Department of Geriatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Avenue, Wuhan, 430022, Hubei, China.
Department of Rehabilitative Medicine, Wuhan NO.1 Hospital, Wuhan, 430022, Hubei Province, China.
Calcif Tissue Int. 2021 Aug;109(2):203-214. doi: 10.1007/s00223-021-00833-4. Epub 2021 Mar 13.
Vascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.
血管钙化（VC）在心血管疾病、糖尿病和慢性肾脏病中高度流行，并且当存在时，与心血管事件和死亡率相关。血管平滑肌细胞（VSMCs）的成骨分化被认为是介导 VC 的基础。相关转录增强因子（RTEF-1），也称为转录增强相关结构域（TEAD）4 或转录增强因子-3（TEF-3），是一种核转录因子，除了在经典 Hippo 通路中具有致癌作用外，对心血管疾病也有很强的作用。然而，RTEF-1 在 VC 中的作用和机制，特别是在 VSMCs 的钙化中，知之甚少。我们的结果表明，RTEF-1 在钙化的 VSMCs 中减少。RTEF-1 显著改善了β-甘油磷酸（β-GP）诱导的 VSMCs 钙化，通过茜素红染色和钙含量测定检测到。此外，RTEF-1 降低碱性磷酸酶（ALP）活性，降低成骨细胞标志物如骨钙素和 Runt 相关转录因子-2（Runx2）的表达，但增加收缩蛋白的表达，包括 SM α-肌动蛋白（α-SMA）。此外，RTEF-1 抑制了在 VSMCs 钙化和成骨分化中起关键作用的 Wnt/β-连环蛋白通路。具体而言，RTEF-1 降低了 Wnt3a、p-β-连环蛋白（Ser675）、糖原合酶激酶-3β（GSK-3β）和 p-GSK-3β（Ser9）的水平，但增加了 p-β-连环蛋白（Ser33/37）的水平。此外，RTEF-1 增加了 p-β-连环蛋白（Ser33/37）与β-连环蛋白蛋白的比值，并降低了 p-GSK-3β（Ser9）与 GSK-3β 蛋白的比值。Wnt/β-连环蛋白信号激活剂 LiCl 观察到逆转 RTEF-1 过表达对β-GP 诱导的 VSMCs 钙化的保护作用。相应地，Wnt 拮抗剂 Dickkopf-1（Dkk1）减弱了 RTEF-1 缺失在β-GP 诱导的 VSMCs 钙化中的作用。总之，我们得出结论，RTEF-1 通过抑制 Wnt/β-连环蛋白信号通路来改善β-GP 诱导的 VSMCs 钙化和成骨分化。
