Protective role of 17ß-estradiol in alcohol-associated liver fibrosis is mediated by suppression of integrin signaling.

BACKGROUND: Alcohol-associated liver disease is a complex disease regulated by genetic and environmental factors such as diet and sex. The combination of high-fat diet and alcohol consumption has synergistic effects on liver disease progression. Female sex hormones are known to protect females from liver disease induced by high-fat diet. In contrast, they promote alcohol-mediated liver injury. We aimed to define the role of female sex hormones on liver disease induced by a combination of high-fat diet and alcohol. METHODS: Wild-type and protein arginine methyltransferase (Prmt)6 knockout female mice were subjected to gonadectomy (ovariectomy, OVX) or sham surgeries and then fed western diet and alcohol in the drinking water. RESULTS: We found that female sex hormones protected mice from western diet/alcohol-induced weight gain, liver steatosis, injury, and fibrosis. Our data suggest that these changes are, in part, mediated by estrogen-mediated induction of arginine methyltransferase PRMT6. Liver proteome changes induced by OVX strongly correlated with changes induced by Prmt6 knockout. Using Prmt6 knockout mice, we confirmed that OVX-mediated weight gain, steatosis, and injury are PRMT6 dependent, while OVX-induced liver fibrosis is PRMT6 independent. Proteomic and gene expression analyses revealed that estrogen signaling suppressed the expression of several components of the integrin pathway, thus reducing integrin-mediated proinflammatory (Tnf, Il6) and profibrotic (Tgfb1, Col1a1) gene expression independent of PRMT6 levels. Integrin signaling inhibition using Arg-Gly-Asp peptides reduced proinflammatory and profibrotic gene expression in mice, suggesting that integrin suppression by estrogen is protective against fibrosis development. CONCLUSIONS: Taken together, estrogen signaling protects mice from liver disease induced by a combination of alcohol and high-fat diet through upregulation of Prmt6 and suppression of integrin signaling.

Kupffer cell diversity maintains liver function in alcohol-associated liver disease.

BACKGROUND AND AIMS: Liver macrophages are heterogeneous and play an important role in alcohol-associated liver disease (ALD) but there is limited understanding of the functions of specific macrophage subsets in the disease. We used a Western diet alcohol (WDA) mouse model of ALD to examine the hepatic myeloid cell compartment by single cell RNAseq and targeted KC ablation to understand the diversity and function of liver macrophages in ALD. APPROACH AND RESULTS: In the WDA liver, KCs and infiltrating monocytes/macrophages each represented about 50% of the myeloid pool. Five major KC clusters all expressed genes associated with receptor-mediated endocytosis and lipid metabolism, but most were predicted to be noninflammatory and antifibrotic with 1 minor KC cluster having a proinflammatory and extracellular matrix degradation gene signature. Infiltrating monocyte/macrophage clusters, in contrast, were predicted to be proinflammatory and profibrotic. In vivo, diphtheria toxin-based selective KC ablation during alcohol exposure resulted in a liver failure phenotype with increases in PT/INR and bilirubin, loss of differentiated hepatocyte gene expression, and an increase in expression of hepatocyte progenitor markers such as EpCAM, CK7, and Igf2bp3. Gene set enrichment analysis of whole-liver RNAseq from the KC-ablated WDA mice showed a similar pattern as seen in human alcoholic hepatitis. CONCLUSIONS: In this ALD model, KCs are anti-inflammatory and are critical for the maintenance of hepatocyte differentiation. Infiltrating monocytes/macrophages are largely proinflammatory and contribute more to liver fibrosis. Future targeting of specific macrophage subsets may provide new approaches to the treatment of liver failure and fibrosis in ALD.

Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Alcohol-induced epigenetic changes prevent fibrosis resolution after alcohol cessation in miceresolution.

BACKGROUND AND AIMS: Alcohol-associated liver disease is a major cause of alcohol-associated mortality. Recently, we identified hepatic demethylases lysine demethylase (KDM)5B and KDM5C as important epigenetic regulators of alcohol response in the liver. In this study, we aimed to investigate the role of KDM5 demethylases in alcohol-associated liver disease resolution. APPROACH AND RESULTS: We showed that alcohol-induced liver steatosis rapidly resolved after alcohol cessation. In contrast, fibrosis persisted in the liver for up to 8 weeks after the end of alcohol exposure. Defects in fibrosis resolution were in part due to alcohol-induced KDM5B and KDM5C-dependent epigenetic changes in hepatocytes. Using cell-type-specific knockout mice, we found that adeno-associated virus-mediated knockout of KDM5B and KDM5C demethylases in hepatocytes at the time of alcohol withdrawal promoted fibrosis resolution. Single-cell ATAC sequencing analysis showed that during alcohol-associated liver disease resolution epigenetic cell states largely reverted to control conditions. In addition, we found unique epigenetic cell states distinct from both control and alcohol states and identified associated transcriptional regulators, including liver X receptor (LXR) alpha (α). In vitro and in vivo analysis confirmed that knockout of KDM5B and KDM5C demethylases promoted LXRα activity, likely through regulation of oxysterol biosynthesis, and this activity was critical for the fibrosis resolution process. Reduced LXR activity by small molecule inhibitors prevented fibrosis resolution in KDM5-deficient mice. CONCLUSIONS: In summary, KDM5B and KDM5C demethylases prevent liver fibrosis resolution after alcohol cessation in part through suppression of LXR activity.

Sepsis-induced endothelial dysfunction drives acute-on-chronic liver failure through Angiopoietin-2-HGF-C/EBPß pathway.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is an acute liver and multisystem failure in patients with previously stable cirrhosis. A common cause of ACLF is sepsis secondary to bacterial infection. Sepsis-associated ACLF involves a loss of differentiated liver function in the absence of direct liver injury, and its mechanism is unknown. We aimed to study the mechanism of sepsis-associated ACLF using a novel mouse model. APPROACH AND RESULTS: Sepsis-associated ACLF was induced by cecal ligation and puncture procedure (CLP) in mice treated with thioacetamide (TAA). The combination of TAA and CLP resulted in a significant decrease in liver synthetic function and high mortality. These changes were associated with reduced metabolic gene expression and increased CCAAT enhancer binding protein beta (C/EBPß) transcriptional activity. We found that C/EBPß binding to its target gene promoters was increased. In humans, C/EBPß chromatin binding was similarly increased in the ACLF group compared with control cirrhosis. Hepatocyte-specific Cebpb knockout mice had reduced mortality and increased gene expression of hepatocyte differentiation markers in TAA/CLP mice, suggesting that C/EBPß promotes liver failure in these mice. C/EBPß activation was associated with endothelial dysfunction, characterized by reduced Angiopoietin-1/Angiopoietin-2 ratio and increased endothelial production of HGF. Angiopoietin-1 supplementation or Hgf knockdown reduced hepatocyte C/EBPß accumulation, restored liver function, and reduced mortality, suggesting that endothelial dysfunction induced by sepsis drives ACLF through HGF-C/EBPß pathway. CONCLUSIONS: The transcription factor C/EBPß is activated in both mouse and human ACLF and is a potential therapeutic target to prevent liver failure in patients with sepsis and cirrhosis.

A Pathogenic Role of Non-Parenchymal Liver Cells in Alcohol-Associated Liver Disease of Infectious and Non-Infectious Origin.

Now, much is known regarding the impact of chronic and heavy alcohol consumption on the disruption of physiological liver functions and the induction of structural distortions in the hepatic tissues in alcohol-associated liver disease (ALD). This review deliberates the effects of alcohol on the activity and properties of liver non-parenchymal cells (NPCs), which are either residential or infiltrated into the liver from the general circulation. NPCs play a pivotal role in the regulation of organ inflammation and fibrosis, both in the context of hepatotropic infections and in non-infectious settings. Here, we overview how NPC functions in ALD are regulated by second hits, such as gender and the exposure to bacterial or viral infections. As an example of the virus-mediated trigger of liver injury, we focused on HIV infections potentiated by alcohol exposure, since this combination was only limitedly studied in relation to the role of hepatic stellate cells (HSCs) in the development of liver fibrosis. The review specifically focusses on liver macrophages, HSC, and T-lymphocytes and their regulation of ALD pathogenesis and outcomes. It also illustrates the activation of NPCs by the engulfment of apoptotic bodies, a frequent event observed when hepatocytes are exposed to ethanol metabolites and infections. As an example of such a double-hit-induced apoptotic hepatocyte death, we deliberate on the hepatotoxic accumulation of HIV proteins, which in combination with ethanol metabolites, causes intensive hepatic cell death and pro-fibrotic activation of HSCs engulfing these HIV- and malondialdehyde-expressing apoptotic hepatocytes.

Arginine Methylation of Integrin Alpha-4 Prevents Fibrosis Development in Alcohol-Associated Liver Disease.

BACKGROUND & AIMS: Alcohol-associated liver disease (ALD) comprises a spectrum of disorders including steatosis, steatohepatitis, fibrosis, and cirrhosis. We aimed to study the role of protein arginine methyltransferase 6 (PRMT6), a new regulator of liver function, in ALD progression. METHODS: Prmt6-deficient mice and wild-type littermates were fed Western diet with alcohol in the drinking water for 16 weeks. Mice fed standard chow diet or Western diet alone were used as a control. RESULTS: We found that PRMT6 expression in the liver is down-regulated in 2 models of ALD and negatively correlates with disease severity in mice and human liver specimens. Prmt6-deficient mice spontaneously developed liver fibrosis after 1 year and more advanced fibrosis after high-fat diet feeding or thioacetamide treatment. In the presence of alcohol Prmt6 deficiency resulted in a dramatic increase in fibrosis development but did not affect lipid accumulation or liver injury. In the liver PRMT6 is primarily expressed in macrophages and endothelial cells. Transient replacement of knockout macrophages with wild-type macrophages in Prmt6 knockout mice reduced profibrotic signaling and prevented fibrosis progression. We found that PRMT6 decreases profibrotic signaling in liver macrophages via methylation of integrin α-4 at R464 residue. Integrin α-4 is predominantly expressed in infiltrating monocyte derived macrophages. Blocking monocyte infiltration into the liver with CCR2 inhibitor reduced fibrosis development in knockout mice and abolished differences between genotypes. CONCLUSIONS: Taken together, our data suggest that alcohol-mediated loss of Prmt6 contributes to alcohol-associated fibrosis development through reduced integrin methylation and increased profibrotic signaling in macrophages.

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

Alcohol-associated liver disease is a major cause of alcohol-related mortality. However, the mechanisms underlying disease progression are not fully understood. Recently we found that liver molecular pathways are altered by alcohol consumption differently in males and females. We were able to associate these sex-specific pathways with two upstream regulators: H3K4-specific demethylase enzymes KDM5B and KDM5C. Mice were fed the Lieber-DeCarli alcohol liquid diet for 3 weeks or a combination of a high-fat diet with alcohol in water for 16 weeks (western diet alcohol model [WDA] model). To assess the role of histone demethylases, mice were treated with AAV-shControl, AAV-shKdm5b, and/or AAV-shKdm5c and/or AAV-shAhR vectors. Gene expression and epigenetic changes after Kdm5b/5c knockdown were assessed by RNA-sequencing and H3K4me3 chromatin immunoprecipitation analysis. We found that less than 5% of genes affected by Kdm5b/Kdm5c knockdown were common between males and females. In females, Kdm5b/Kdm5c knockdown prevented fibrosis development in mice fed the WDA alcohol diet for 16 weeks and decreased fibrosis-associated gene expression in mice fed the Lieber-DeCarli alcohol liquid diet. In contrast, fibrosis was not affected by Kdm5b/Kdm5c knockdown in males. We found that KDM5B and KDM5C promote fibrosis in females through down-regulation of the aryl hydrocarbon receptor (AhR) pathway components in hepatic stellate cells. Kdm5b/Kdm5c knockdown resulted in an up-regulation of Ahr, Arnt, and Aip in female but not in male mice, thus preventing fibrosis development. Ahr knockdown in combination with Kdm5b/Kdm5c knockdown restored profibrotic gene expression. Conclusion: KDM5 demethylases contribute to differences between males and females in the alcohol response in the liver. The KDM5/AhR axis is a female-specific mechanism of fibrosis development in alcohol-fed mice.

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

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.

A Western diet with alcohol in drinking water recapitulates features of alcohol-associated liver disease in mice.

BACKGROUND: Mouse models of alcohol-associated liver disease vary greatly in their ease of implementation and the pathology they produce. Effects range from steatosis and mild inflammation with the Lieber-DeCarli liquid diet to severe inflammation, fibrosis, and pyroptosis seen with the Tsukamoto-French intragastric feeding model. Implementation of all of these models is limited by the labor-intensive nature of the protocols and the specialized skills necessary for successful intragastric feeding. We thus sought to develop a new model to reproduce features of alcohol-induced inflammation and fibrosis with minimal operational requirements. METHODS: Over a 16-week period, mice were fed ad libitum with a pelleted high-fat Western diet (WD; 40% calories from fat) and alcohol added to the drinking water. We found the optimal alcohol consumption to be that at which the alcohol concentration was 20% for 4 days and 10% for 3 days per week. Control mice received WD pellets with water alone. RESULTS: Alcohol consumption was 18 to 20 g/kg/day in males and 20 to 22 g/kg/day in females. Mice in the alcohol groups developed elevated serum transaminase levels after 12 weeks in males and 10 weeks in females. At 16 weeks, both males and females developed liver inflammation, steatosis, and pericellular fibrosis. Control mice on WD without alcohol had mild steatosis only. Alcohol-fed mice showed reduced HNF4α mRNA and protein expression. HNF4α is a master regulator of hepatocyte differentiation, down-regulation of which is a known driver of hepatocellular failure in alcoholic hepatitis. CONCLUSION: A simple-to-administer, 16-week WD alcohol model recapitulates the inflammatory, fibrotic, and gene expression aspects of human alcohol-associated steatohepatitis.

Arginine Methylation of Hepatic hnRNPH Suppresses Complement Activation and Systemic Inflammation in Alcohol-Fed Mice.

Protein arginine methyl transferase 1 (PRMT1) is the main enzyme for cellular arginine methylation. It regulates many aspects of liver biology including inflammation, lipid metabolism, and proliferation. Previously we identified that PRMT1 is necessary for protection from alcohol-induced liver injury. However, many PRMT1 targets in the liver after alcohol exposure are not yet identified. We studied the changes in the PRMT1-dependent arginine methylated proteome after alcohol feeding in mouse liver using mass spectrometry. We found that arginine methylation of the RNA-binding protein (heterogeneous nuclear ribonucleoprotein [hnRNP]) H1 is mediated by PRMT1 and is altered in alcohol-fed mice. PRMT1-dependent methylation suppressed hnRNP H1 binding to several messenger RNAs of complement pathway including complement component C3. We found that PRMT1-dependent hnRNP H methylation suppressed complement component expression in vitro, and phosphorylation is required for this function of PRMT1. In agreement with that finding, hepatocyte-specific PRMT1 knockout mice had an increase in complement component expression in the liver. Excessive complement expression in alcohol-fed PRMT1 knockout mice resulted in further complement activation and an increase in serum C3a and C5a levels, which correlated with inflammation in multiple organs including lung and adipose tissue. Using specific inhibitors to block C3aR and C5aR receptors, we were able to prevent lung and adipose tissue inflammation without affecting inflammation in the liver or liver injury. Conclusion: Taken together, these data suggest that PRMT1-dependent suppression of complement production in the liver is necessary for prevention of systemic inflammation in alcohol-fed mice. C3a and C5a play a role in this liver-lung and liver-adipose interaction in alcohol-fed mice deficient in liver arginine methylation.

Hepatocellular Protein Arginine Methyltransferase 1 Suppresses Alcohol-Induced Hepatocellular Carcinoma Formation by Inhibition of Inducible Nitric Oxide Synthase.

Alcohol is a well-established risk factor for hepatocellular carcinoma (HCC), but the mechanisms by which alcohol promotes liver cancer are not well understood. Studies suggest that ethanol may enhance tumor progression by increasing hepatocyte proliferation and through alcohol-induced liver inflammation. Protein arginine methyltransferase 1 (PRMT1) is the main enzyme responsible for cellular arginine methylation. Asymmetric dimethyl arginine, produced by PRMT1, is a potent inhibitor of nitric oxide synthases. PRMT1 is implicated in the development of several types of tumors and cardiovascular disease. Our previous work has shown that PRMT1 in the liver regulates hepatocyte proliferation and oxidative stress and protects from alcohol-induced liver injury. However, its role in HCC development remains controversial. In this study, we found that hepatocyte-specific PRMT1-knockout mice develop an increased number of tumors in an N-nitrosodiethylamine (DEN) alcohol model of liver tumorigenesis in mice. This effect was specific to the alcohol-related component because wild-type and knockout mice developed similar tumor numbers in the DEN model without the addition of alcohol. We found that in the presence of alcohol, the increase in tumor number was associated with increased proliferation in liver and tumor, increased WNT/ß-catenin signaling, and increased inflammation. We hypothesized that increased inflammation was due to increased oxidative and nitrosative stress in knockout mice. By blocking excess nitric oxide production using an inducible nitric oxide synthase inhibitor, we reduced hepatocyte death and inflammation in the liver and prevented the increase in WNT/ß-catenin signaling, proliferation, and tumor number in livers of knockout mice. Conclusion: PRMT1 is an important protection factor from alcohol-induced liver injury, inflammation, and HCC development.

The polymorphism rs975484 in the protein arginine methyltransferase 1 gene modulates expression of immune checkpoint genes in hepatocellular carcinoma.

Protein arginine methyltransferase 1 (PRMT1) is a key regulator of hepatic immune responses. Recently, we reported that PRMT1 regulates the tumor immune response in hepatocellular carcinoma (HCC). Here we found that PRMT1 expression in human HCC correlates with that of programmed cell death 1 ligand 1 (PD-L1), PD-L2, and other checkpoint genes. PRMT1 deletion in mice reduced PD-L1 and PD-L2 expression in tumors and reduced the efficiency of PD-1 antibody treatment in a diethylnitrosamine-induced HCC mouse model, suggesting that PRMT1 regulates the hepatic immune checkpoint. Mice had reduced PD-L1 and PD-L2 expression when PRMT1 was specifically deleted in tumor cells or macrophages, but PRMT1 deletion in dendritic cells did not alter PD-L1 and PD-L2 expression. rs975484 is a common polymorphism in the human PRMT1 gene promoter, and we found that it alters PRMT1 expression in blood monocytes and tumor-associated macrophages in human HCC. PRMT1 expression was higher in individuals with a GG genotype than in individuals with a CC genotype, and heterozygous carriers had intermediate expression. Luciferase reporter assays indicated that this differential expression is due to an extra C/EBPß-binding site in the PRMT1 promoter of individuals carrying the minor G allele. The rs975484 genotype also correlated with PRMT1 target expression in HCC. Individuals with the GG genotype had significantly higher levels of the PRMT1 targets PD-L1, PD-L2, and VISTA than those with the CC genotype. We conclude that PRMT1 critically controls immune checkpoints in mice and humans and that the PRMT1 polymorphism rs975484 affects checkpoint gene expression in HCC.

Hepatocyte PRMT1 protects from alcohol induced liver injury by modulating oxidative stress responses.

Protein Arginine methyltransferase 1 (PRMT1) is the main enzyme of cellular arginine methylation. Previously we found that PRMT1 activity in the liver is altered after alcohol exposure resulting in epigenetic changes. To determine the impact of these PRMT1 changes on the liver's response to alcohol, we induced a hepatocyte specific PRMT1 knockout using AAV mediated Cre delivery in mice fed either alcohol or control Lieber-DeCarli liquid diet. We found that in alcohol fed mice, PRMT1 prevents oxidative stress and promotes hepatocyte survival. PRMT1 knockout in alcohol fed mice resulted in a dramatic increase in hepatocyte death, inflammation and fibrosis. Additionally, we found that alcohol promotes PRMT1 dephosphorylation at S297. Phosphorylation at this site is necessary for PRMT1-dependent protein arginine methylation. PRMT1 S297A, a dephosphorylation mimic of PRMT1 had reduced ability to promote gene expression of pro-inflammatory cytokines, pro-apoptotic genes BIM and TRAIL and expression of a suppressor of hepatocyte proliferation, Hnf4α. On the other hand, several functions of PRMT1 were phosphorylation-independent, including expression of oxidative stress response genes, Sod1, Sod2 and others. In vitro, both wild type and S297A PRMT1 protected hepatocytes from oxidative stress induced apoptosis, however S297D phosphorylation mimic PRMT1 promoted cell death. Taken together these data suggest that PRMT1 is an essential factor of liver adaptation to alcohol; alcohol-induced dephosphorylation shifts PRMT1 toward a less pro-inflammatory, more pro-proliferative and pro-survival form.

PRMT1-Dependent Macrophage IL-6 Production Is Required for Alcohol-Induced HCC Progression.

Alcohol is a well-established risk factor for hepatocellular carcinoma, but the mechanisms are not well understood. Several studies suggested that alcohol promotes tumor growth by altering immune cell phenotypes in the liver. Arginine methylation is a common posttranslational modification generated mostly by a single protein, PRMT1. In myeloid cells PRMT1 is a key regulator of immune response. Myeloid-specific PRMT1 knockout mice are hyperresponsive to LPS and deficient in PPARγ-dependent macrophage M2 polarization. We aimed to define the role of myeloid PRMT1 in alcohol-associated liver tumor progression using a mouse model of DEN injection followed by Lieber-DeCarli alcohol liquid diet feeding. We found that PRMT1 knockout mice showed significantly lower expression of IL-10 and IL-6 cytokines in the liver and downstream STAT3 activation, which correlated with reduced number of surface tumors, reduced proliferation, and reduced number of M2 macrophages in the liver as well as within proliferating nodules. We found that blocking IL-6 signaling in alcohol-fed mice reduced the number of tumors and liver proliferation in wild-type mice but not in knockout mice suggesting that reduced IL-6 in PRMT1 knockout mice contributes to the protection from alcohol. Additionally, PRMT1 knockout did not show any protection in tumor formation in the absence of alcohol. Finally, we confirmed that this mechanism is relevant in humans. We found that PRMT1 expression in tumor-associated macrophages correlated with STAT3 activation in human HCC specimens. Taken together, these data suggest that the PRMT1-IL-6-STAT3 axis is an important mechanism of alcohol-associated tumor progression.

Demethylase JMJD6 as a New Regulator of Interferon Signaling: Effects of HCV and Ethanol Metabolism.

BACKGROUND & AIMS: Alcohol-induced progression of hepatitis C virus (HCV) infection is related to dysfunction of innate immunity in hepatocytes. Endogenously produced interferon (IFN)α induces activation of interferon-stimulated genes (ISGs) via triggering of the Janus kinase-signal transducer and activator of transcription 1 (STAT1) pathway. This activation requires protein methyltransferase 1-regulated arginine methylation of STAT1. Here, we aimed to study whether STAT1 methylation also depended on the levels of demethylase jumonji domain-containing 6 protein (JMJD6) and whether ethanol and HCV affect JMJD6 expression in hepatocytes. METHODS: Huh7.5-CYP (RLW) cells and hepatocytes were exposed to acetaldehyde-generating system (AGS) and 50 mmol/L ethanol, respectively. JMJD6 messenger RNA and protein expression were measured by real-time polymerase chain reaction and Western blot. IFNα-activated cells either overexpressing JMJD6 or with knocked-down JMJD6 expression were tested for STAT1 methylation, ISG activation, and HCV RNA. In vivo studies have been performed on C57Bl/6 mice (expressing HCV structural proteins or not) or chimeric mice with humanized livers fed control or ethanol diets. RESULTS: AGS exposure to cells up-regulated JMJD6 expression in RLW cells. These results were corroborated by ethanol treatment of primary hepatocytes. The promethylating agent betaine reversed the effects of AGS/ethanol. Similar results were obtained in vivo, when mice were fed control/ethanol with and without betaine supplementation. Overexpression of JMJD6 suppressed STAT1 methylation, IFNα-induced ISG activation, and increased HCV-RNA levels. In contrast, JMJD6 silencing enhanced STAT1 methylation, ISG stimulation by IFNα, and attenuated HCV-RNA expression in Huh7.5 cells. CONCLUSIONS: We conclude that arginine methylation of STAT1 is suppressed by JMJD6. Both HCV and acetaldehyde increase JMJD6 levels, thereby impairing STAT1 methylation and innate immunity protection in hepatocytes exposed to the virus and/or alcohol.

Protein arginine methyl transferase 1- and Jumonji C domain-containing protein 6-dependent arginine methylation regulate hepatocyte nuclear factor 4 alpha expression and hepatocyte proliferation in mice.

Alcohol is a well-established risk factor for hepatocellular carcinoma (HCC), but the mechanisms by which it promotes liver cancer are not well understood. Several studies have shown that cellular protein arginine methylation is inhibited by alcohol. Arginine methylation is controlled by the reciprocal activity of protein arginine methyltransferases, primarily protein arginine methyl transferase 1 (PRMT1), and a demethylase Jumonji C domain-containing protein 6 (JMJD6). The aim of this study was to explore the role of arginine methylation changes in alcohol pathogenesis. We found that PRMT1 activity is inhibited in livers of mice fed with alcohol compared to pair-fed mice. Using hepatocyte-specific PRMT1 knockout mice, we identified that loss of PRMT1 results in enhanced hepatocyte proliferation and a 33% increase in liver size. This increased hepatocyte proliferation was associated with reduced expression of hepatocyte nuclear factor 4 alpha (Hnf4α), an important regulator of liver tumorigenesis. We found that PRMT1 regulates Hnf4α expression directly through arginine methylation at the (Hnf4α) promoter. In the absence of PRMT1, JMJD6 can demethylate the Hnf4α promoter and suppress its expression. We were able to restore Hnf4α expression and abolish the increase in hepatocyte proliferation by knockdown of JMJD6 in PRMT1 knockout mice. Knockdown of JMJD6 in alcohol-fed mice similarly increased Hnf4α expression. We then examined whether loss of arginine methylation might play a role in alcohol-associated liver cancers. We examined 25 human HCC specimens and found a strong correlation (R = 0.8; P < 0.01) between arginine methylation levels and Hnf4α expression in these specimens, suggesting that the above mechanism is relevant in patients. CONCLUSION: Taken together, these data suggest that PRMT1 inhibition, such as induced by alcohol, may result in epigenetic changes leading to loss of Hnf4α. This effect may contribute to alcohol's ability to promote liver tumors. (Hepatology 2018;67:1109-1126).

Arginine methylation regulates c-Myc-dependent transcription by altering promoter recruitment of the acetyltransferase p300.

Protein arginine methyltransferase 1 (PRMT1) is an essential enzyme controlling about 85% of the total cellular arginine methylation in proteins. We have shown previously that PRMT1 is an important regulator of innate immune responses and that it is required for M2 macrophage differentiation. c-Myc is a transcription factor that is critical in regulating cell proliferation and also regulates the M2 transcriptional program in macrophages. Here, we sought to determine whether c-Myc in myeloid cells is regulated by PRMT1-dependent arginine methylation. We found that PRMT1 activity was necessary for c-Myc binding to the acetyltransferase p300. PRMT1 inhibition decreased p300 recruitment to c-Myc target promoters and increased histone deacetylase 1 (HDAC1) recruitment, thereby decreasing transcription at these sites. Moreover, PRMT1 inhibition blocked c-Myc-mediated induction of several of its target genes, including peroxisome proliferator-activated receptor γ (PPARG) and mannose receptor C-type 1 (MRC1), suggesting that PRMT1 is necessary for c-Myc function in M2 macrophage differentiation. Of note, in primary human blood monocytes, p300-c-Myc binding was strongly correlated with PRMT1 expression, and in liver sections, PRMT1, c-Myc, and M2 macrophage levels were strongly correlated with each other. Both PRMT1 levels and M2 macrophage numbers were significantly lower in livers from individuals with a history of spontaneous bacterial peritonitis, known to have defective cellular immunity. In conclusion, our findings demonstrate that PRMT1 is an important regulator of c-Myc function in myeloid cells. PRMT1 loss in individuals with cirrhosis may contribute to their immune defects.

Protein arginine methyltransferase 1 modulates innate immune responses through regulation of peroxisome proliferator-activated receptor γ-dependent macrophage differentiation.

Arginine methylation is a common posttranslational modification that has been shown to regulate both gene expression and extranuclear signaling events. We recently reported defects in protein arginine methyltransferase 1 (PRMT1) activity and arginine methylation in the livers of cirrhosis patients with a history of recurrent infections. To examine the role of PRMT1 in innate immune responses in vivo, we created a cell type-specific knock-out mouse model. We showed that myeloid-specific PRMT1 knock-out mice demonstrate higher proinflammatory cytokine production and a lower survival rate after cecal ligation and puncture. We found that this defect is because of defective peroxisome proliferator-activated receptor γ (PPARγ)-dependent M2 macrophage differentiation. PPARγ is one of the key transcription factors regulating macrophage polarization toward a more anti-inflammatory and pro-resolving phenotype. We found that PRMT1 knock-out macrophages failed to up-regulate PPARγ expression in response to IL4 treatment resulting in 4-fold lower PPARγ expression in knock-out cells than in wild-type cells. Detailed study of the mechanism revealed that PRMT1 regulates PPARγ gene expression through histone H4R3me2a methylation at the PPARγ promoter. Supplementing with PPARγ agonists rosiglitazone and GW1929 was sufficient to restore M2 differentiation in vivo and in vitro and abrogated the difference in survival between wild-type and PRMT1 knock-out mice. Taken together these data suggest that PRMT1-dependent regulation of macrophage PPARγ expression contributes to the infection susceptibility in PRMT1 knock-out mice.