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AGE/RAGE promotes thecalcification of human aortic smooth muscle cells via the Wnt/?-catenin axis.

ABSTRACT: OBJECTIVE:The present study aims to investigate whether RAGE promotes the calcification of human arterial smooth muscle cells (HASMCs) and determine the relationshipbetween RAGE and the Wnt/?-catenin signaling pathway in this process. METHOD:In this study,there were four groups, namelythe blank control group, the non-transfection group, the empty vector group, and the RAGE transfection group.Cells were co-cultured with 10 mmol/L ?-glycerophosphoric acid, pyruvate and 20 mg/L AGE. The expression of osteogenic proteins in each group before and after the intervention wasdetected using Western blotting. Short interfering RNA (siRNA) targeting ?-catenin was used toinhibitthe expression of ?-catenin. HASMCs cultured under normal conditions were usedas the blank control. RESULTS:(1) High RAGE expression was successfully induced in HASMCs according to the results of GFP detection, flow cytometry and Western blotting. (2) Compared with the blank control group, non-transfection group and empty vector group, RAGE transfection enhanced the calcification of cells when incubated with calcification medium plus AGE. (3) The expression of RAGE, ?-catenin, OPG and Cbfa1 proteins in the blank control group, empty vector group and RAGE transfection group wasnot significantly enhanced after intervention. However, expression of the proteins in the RAGE transfection group was much higher than those of the other groups. (4) Compared with the RAGE transfection group and control siRNA group, the cells transfected with ?-catenin siRNA and cultured with interventional drugs showed significant inhibition of the expression of the downstream Cbfa1 and OPG genes. CONCLUSION:Increased expression of RAGE promoted calcification in HASMCs and up regulated the ?-catenin, OPG and Cbfa1 genes. RAGE may activate the downstream genes via the Wnt?-catenin pathway, thereby promoting HASMC differentiation into osteogenic cells and calcification.

SUBMITTER: Liu Y

PROVIDER: S-EPMC5126310 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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AGE/RAGE promotes thecalcification of human aortic smooth muscle cells via the Wnt/β-catenin axis.

Liu Yong Y Wang Wei-Ming WM Zhang Xue-Lin XL He Hu-Qiang HQ Sun Xiao-Lei XL Zeng Hong H Xu Xiong-Fei XF Huang Liang L Zhu Zhi Z Zhang Lei L Zhou Xiang-Yu XY He Yan-Zheng YZ

American journal of translational research 20161115 11

<h4>Objective</h4>The present study aims to investigate whether RAGE promotes the calcification of human arterial smooth muscle cells (HASMCs) and determine the relationshipbetween RAGE and the Wnt/β-catenin signaling pathway in this process.<h4>Method</h4>In this study,there were four groups, namelythe blank control group, the non-transfection group, the empty vector group, and the RAGE transfection group.Cells were co-cultured with 10 mmol/L β-glycerophosphoric acid, pyruvate and 20 mg/L AGE. ...[more]

PMID: 27904668

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Project description: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.

Dataset Information

AGE/RAGE promotes thecalcification of human aortic smooth muscle cells via the Wnt/?-catenin axis.

Publications

AGE/RAGE promotes thecalcification of human aortic smooth muscle cells via the Wnt/β-catenin axis.

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