RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/β-Catenin Signaling Pathway

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.

Neuromedin B modulates phosphate-induced vascular calcification

“…Wnt/β-cate-nin signaling could play a crucial role in the regulation of high phosphate-induced calcification of VSMCs via the induction of osteogenic genes such as Runx2 ( 15 ). The present study and other reports ( 27 , 28 ) demonstrated that high phosphate induces two active forms of β-catenin, β-catenin dephosphorylated at Ser37/Thr41 via the inactivation of GSK-3β through Ser9-phosphorylation and β-catenin phosphorylated at Ser675, resulting in promotion its stability and transcriptional activity. These changes were completely reversed by treatment with NMB siRNA or PD168368 ( Fig.…”

Hsa_circRNA_0008028 Deficiency Ameliorates High Glucose-Induced Proliferation, Calcification, and Autophagy of Vascular Smooth Muscle Cells via miR-182-5p/TRIB3 Axis

“…To further verify the effect of hsa_circRNA_0008028 on cell calcification and osteogenic differentiation, HG-treated VSMCs were coincubated with 10 mmol/L β -glycerophosphate ( β -GP) for 12 days after transfection [ 21 ]. As expected, obvious calcification, characterized by increased alkaline phosphatase (ALP) activity ( Figure 2(e) ) can be observed in cells in the HG group and the HG and si-NC group.…”

“…Before the subsequent experiments, all cells were kept quiescent by starvation for 24 hours and then treated with 5.5 mmol/L D-glucose (normal glucose (NG)) or 30 mmol/L D-glucose (HG) for 48 hours. All VSMCs to be tested for calcification and osteogenic differentiation were treated with HG and coincubated with 10 mmol/L β -glycerophosphate ( β -GP, Sigma-Aldrich, USA) for 12 days after siRNA transfection or overexpression [ 21 ]. VSMCs exposed to 30 mmol/L mannitol (MA, Aladdin, China) was chosen as the negative control.…”

Identification of potential biomarkers of vascular calcification using bioinformatics analysis and validation in vivo

“…The transcription factor XBP1, synergistically with Runx2, is involved in VC due to oxidative stress ( Liberman et al, 2011 ). TEAD4 is a nuclear transcription factor that inhibits vascular calcification by regulating the Wnt/β-Catenin signaling pathway ( Cong et al, 2021 ). By intervening in VC hub genes, it may be a new direction for individualized genomic therapy of VC.…”