Alcohol directly stimulates epigenetic modifications in hepatic stellate cells

Background & Aims

Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol.

Methods

HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patient’s liver and through precision cut liver slices.

Results

HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol.

Conclusions

Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.

Introduction

Chronic alcohol consumption is both a direct cause of liver disease as well as a major co-morbidity factor in the progression of liver disease, resulting from other primary causes such as viral hepatitis [1], [2]. Mechanisms explaining the hepatotoxicity of alcohol are beginning to be understood and help to explain its impact on liver disease. Alcohol and its metabolites, in particular reactive oxygen species (ROS), such as the hydroxyethyl radical and nitric oxide are a major cause of hepatocellular damage [3]. Furthermore these metabolites can induce hepatic inflammation, which via cytokines such as TNFα are an important indirect cause of alcohol-induced hepatocyte damage and death. In addition, ROS-mediated lipid peroxidation has been proposed to cause damage to hepatocyte proteins, which can drive antigenic responses that perpetuate inflammation and liver damage [4].

Hepatocellular damage and inflammation stimulate the transdifferentiation of resident perisinusoidal HSCs into α-smooth muscle actin (αSMA)-positive myofibroblasts. These so-called “activated” HSCs (aHSCs) are the major hepatocellular source of fibrotic ECM proteins and promote the net deposition of fibrotic ECM in chronic liver disease [5]. The molecular processes that promote the progression of liver disease to severe fibrosis remain poorly defined, however, the continued production of ECM proteins by aHSCs is a major contributory factor. In addition, aHSCs secrete proteins that promote cross-linking, maturation and insolubility of the fibrotic ECM, such as elastin. Elastin is a target for cross-linking, catalysed by lysyl-oxidase or tissue transglutaminase [6], and its accumulation in the fibrotic ECM limits its potential for degradation and impacts on the degree of reversibility of the fibrotic tissue. Hence, factors that stimulate the expression of ECM maturation proteins, such as elastin, are likely to determine fibrosis progression in liver disease.

Recent studies from our laboratory and other investigators have revealed the importance of epigenetic signalling events in HSC transdifferentiation and fibrogenesis. Experimental manipulation of epigenetic signatures, such as DNA methylation, histone acetylation/methylation, and the activities of proteins that either annotate or interpret these epigenetic marks, can have profound effects on the HSC phenotype [7]. The concept that alcohol can stimulate epigenetic changes in liver tissue is already well established. Ethanol impairs the normal metabolism of methionine and in turn affects the availability of methyl groups in form of S-adenosylmethionine (SAMe) for DNA and histone methylation [8]. Kendrick and colleagues showed that ethanol or acetate reduced histone deacetylase activity and upregulated expression of acetyl-CoA synthetases, resulting in increases in histone acetylation and transcriptional activity at inflammatory genes [9]. Less is understood concerning the potential for alcohol to influence the HSC epigenome and expression of ECM proteins.

Here, we investigated the impact of alcohol on the expression of epigenetic regulators during HSC transdifferentiation and report that histone-modifying enzymes, such as the H3K4 methyltransferase MLL1, are upregulated in HSC exposed to ethanol and lead to the altered expression of profibrogenic genes including elastin. We provide combinatorial chromatin immunoprecipitation sequencing (ChIPseq) analysis on H3K4me3 and MLL1 signature in ethanol-stimulated HSCs that reveals widespread changes in chromatin structure, confirming a profound genome-wide effect of alcohol on HSC transdifferentiation. We suggest that our findings provide new insights into the mechanisms by which alcohol acts as a co-morbidity factor and a stimulator of fibrosis in chronic liver disease.