic marker gene expression. Protein expression assays and ChIP assays showed that overexpression of SOX5 led to increased recruitment of SOX5 to the SOX9 promoter and resulted in increased mRNA and protein expression of SOX9. Coimmunoprecipitation and immunofluorescence showed that SOX5 binds to SOX9 with its HMG domain in nucleus. Blue Native PAGE showed overexpression of SOX5 led to multimeric complex formation of SOX5 and resulted in decreased binding of SOX5 to SOX9 similar to the results of knockdown of SOX5. Further ChIP and western blotting assays showed that both knockdown and overexpression of SOX5 resulted in SOX9 initiating transcription of anti-calcific gene LRP6 in warfarin-treated pAVICs. Knockdown of LRP6 rescues the anti-calcification effect of SOX5 overexpression. We found that both loss and gain of function of SOX5 lead to the same phenotype: decreased warfarin induced calcification. The stoichiometry of SOX5 is crucial for cooperation with SOX9, SOX9 nuclear localization and subsequent binding of SOX9 to LRP6 promoter. These results suggest that SOX5 is a potential target for the development of anti-calcification therapy.

钙化性主动脉瓣疾病 (CAVD) 是一种重要的健康负担，因为其患病率不断增加且缺乏可用的治疗方法。主动脉瓣间质细胞 (AVIC) 的成骨转分化导致瓣膜钙化。SRY 相关的 HMG-box 转录因子 5 (SOX5) 对软骨发育至关重要。SOX5 是否参与 AVIC 钙化尚未确定。本研究旨在探讨 SOX5 在华法林诱导的 AVIC 钙化中的作用。与人非钙化 AV 和对照小鼠 AV 组织相比，免疫染色显示人钙化 AV 和华法林诱导的小鼠钙化 AV 组织中的 SOX5 降低。在钙化的人类 AVICs (hAVICs) 和猪 AVICs (pAVICs) 中，SOX5 的敲低和过表达都抑制了钙沉积和成骨标记基因的表达。SOX9启动子并导致 SOX9 的 mRNA 和蛋白质表达增加。共免疫沉淀和免疫荧光显示 SOX5 与 SOX9 结合，其 HMG 结构域位于细胞核中。Blue Native PAGE 显示 SOX5 的过表达导致 SOX5 的多聚体复合物形成，并导致 SOX5 与 SOX9 的结合减少，类似于 SOX5 的敲低结果。进一步的 ChIP 和蛋白质印迹分析表明，SOX5 的敲低和过表达均导致 SOX9 启动抗钙化基因LRP6的转录在华法林治疗的 pAVIC 中。LRP6 的敲低挽救了 SOX5 过表达的抗钙化作用。我们发现 SOX5 功能的丧失和获得都导致相同的表型：华法林诱导的钙化减少。SOX5 的化学计量对于与 SOX9 的合作、SOX9 核定位以及随后 SOX9 与LRP6启动子的结合至关重要。这些结果表明，SOX5 是开发抗钙化疗法的潜在靶点。