ALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease
Liu Ouyang, Xiaoyan Su, Wenxin Li, Liangqiu Tang, Mengbi Zhang, Yongjun Zhu, Changming Xie, Puhua Zhang, Jie Chen, Hui Huang
Liu Ouyang, Xiaoyan Su, Wenxin Li, Liangqiu Tang, Mengbi Zhang, Yongjun Zhu, Changming Xie, Puhua Zhang, Jie Chen, Hui Huang
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Research Article Cardiology Vascular biology

ALKBH1-demethylated DNA N6-methyladenine modification triggers vascular calcification via osteogenic reprogramming in chronic kidney disease

Abstract

Vascular calcification (VC) predicts cardiovascular morbidity and mortality in chronic kidney disease (CKD). To date, the underlying mechanisms remain unclear. We detected leukocyte DNA N6-methyladenine (6mA) levels in patients with CKD with or without aortic arch calcification. We used arteries from CKD mice infected with vascular smooth muscle cell–targeted (VSMC-targeted) adeno-associated virus encoding alkB homolog 1 (Alkbh1) gene or Alkbh1 shRNA to evaluate features of calcification. We identified that leukocyte 6mA levels were significantly reduced as the severity of VC increased in patients with CKD. Decreased 6mA demethylation resulted from the upregulation of ALKBH1. Here, ALKBH1 overexpression aggravated whereas its depletion blunted VC progression and osteogenic reprogramming in vivo and in vitro. Mechanistically, ALKBH1-demethylated DNA 6mA modification could facilitate the binding of octamer-binding transcription factor 4 (Oct4) to bone morphogenetic protein 2 (BMP2) promoter and activate BMP2 transcription. This resulted in osteogenic reprogramming of VSMCs and subsequent VC progression. Either BMP2 or Oct4 depletion alleviated the procalcifying effects of ALKBH1. This suggests that targeting ALKBH1 might be a therapeutic method to reduce the burden of VC in CKD.

Authors

Liu Ouyang, Xiaoyan Su, Wenxin Li, Liangqiu Tang, Mengbi Zhang, Yongjun Zhu, Changming Xie, Puhua Zhang, Jie Chen, Hui Huang

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Figure 8

ALKBH1-dependent 6mA demethylation promotes Oct4 binding to the BMP2 promoter and activates BMP2 transcription.

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ALKBH1-dependent 6mA demethylation promotes Oct4 binding to the BMP2 pro...
(A) Integrative genomics viewer plots showing the increasing 6mA peaks (selected one marked as ChIP1-3) in human BMP2 gene (hg19) region with ALKBH1 knockdown via shRNA lentiviral constructs. (B) ChIP-qPCR assay displaying the 6mA enrichment on the 3 BMP2 fragments in treated HASMCs (n = 4 per group). (C and D) Quantitative Western blot (C) and real-time PCR analysis of BMP2 expression (D) in HASMCs with scramble or OCT4 siRNA transfection under calcifying condition (n = 3 per group). (E) ChIP-qPCR assay with Oct4 or IgG antibody for the ChIP-1 enrichment in treated HASMCs (n = 4 per group). (F) Western blot analysis of Oct4 in HASMCs incubated with osteogenic medium after transfection with scrambled siRNA (si-Scr) or ALKBH1 siRNA (si-ALKBH1). (G) Logos of the standard Oct4 motif and schematic of human BMP2 promoter showing wide-type (WT) and deleted (Del) binding sites for Oct4 within the first 6mA peak. (H) Bar graphs representative of the luciferase activity analyzed in HASMCs after cotransfection with control Renilla luciferase plasmid and serial deletion constructs of BMP2 promoter-driven luciferase reporters containing WT or Del Oct4 site (n = 5 per group). (I) Relative promoter activities measured by dual-luciferase reporter assay in HASMCs, which pretreated with indicated siRNA and then infected with pGL3-Oct4-WT or pGL3-Oct4-Del under calcifying conditions (n = 4 per group). Statistical significance was assessed using 1-way ANOVA followed by Bonferroni’s test. All values are presented as mean ± SD. *P < 0.05.