The role of SHP/REV-ERBα/CYP4A axis in the pathogenesis of alcohol-associated liver disease
Zhihong Yang, Rana V. Smalling, Yi Huang, Yanchao Jiang, Praveen Kusumanchi, Will Bogaert, Li Wang, Don A. Delker, Nicholas J. Skill, Sen Han, Ting Zhang, Jing Ma, Nazmul Huda, Suthat Liangpunsakul
Zhihong Yang, Rana V. Smalling, Yi Huang, Yanchao Jiang, Praveen Kusumanchi, Will Bogaert, Li Wang, Don A. Delker, Nicholas J. Skill, Sen Han, Ting Zhang, Jing Ma, Nazmul Huda, Suthat Liangpunsakul
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Research Article Hepatology

The role of SHP/REV-ERBα/CYP4A axis in the pathogenesis of alcohol-associated liver disease

Abstract

Alcohol-associated liver disease (ALD) represents a spectrum of histopathological changes, including alcoholic steatosis, steatohepatitis, and cirrhosis. One of the early responses to excessive alcohol consumption is lipid accumulation in the hepatocytes. Lipid ω-hydroxylation of medium- and long-chain fatty acid metabolized by the cytochrome P450 4A (CYP4A) family is an alternative pathway for fatty acid metabolism. The molecular mechanisms of CYP4A in ALD pathogenesis have not been elucidated. In this study, WT and Shp−/− mice were fed with a modified ethanol-binge, National Institute on Alcohol Abuse and Alcoholism model (10 days of ethanol feeding plus single binge). Liver tissues were collected every 6 hours for 24 hours and analyzed using RNA-Seq. The effects of REV-ERBα agonist (SR9009, 100 mg/kg/d) or CYP4A antagonist (HET0016, 5 mg/kg/d) in ethanol-fed mice were also evaluated. We found that hepatic Cyp4a10 and Cyp4a14 expression were significantly upregulated in WT mice, but not in Shp−/− mice, fed with ethanol. ChIP quantitative PCR and promoter assay revealed that REV-ERBα is the transcriptional repressor of Cyp4a10 and Cyp4a14. Rev-Erbα−/− hepatocytes had a marked induction of both Cyp4a genes and lipid accumulation. REV-ERBα agonist SR9009 or CYP4A antagonist HET0016 attenuated Cyp4a induction by ethanol and prevented alcohol-induced steatosis. Here, we have identified a role for the SHP/REV-ERBα/CYP4A axis in the pathogenesis of ALD. Our data also suggest REV-ERBα or CYP4A as the potential therapeutic targets for ALD.

Authors

Zhihong Yang, Rana V. Smalling, Yi Huang, Yanchao Jiang, Praveen Kusumanchi, Will Bogaert, Li Wang, Don A. Delker, Nicholas J. Skill, Sen Han, Ting Zhang, Jing Ma, Nazmul Huda, Suthat Liangpunsakul

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Figure 2

REV-ERBα is the potential transcriptional regulator of Cyp4a10 and Cyp4a14.

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REV-ERBα is the potential transcriptional regulator of Cyp4a10 and Cyp4a...
(A) Proximity ligation assay (PLA, red) demonstrated the interaction between GFP-SHP (green) and FLAG-REV-ERBα proteins in HEK293T cells. Scale bar: 100 μm. (B) Three RNA-Seq data sets, Rev-Erbα−/− versus WT (GSE59486, GSE59460), Shp−/− versus WT (GSE43893), and WE versus WC (ZT 12), were integrated to identify overlapping genes, which were coregulated by each pathway. Venn diagram indicated 9 overlapping genes, including both Cyp4a10 and Cyp4a14 (red). WC, WT control; WE, WT treated with ethanol. (C) ChIP-Seq (GSE67962) revealed the location of REV-ERBα binding peaks on Cyp4a10 and Cyp4a14 promoters in mouse livers (red arrows). The mutated REV-ERBα DNA-binding domain (DBD-mut) served as negative controls. The binding peak indicated by the purple arrow could be the binding independent from REV-ERBα DNA-binding domain. (D) The diagram of Cyp4a10 and Cyp4a14 promoter with distance from transcription start site (TSS) and REV-ERBα binding sites. Black arrows indicated location of the ChIP primers. Mut, mutant on the REV-ERBα binding site in the promoter constructs. (E) ChIP assay with anti-REV-ERBα Ab or IgG (negative control) from mouse liver at ZT 12. WT-Con, WT control; WT-Etoh, WT-ethanol. ****P < 0.0001 versus IgG; ###P < 0.001, ####P < 0.0001 versus WT-Con. One-way ANOVA. (F) Luciferase assay with Cyp4a10 or Cyp4a14 promoter and cotransfected with 0, 50, or 100 ng/well of REV-ERBα plasmids in 24-well plates. *P < 0.05, **P < 0.01 versus control. One-way ANOVA. (G) Luciferase assay with indicated promoters and cotransfected with 0, 50, 100 ng/well of pLKO-shRNA-REV-ERBα plasmids (sh-Rev-Erbα). *P < 0.05, **P < 0.01, ***P < 0.001 versus control. One-way ANOVA. (H) Luciferase assay with indicated promoters and cotransfected with 100 ng/well pcDNA3 (p) or REV-ERBα (Rev100) plasmids. **P < 0.01 versus pcDNA3. Two-tailed Student’s t test. (I) Luciferase assay with indicated promoters and plasmids (100 ng/each). **P < 0.01, ***P < 0.001 versus control. One-way ANOVA.