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 5

SIRT6 promotes Runx2 degradation via the ubiquitin-proteasome system.

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SIRT6 promotes Runx2 degradation via the ubiquitin-proteasome system. (A...
(A) WT and SIRT6-Tg VSMCs were treated with Pi (3.0 mM) for 7 days and incubated with the protein translation inhibitor CHX (0.2 mM) for the indicated times before harvest, followed by immunoblotting with the anti-Runx2 antibody and anti-GAPDH anti-body. The curve shows the stability of Runx2 protein. (B and C) SIRT6 was decreased in primary VSMCs via siRNA (B) or specific inhibitor (C) together with Pi (3.0 mM) incubation for 7 days. The protein translation inhibitor CHX (0.2 mM) was added for indicated times before harvest, followed by immunoblotting with the anti-Runx2 antibody and anti-GAPDH antibody. The curve shows the stability of Runx2 protein. (D and E) SIRT6-Tg VSMCs were incubated with Pi (3.0 mM) together with the leupeptin (1.5 μM) (D) or MG132 (10 μM) (E) for 7 days, and then the protein translation inhibitor CHX (0.2 mM) was added for the indicated times before harvest, followed by immunoblotting with the anti-Runx2 antibody and anti-GAPDH antibody. The curve shows the stability of Runx2 protein. (F) WT and SIRT6-Tg VSMC lysates were immunoprecipitated with anti-Runx2 antibody and immunoblotted with anti-ubiquitin (anti-Ub) antibody. (G) HEK-293T cells were transfected with His-Ub together with HA-Runx2 plasmid, flag-SIRT6 plasmid, or both. The anti-HA IP was followed by Western blot with anti-Ub antibody and anti-HA antibody. (H) SIRT6-Tg VSMCs were pretransfected with siSIRT6 or siNC together with Pi (3.0 mM) for 7 days, and OSS-128167 or DMSO were incubated with Pi (3.0 mM) for 7 days. The cell lysates were immunoprecipitated with anti-Runx2 antibody and immunoblotted with anti-Ub antibody and anti-Runx2 antibody. Statistical significance was assessed using 2-way ANOVA (A–E). All the above experimental processing was duplicated 3 times.