The aging of the population has led to an annual increase in the incidence of vascular calcification (VC). Specific protein 1 (Sp1) is a transcriptional activator that serves an important role in VC. The deacetylation of transcription factors represses their binding to the promoters of downstream genes, thereby causing their downregulation. The present study aimed to investigate the role of deacetylated Sp1 in the development of VC. In the present study, western blotting and immunoprecipitation (IP) were performed to detect the protein levels of acetylated Sp1. Western blotting and immunofluorescence staining were used to analyze phenotypic switching in vascular smooth muscle cells (VSMCs). Alizarin red S, alkaline phosphatase (ALP) activity and calcium content assays were used to assess calcium deposition in VSMCs. Western blotting, flow cytometry, TUNEL staining and caspase3 activity assay were used to evaluate apoptosis of VSMCs. Chromatin immunoprecipitation (ChIP) assay was used to detect Sp1 binding to the BMP2 promoter. The results indicated that, in a β-glycerophosphate (β-GP)-induced VSMC calcification model, the level of acetylated Sp1 was increased. Western blotting and immunofluorescence staining results showed that, compared with the Sp1 overexpression group (Sp1-WT), deacetylated Sp1 (Sp1-K704A) downregulated the expression of osteogenic markers runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and upregulated the expression of contraction marker α-smooth muscle actin (α-SMA) and calponin 1. In addition, deacetylated Sp1 also reduced the ALP activity and calcium content of calcified VSMCs, and the Alizarin red S assay revealed that the calcium crystallization of Sp1-K704A group was markedly decreased. Western blotting, flow cytometry, TUNEL staining and caspase-3 activity assay were detected to indicate that the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein ratio was increased, and caspase-3 activity and the apoptotic rate of VSMCs were decreased, in the Sp1-K704A group, as compared with the Sp1-WT group. ChIP assay revealed that Sp1 binding to the BMP2 promoter was downregulated in the Sp1-K704A group, compared with that in theSp1-WT group. In conclusion, a deacetylated mutant of Sp1 decreased Sp1 binding to the BMP2 promoter, thus decreasing apoptosis, phenotypic switching and calcium deposition in calcified VSMCs. This finding may indicate potential therapeutic targets for VC.
Keywords: BMP2; Sp1; apoptosis; deacetylation; phenotype switching; vascular calcification.
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