Li Shao-Jung, Kao Yu-Hsun, Chung Cheng-Chih, Cheng Wan-Li, Chen Yi-Jen
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University Taipei, Taiwan.
Division of Cardiovascular Surgery, Department of Surgery, Wan Fang Hospital, Taipei Medical University Taipei, Taiwan.
Am J Transl Res. 2019 Feb 15;11(2):744-754. eCollection 2019.
The cellular mechanisms of calcific aortic valve (AV) disease and optimal medications for its treatment are poorly elucidated. Glycogen synthase kinase (GSK)-3β and non-canonical wingless-related integration site (Wnt) signaling play crucial roles in regulating the pathogenesis of valvular interstitial cell (VIC) calcification. Histone acetylation was found to regulate VIC calcification. However, whether histone deacetylases (HDACs) modulate the pathophysiology of AV calcification is unclear. Different HDAC isoforms have dissimilar cardiovascular effects. We hypothesized that distinctive HDAC inhibitors modulate runt-related transcription factor 2 (RUNX2) in aortic VICs through the regulation of Wnt signaling.
Western blotting, real-time polymerase chain reaction, and proliferation assay were used to analyze osteogenesis marker expression, Wnt signaling, bone morphogenetic protein (BMP) signaling, and proliferation in porcine VICs treated with osteogenic (OST) medium alone or in combination with HDAC inhibitors.
VICs treated with OST medium for 5 days exhibited higher RUNX2 and GSK-3β expression levels than did control cells. A class I HDAC inhibitor (MS-275 at 1 μM) reduced the RUNX2 mRNA and protein expression levels and alkaline phosphatase activity and downregulated non-canonical Wnt/GSK-3β signaling, canonical Wnt/β-catenin signaling, and BMP signaling. By contrast, a combined class IIa (MC1568) and IIb HDAC (tubacin) inhibitor (0.1 μM) increased RUNX2 expression. MS-275, MC1568, and tubacin reduced VIC proliferation; however, the extent of reduction differed. MS-275 reduced RUNX2 and osteocalcin expression in VICs treated with OST medium for an extended period (14 days).
MS-275 critically regulates RUNX2 transactivation in VICs through both canonical and non-canonical Wnt signaling pathways.
钙化性主动脉瓣疾病的细胞机制及其最佳治疗药物尚未完全阐明。糖原合酶激酶（GSK）-3β和非经典无翅相关整合位点（Wnt）信号传导在调节瓣膜间质细胞（VIC）钙化的发病机制中起关键作用。已发现组蛋白乙酰化可调节VIC钙化。然而，组蛋白脱乙酰酶（HDAC）是否调节主动脉瓣钙化的病理生理学尚不清楚。不同的HDAC亚型具有不同的心血管效应。我们假设不同的HDAC抑制剂通过调节Wnt信号传导来调节主动脉VIC中的 runt相关转录因子2（RUNX2）。
采用蛋白质印迹法、实时聚合酶链反应和增殖试验分析单独用成骨（OST）培养基或与HDAC抑制剂联合处理的猪VIC中骨生成标志物表达、Wnt信号传导、骨形态发生蛋白（BMP）信号传导和增殖情况。
用OST培养基处理5天的VIC比对照细胞表现出更高的RUNX2和GSK-3β表达水平。I类HDAC抑制剂（1 μM的MS-275）降低了RUNX2 mRNA和蛋白表达水平以及碱性磷酸酶活性，并下调了非经典Wnt/GSK-3β信号传导、经典Wnt/β-连环蛋白信号传导和BMP信号传导。相比之下，IIa类（MC1568）和IIb类HDAC（tubacin）抑制剂联合使用（0.1 μM）增加了RUNX2表达。MS-275、MC1568和tubacin降低了VIC增殖；然而，降低程度有所不同。MS-275降低了用OST培养基长期（第14天）处理的VIC中RUNX2和骨钙素的表达。
MS-275通过经典和非经典Wnt信号通路关键地调节VIC中RUNX2的反式激活。
