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.

Alcohol‐associated fibrosis in females is mediated by female‐specific activation of lysine demethylases KDM5B and KDM5C

“…[ 27 ] We found that males and females dramatically differ in their response to alcohol, and we identified KDM5B and KDM5C as regulators of male/female differences, specifically male‐specific alcohol response in hepatocytes. [ 27 ]…”

“…Recently we performed scRNA‐seq analysis to identify sex‐specific pathways affected by alcohol in individual subsets of liver cells. [ 27 ] We found that males and females dramatically differ in their response to alcohol, and we identified KDM5B and KDM5C as regulators of male/female differences, specifically male‐specific alcohol response in hepatocytes. [ 27 ]…”

“…To determine which cell types contributed to gene‐expression changes after Kdm5b/Kdm5c knockdown, we assessed gene expression of fibrosis and inflammation‐associated genes in individual cell clusters using the scRNA‐seq data set we previously obtained. [ 27 ] We reclustered cells from females fed alcohol diet and analyzed gene expression in individual clusters. We found that top differentially regulated fibrosis and inflammation associated genes such as Col3a1 , Itgav , Cfh , C1s1 , and Gabarapl1 were predominantly expressed in HSCs, whereas a few of the others were more specific to macrophages ( Tnfrsf1b ) or endothelial cells ( Lifr ) (Figure 3B ).…”

“…Single‐cell RNA sequencing (scRNA‐seq) analysis was performed as previously described. [ 27 ] Live hepatocytes and NPC cells were purified using the dead cell removal kit (MiltenyiBiotec). Liver cells were immediately used to generate barcoded complementary DNA cDNA libraries using a 10× Genomics Chromium platform (10× Genomics, Pleasanton, CA) with a total input of 10,000 cells per condition.…”

“…[ 26 ] Recently we have shown that they regulate alcohol response in the liver in a sex‐specific way. [ 27 ]…”