Abstract 1463: Impaired reciprocal regulation between SIRT6 and TGF-β signaling as a potential mechanism for development and progression of fatty liver
Authors: Ohshiro Kazufumi , Xiang Xiyan , Bernstein David , Crawford James M. , Mishra Bibhuti , Latham Patricia S. , Gough Nancy R. , Rao Shuyun , Mishra Lopa 
Published: June 2022

Background: Non-alcoholic fatty liver disease (NAFLD) is associated with an increased risk of fibrotic liver disease and hepatocellular carcinoma (HCC). Dysregulated TGF-β signaling and loss of SIRT6 activity are implicated in fatty liver disease. SIRT6 limits fibrosis by inhibiting SMAD3 activity and limits de novo lipogenesis by inhibiting SREBP1 and SREBP2 activity. We hypothesized that altered reciprocal regulation between TGF-β signaling and SIRT6 contributes to NAFLD and its progression. The goal of this study was to identify regulatory crosstalk between SIRT6 and SMAD3 and SPTBN1, a regulator of SMAD3 activity.

Methods: We used bioinformatics and chromatin immunoprecipitation (ChIP) with HepG2 cells to examine the binding of SMAD3 or CTCF to the SIRT6 gene in the presence or absence of TGF-β. Using cultured cells, we examined the effect of altering SPTBN1 or SMAD3 on SIRT6 abundance, and of altering SIRT6 on SPTBN1 and SMAD3 abundance. We examined liver phenotypes of SPTBN1+/- mice fed either a normal chow diet or a high-fat diet (HFD) and monitored body weight and serum total cholesterol and triglyceride concentrations, as well as analyzed liver tissue for SIRT6 abundance.

Results: We identified two consensus SMAD-binding elements and two consensus CTCF binding sites in the SIRT6 promoter and showed by ChIP that TGF-β stimulated SMAD3 and CTCF binding to the promoter region of SIRT6. We found that deficiency in SMAD3 or SPTBN1 reduced SIRT6 mRNA and protein abundance. Overexpression of SIRT6 reduced expression of selected TGF-β-induced genes. Knockdown of SIRT6 increased SPTBN1 but not SMAD3 abundance and overexpression of SIRT6 reduced only SPTBN1 abundance. We found that fatty liver and associated metabolic changes induced by HFD is worse in SPTBN1+/- mice than in control mice. Furthermore, this condition was associated with reduced SIRT6 protein abundance in the liver.

Conclusions: We found a reciprocal regulatory mechanism involving SPTBN1 through which SIRT6 can influence TGF-β signaling and identified SIRT6 as a target of TGF-β-SMAD3 signaling. The development of liver steatosis with reduced SIRT6 in SPTBN1+/- mice suggested that impaired induction of SIRT6 contributes to the severe liver phenotype, which resembles nonalcoholic steatohepatitis (NASH). Future investigation may yield opportunities to intervene and prevent NAFLD from progressing to NASH and thus reduce the risk of HCC.

Citation Format: Kazufumi Ohshiro, Xiyan Xiang, David Bernstein, James M. Crawford, Bibhuti Mishra, Patricia S. Latham, Nancy R. Gough, Shuyun Rao, Lopa Mishra. Impaired reciprocal regulation between SIRT6 and TGF-β signaling as a potential mechanism for development and progression of fatty liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1463.