SIRT6 protects vascular smooth muscle cells from osteogenic transdifferentiation via Runx2 in chronic kidney disease
Wenxin Li, … , Baohua Liu, Hui Huang
Wenxin Li, … , Baohua Liu, Hui Huang
Published November 18, 2021
Citation Information: J Clin Invest. 2022;132(1):e150051. https://doi.org/10.1172/JCI150051.
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Research Article Cell biology Vascular biology

SIRT6 protects vascular smooth muscle cells from osteogenic transdifferentiation via Runx2 in chronic kidney disease

Abstract

Vascular calcification (VC) is regarded as an important pathological change lacking effective treatment and associated with high mortality. Sirtuin 6 (SIRT6) is a member of the Sirtuin family, a class III histone deacetylase and a key epigenetic regulator. SIRT6 has a protective role in patients with chronic kidney disease (CKD). However, the exact role and molecular mechanism of SIRT6 in VC in patients with CKD remain unclear. Here, we demonstrated that SIRT6 was markedly downregulated in peripheral blood mononuclear cells (PBMCs) and in the radial artery tissue of patients with CKD with VC. SIRT6-transgenic (SIRT6-Tg) mice showed alleviated VC, while vascular smooth muscle cell–specific (VSMC-specific) SIRT6 knocked-down mice showed severe VC in CKD. SIRT6 suppressed the osteogenic transdifferentiation of VSMCs via regulation of runt-related transcription factor 2 (Runx2). Coimmunoprecipitation (co-IP) and immunoprecipitation (IP) assays confirmed that SIRT6 bound to Runx2. Moreover, Runx2 was deacetylated by SIRT6 and further promoted nuclear export via exportin 1 (XPO1), which in turn caused degradation of Runx2 through the ubiquitin-proteasome system. These results demonstrated that SIRT6 prevented VC by suppressing the osteogenic transdifferentiation of VSMCs, and as such targeting SIRT6 may be an appealing therapeutic target for VC in CKD.

Authors

Wenxin Li, Weijing Feng, Xiaoyan Su, Dongling Luo, Zhibing Li, Yongqiao Zhou, Yongjun Zhu, Mengbi Zhang, Jie Chen, Baohua Liu, Hui Huang

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Figure 6

SIRT6 mediates Runx2 nuclear export depending on XPO1.

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SIRT6 mediates Runx2 nuclear export depending on XPO1. (A) Runx2 IF stai...
(A) Runx2 IF staining was performed in abdominal arteries. Scale bar: 50 μm. Statistical significance was assessed using 2-tailed t tests, *P < 0.05. (B) VSMCs were incubated with Pi for 7 days. IF staining was performed for Runx2. Scale bars: 50 μm. (C) VSMCs were incubated with Pi for 7 days. Cells were harvested and immunoblotted for the indicated proteins. (D) SIRT6-Tg VSMCs were incubated with Pi for 7 days after posttransfection of siSIRT6. Cells were harvested and immunoblotted for the indicated proteins. (E) SIRT6-Tg VSMCs were incubated with Pi together with nicotinamide for 7 days. Cells were harvested and immunoblotted for the indicated proteins. (F) SIRT6-Tg VSMCs were transfected with shRNA targeting XPO1, XPO4, XPO7, or their vector control, and then incubated with Pi for 7 days after transfection. Nuclear extracts were immunoblotted for Runx2. (G and H) SIRT6-Tg VSMCs were incubated with Pi together with Leptomycin A (0.5 nM) for 7 days. Cells were harvested and immunoblotted for the indicated proteins (G). IF staining was performed for Runx2. Scale bars: 50 μm (H). (I) Anti-XPO1 IP followed by Western blot with anti-Runx2 or anti-XPO1 antibody in SIRT6-Tg VSMCs after treatment with Pi for 7 days. Anti-rabbit IgG IP was used as negative control. (J) Anti-Runx2 IP in SIRT6-Tg VSMCs after treatment with Pi for 7 days. Western blot was carried out with anti-XPO1 or anti-Runx2 antibody. Anti-mouse IgG IP was used as negative control. (K) SIRT6-Tg VSMCs were incubated with Pi together with Leptomycin A for 7 days, and then CHX (0.2 mM) was added for the indicated times before harvest, followed by immunoblotting for the indicated proteins. (L) Curve shows the stability of Runx2 and was assessed using 2-way ANOVA. Pi treatment is 3.0 mM. All the above experimental processing was duplicated 3 times.