Abstract
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.
Highlights
Vascular calcification (VC) is a highly prevalent pathophysiological process that has been associated with aging, atherothrombotic cardiovascular disease, diabetes mellitus, and Jingjing Cong and Bei Cheng have contributed as co-first authors.1 3 Vol.:(0123456789)Related transcriptional enhancer factor (RTEF-1), named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a key member of the TEAD family
The results showed that β-GP-increased calcium content, as well as Runx2 and Osteocalcin mRNA levels, became detectable after culture of 6 days, whereas β-GPenhanced alkaline phosphatase (ALP) activity became detectable after culture of 9 days, indicating that ALP activity may be not suitable as an early indicator for predicting vascular smooth muscle cells (VSMCs) calcification
We revealed that RTEF-1 was present in low levels in calcified VSMCs, this was accompanied by a decrease in α-SMA expression and increased levels of osteogenic markers including ALP, osteocalcin, and Runx2
Summary
Vascular calcification (VC) is a highly prevalent pathophysiological process that has been associated with aging, atherothrombotic cardiovascular disease, diabetes mellitus, and Jingjing Cong and Bei Cheng have contributed as co-first authors.1 3 Vol.:(0123456789)Related transcriptional enhancer factor (RTEF-1), named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a key member of the TEAD family. TEAD transcription factors are downstream effectors of the Hippo pathway, and they regulate cell proliferation, tissue regeneration, and metastasis [5]. The other three members of the TEAD family, such as TEAD1, TEAD2, and TEAD3, have the same domain structure: a DNA-binding TEA/ATTS domain in the N-terminus, but show tissue-specific expression, indicating that they have both common functions (regulating cell proliferation and contact inhibition) and distinct functions (heart development, neural development, and trophectoderm lineage determination) [6]. RTEF-1 is highly expressed in skeletal muscle; initial studies focused on its role in blastocyst formation and reported that TEAD4 is required for specification of the trophectoderm lineage in preimplantation embryos [7, 8]. The role and mechanism of RTEF-1 in VC, in calcification of VSMCs, are poorly understood
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