Rtef-1 Inhibits Vascular Smooth Muscle Cell Calcification Through Regulating Wnt/Beta-Catenin Signaling Pathway

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 beta-glycerophosphate (beta-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 alpha-actin (alpha-SMA). Additionally, RTEF-1 inhibited beta-GP-activated Wnt/beta-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-beta-catenin (Ser675), glycogen synthase kinase-3 beta (GSK-3 beta), and p-GSK-3 beta (Ser9), but increased the levels of p-beta-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-beta-catenin (Ser33/37) to beta-catenin proteins and decreased the ratio of p-GSK-3 beta (Ser9) to GSK-3 beta protein. LiCl, a Wnt/beta-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by beta-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in beta-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated beta-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/beta-catenin signaling pathway.