Male-Specific Activation of Lysine Demethylases 5B and 5C Mediates Alcohol-Induced Liver Injury and Hepatocyte Dedifferentiation

Abstract

Alcohol-associated liver disease (ALD) is a major cause of alcohol-related mortality. Sex differences in sensitivity to ALD are well described, but these are often disregarded in studies of ALD development. We aimed to define sex-specific pathways in liver exposed to alcohol. Mice were fed the Lieber-DeCarli alcohol liquid diet or a combination of a high-fat diet with alcohol in water. Single-cell RNA sequencing (scRNA-Seq) was performed on liver cells from male and female mice. Mice were treated with adeno-associated virus (AAV)-short hairpin (sh)Control or AAV-sh lysine demethylase 5b (shKdm5b) and/or AAV-shKdm5c vectors. Changes after Kdm5b/5c knockdown were assessed by RNA-Seq and histone H3 lysine K4 (H3K4)me3 chromatin immunoprecipitation-Seq analysis. Using scRNA-Seq analysis, we found several sex-specific pathways induced by alcohol, including pathways related to lipid metabolism and hepatocyte differentiation. Bioinformatic analysis suggested that two epigenetic regulators, H3K4-specific lysine demethylases KDM5B and KDM5C, contribute to sex differences in alcohol effects. We found that in alcohol-fed male mice, KDM5B and KDM5C are involved in hepatocyte nuclear factor 4 alpha (Hnf4a) down-regulation, hepatocyte dedifferentiation, and an increase in fatty acid synthesis. This effect is mediated by alcohol-induced KDM5B and KDM5C recruitment to Hnf4a and other gene promoters in male but not in female mice. Kdm5b and Kdm5c knockdown or KDM5-inhibitor treatment prevented alcohol-induced lipid accumulation and restored levels of Hnf4a and other hepatocyte differentiation genes in male mice. In addition, Kdm5b knockdown prevented hepatocellular carcinoma development in male mice by up-regulating Hnf4a and decreasing tumor cell proliferation. Conclusion: Alcohol specifically activates KDM5 demethylases in male mice to promote alcohol-induced hepatocyte dedifferentiation and tumor development.