Sestrin 3 Protects Against Diet-Induced Nonalcoholic Steatohepatitis in Mice Through Suppression of Transforming Growth Factor β Signal Transduction

Abstract

Sestrin 3 (Sesn3) belongs to the three-member sestrin protein family. Sestrins have been implicated in antioxidative stress, adenosine monophosphate-activated protein kinase and mammalian target of rapamycin signal transduction, and metabolic homeostasis. However, the role of Sesn3 in the development of nonalcoholic steatohepatitis (NASH) has not been previously studied. In this work, we generated Sesn3 whole-body knockout and liver-specific transgenic mice to investigate the hepatic function of Sesn3 in diet-induced NASH. With only 4 weeks of dietary treatment, Sesn3 knockout mice developed severe NASH phenotype as characterized by hepatic steatosis, inflammation, and fibrosis. Strikingly, after 8-week feeding with a NASH-inducing diet, Sesn3 transgenic mice were largely protected against NASH development. Transcriptomic analysis revealed that multiple extracellular matrix-related processes were up-regulated, including transforming growth factor β (TGF-β) signaling and collagen production. Further biochemical and cell biological analyses have illustrated a critical control of the TGF-β-mothers against decapentaplegic homolog (Smad) pathway by Sesn3 at the TGF-β receptor and Smad3 levels. First, Sesn3 inhibits the TGF-β receptor through an interaction with Smad7; second, Sesn3 directly inhibits the Smad3 function through protein-protein interaction and cytosolic retention. Conclusion: Sesn3 is a critical regulator of the extracellular matrix and hepatic fibrosis by suppression of TGF-β-Smad3 signaling.

Figure 1.. SESN3 protein expression in human NASH and characterization of Sesn3 knockout mice.

(A) Western blot analysis and quantification of SESN3 protein in the human liver samples from NASH patients and controls. Data are presented as mean ± SEM (n=3). *P < 0.05. (B, C) Immunohistochemistry and quantification of SESN3 expression in liver sections from human NASH patients (F0-F4). Data are presented as mean ± SEM (n=3). #P< 0.05, ##P< 0.01, ###P< 0.001 vs. F0. (D-F) Body weight, liver weight, and liver to body weight ratios in WT and Sesn3 KO male mice treated with different diets (chow groups: 5-6 months of age; special diet groups: 4-5.5 months of age). Data are presented as mean ± SEM (n=8/group). *P < 0.05 and ***P < 0.001 for Sesn3-KO vs. WT for the respective diet; ##P< 0.01 and ###P< 0.001 for the respective genotype treated with a high-fat diet vs. chow.

Figure 2.. Sesn3 deficient mice are more susceptible to diet-induced hepatic steatosis.

(A) H&E staining of liver sections of WT and Sesn3 KO male mice fed with HFCC, HFC, HFCA and HFD diets for 4 weeks. (B) Macroscopic images of WT and Sesn3 KO male mouse livers. (C-F) Liver TG (C), liver cholesterol (D), serum TG (E), and serum cholesterol (F) in WT and Sesn3 KO male mice fed with HFCC, HFC, HFCA, and HFD diets for 4 weeks (n=8/group; chow groups: 5–6 months of age; special diet groups: 4-5.5 months of age). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 for Sesn3 KO vs. WT on the respective diet; ##P< 0.01, ###P< 0.001 for the respective genotype fed with a high-fat diet vs. chow.

Figure 3.. Sesn3 deficiency exacerbates the diet-induced hepatic inflammation, fibrosis and injury.

(A) Immunohistochemistry analysis of MPO (for neutrophils) and F4/80 (for macrophages) and Sirius Red staining (for fibrosis) of liver sections of WT and Sesn3 KO male mice fed with HFCC, HFC, HFCA, and HFD diets for 4 weeks. (B) Quantitative data for the MPO-positive signals in Panel A. (C) Quantitative data for the F4/80-positive signals in Panel A. (D) Quantitative data of the Sirius Red-positive signals in Panel A. (E) Serum ALT levels in WT and Sesn3 KO male mice fed with HFCC, HFC, HFCA, and HFD diets for 4 weeks (n=6/group; chow group: 5-6 months of age; special diet groups: 4-5.5 months of age). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 for Sesn3 KO vs. WT for the respective diet; #P< 0.05, ##P< 0.01, ###P< 0.001 for the respective genotype fed with a high-fat diet vs. chow.

Figure 4.. Liver-specific Sesn3 overexpression protects against diet-induced NASH.

(A) Immunoblot analysis of Sesn3 in the liver and heart tissues of WT and liver-specific Sesn3 transgenic male mice (Tg). (B) H&E staining, immunohistochemistry analysis of MPO and F4/80, and Sirius Red staining of liver sections of WT and Tg male mice fed with the HFCC diet for 8 weeks. (C) Quantitative data for lipid droplet area, MPO positive signals, F4/80 positive signals, and Sirius Red positive signals in Panel B. (D) serum ALT level in WT and Tg male mice fed with the HFCC diet for 8 weeks (n=6/group; 6-9 months of age). (E) Liver TG and cholesterol measurements in WT and Tg male mice fed with the HFCC diet for 8 weeks (n=6/group; 6-9 months of age). (F) Serum TG and cholesterol measurements in WT and Tg male mice fed with the HFCC diet for 8 weeks (n=6/group; 6-9 months of age). Data are presented as mean ± SEM. ***P < 0.001 for Tg vs. WT for the respective diet; #P< 0.05, ###P< 0.001 for the respective genotype on HFCC vs. chow.

Figure 5.. Transcriptomic analysis reveals upregulated fibrosis genes in the liver of Sesn3 KO mice.

(A) A volcano plot representation of significantly up- and down-regulated genes in the liver of Sesn3 KO mice compared to WT mice fed with HFCC diets for 4 weeks (n=3 males/group). (B) Gene ontology analysis of significantly upregulated genes in top 11 biological processes. (C) Gene ontology analysis of significantly downregulated genes in top 10 biological processes. (D) Heatmap presentation of significantly up-regulated fibrosis related genes in the liver of Sesn3 KO mice compared to WT mice fed with an HFCC diet for 4 weeks.

Figure 6.. Sesn3 inhibits hepatic inflammation and fibrosis.

(A) Real-time PCR analysis of fibrosis- and inflammation- related genes in the liver of WT and Sesn3 KO male mice (n = 4/group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 for Sesn3 KO vs. WT; #P< 0.05, ##P< 0.01 and ###P< 0.001 for HFCC vs. chow for the same genotype. (B) Real-time PCR analysis of fibrosis- and inflammation- related genes in the livers of WT and Tg mice (n = 4/group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 for Tg vs. WT; #P< 0.05, ##P< 0.01 and ###P< 0.001 for HFCC vs. chow for the same genotype. (C) Western blot analysis and quantification of total Smad3 and p-Smad3 (phosphorylated) in the livers of WT and Sesn3 KO mice. Data are presented as mean ± SEM. **P < 0.01 for Sesn3-KO vs. WT; ##P< 0.01 and ###P< 0.001 for HFCC vs. chow for the same genotype. (D, E) LX-2 cells were transduced with adenoviral AdGFP or AdSesn3 in the absence or presence of TGFβ1 (5 ng/ml) for 3 or 24 hrs. Western blot analysis and quantification (D) were performed for total Smad3 and p-Smad3; real-time PCR analysis (E) was performed for fibrosis-related genes. Data are presented as mean ± SEM. *P < 0.05 and ***P < 0.001 for AdSesn3 vs AdGFP; #P<0.05, ##P< 0.001, ###P< 0.001 for TGFβ1 treatment vs. Control.

Figure 7.. Sesn3 interacts with Smad3 and inhibits Smad3 nuclear translocation.

(A-C) LX-2 cells were transfected with Sesn3-HA or vector plasmid for 24 hrs. Immunofluorescence microscopy of Sesn3 and Smad3 in absence of TGFβ1 (A). Immunofluorescence microscopy of Sesn3 and Smad3 after treatment with TGFβ1 (5 ng/ml) for 3 hrs (B). Immunofluorescence microscopy of Sesn3 and p-Smad3 after treatment with TGFβ1 (5 ng/ml) for 3 hrs (C). (D) Co-IP analysis of interactions between Smad3 or p-Smad3 with Sesn3 in LX-2 cells with or without TGFβ1 (5 ng/ml) treatment for 3 hrs.

Figure 8.. Sesn3 interacts with Smad7 and inhibits TGFβ receptors.

(A-C) Co-IP analysis of interactions between Sesn3 and Smad7 or TGFβR1 in LX-2 cells after treatment with TGFβ1 (5 ng/ml) for 3 hrs. (D, E) Western blot analysis of Smad7 and TGFβR1 in the liver of WT, Sesn3 KO, or Tg mice fed with an HFCC diet. Data are presented as mean ± SEM. *P < 0.05 for Sesn3 KO or Tg vs. WT.