Study On RTEF-1 Inhibiting Vascular Smooth Muscle Cell Calcification Through Regulating Wnt/β-catenin Signaling Pathway

[Objective] To investigate vascular smooth muscle cells(VSMCs)calcification induced by β-glycerophosphoricacid(β-GP)and explore the impact of Related Transcriptional Enhancer Factor(RTEF-1)on VSMCs calcification through regulating Wnt/β-catenin signaling pathway.[Methods] Human aortic SMCs were cultured in DMEM,supplemented with 10 m M β-GP for 12 days to induce VSMCs calcification.Calcification assays,bone-related proteins,SM contractile proteins,and RTEF-1 gene expression were detected in human aortic SMCs treated with β-GP or control.To explore the effect of RTEF-1 on Wnt/β-catenin signaling pathway and VSMCs calcification in vitro,β-GP-induced human aortic SMCs were treated with Full-length RTEF-1 c DNA(RTEF-1 o/e)and small interfering RNA against RTEF-1(RTEF-1 si RNA),which subsequently incubated with Wnt/β-catenin signaling pathway activator(Li Cl)and inhibitor(Dkk1),respectively.The deposition of calcium phosphate was examined by Alizarin Red S staining.The levels of intracellular calcium were examined by the methylthymol blue colorimetry method.The activity of ALP was examined by ALP assay kit.Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine the messenger RNA and protein levels of Osteocalcin,Runx2,α-SMA,Wnt-3a,GSK-β,p-GSK-β,β-catenin,and p-β-catenin(Ser675).[Results] β-GP-treated human aortic SMCs markedly increased osteocalcin and Runx2 expressions and enhanced intracellular calcium levels at 6 days,and reduced α-SMA expression and increased ALP activity as well as calcified nodules formation at 9 days.The calcium levels,the activity of ALP,and the expressions of osteocalcin and Runx2 in β-GP group at 9 or 12 days were significantly higher than that at 3 days.Meanwhile,the levels of RTEF-1 m RNA and protein were decreased in human aortic SMCs after β-GP treatment at 12 days compared with the control group,and the expression of RTEF-1 in β-GP group at 6,9,or 12 days were far lower than that at 1 day.Furthermore,RTEF-1 o/e decreased the deposition of calcium phosphate,the levels of intracellular calcium,the activity of ALP,and the expressions of osteocalcin and Runx2,increased α-SMA expression,and decreased the levels of Wnt-3a,GSK-3β,p-GSK-3β,and p-β-catenin/β-catenin under high phosphate conditions.RTEF-1 si RNA had the opposite effects.Whereas stimulation of Li Cl weakened the inhibitory effects of RTEF-1 on β-GP-induced calcification deposition,increased ALP activity,and enhanced the expressions of osteocalcin and Runx2.Similarly,pretreatment of Dkk1 partially reversed the above changes of β-GP-induced RTEF-1 si RNA group.[Conclusion] In vitro β-GP can induce the calcification and osteoblastic differentiation of VSMCs,and these changes become more pronounced over time.The levels of RTEF-1 m RNA and protein are reduced in calcifying VSMCs.RTEF-1 ameliorates high phosphateinduced VSMCs calcification through regulating Wnt/β-catenin signaling pathway.