Role of HNF4alpha-cMyc interaction in liver regeneration and recovery after acetaminophen-induced acute liver injury.

BACKGROUND AND AIMS: Overdose of acetaminophen (APAP) is the major cause of acute liver failure in the western world. We report a novel signaling interaction between hepatocyte nuclear factor 4 alpha (HNF4α) cMyc and nuclear factor erythroid 2-related factor 2 (Nrf2) during liver injury and regeneration after APAP overdose. APPROACH AND RESULTS: APAP-induced liver injury and regeneration were studied in male C57BL/6J (WT) mice, hepatocyte-specific HNF4α knockout mice (HNF4α-KO), and HNF4α-cMyc double knockout mice (DKO). C57BL/6J mice treated with 300 mg/kg maintained nuclear HNF4α expression and exhibited liver regeneration, resulting in recovery. However, treatment with 600-mg/kg APAP, where liver regeneration was inhibited and recovery was delayed, showed a rapid decline in HNF4α expression. HNF4α-KO mice developed significantly higher liver injury due to delayed glutathione recovery after APAP overdose. HNF4α-KO mice also exhibited significant induction of cMyc, and the deletion of cMyc in HNF4α-KO mice (DKO mice) reduced the APAP-induced liver injury. The DKO mice had significantly faster glutathione replenishment due to rapid induction in Gclc and Gclm genes. Coimmunoprecipitation and ChIP analyses revealed that HNF4α interacts with Nrf2 and affects its DNA binding. Furthermore, DKO mice showed significantly faster initiation of cell proliferation resulting in rapid liver regeneration and recovery. CONCLUSIONS: These data show that HNF4α interacts with Nrf2 and promotes glutathione replenishment aiding in recovery from APAP-induced liver injury, a process inhibited by cMyc. These studies indicate that maintaining the HNF4α function is critical for regeneration and recovery after APAP overdose.

Identifying novel mechanisms of per- and polyfluoroalkyl substance-induced hepatotoxicity using FRG humanized mice.

Per- and polyfluoroalkyl substances (PFAS) such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) and perfluoro-2-methyl-3-oxahexanoic acid (GenX), the new replacement PFAS, are major environmental contaminants. In rodents, these PFAS induce several adverse effects on the liver, including increased proliferation, hepatomegaly, steatosis, hypercholesterolemia, nonalcoholic fatty liver disease and liver cancers. Activation of peroxisome proliferator receptor alpha by PFAS is considered the primary mechanism of action in rodent hepatocyte-induced proliferation. However, the human relevance of this mechanism is uncertain. We investigated human-relevant mechanisms of PFAS-induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes. Male FRG humanized mice were treated with 0.067 mg/L of PFOA, 0.145 mg/L of PFOS, or 1 mg/L of GenX in drinking water for 28 days. PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed that all three PFAS induced activation of p53 and inhibition of androgen receptor and NR1D1, a transcriptional repressor important in circadian rhythm. Further biochemical studies confirmed NR1D1 inhibition and in silico modeling indicated potential interaction of all three PFAS with the DNA-binding domain of NR1D1. In conclusion, our studies using FRG humanized mice have revealed new human-relevant molecular mechanisms of PFAS including their previously unknown effect on circadian rhythm.

HNF4α in Hepatocyte Health and Disease.

Hepatocyte nuclear factor 4 α (HNF4α) is a highly conserved member of the nuclear receptor superfamily expressed at high levels in the liver, kidney, pancreas, and gut. In the liver, HNF4α is exclusively expressed in hepatocytes, where it is indispensable for embryonic and postnatal liver development and for normal liver function in adults. It is considered a master regulator of hepatic differentiation because it regulates a significant number of genes involved in hepatocyte-specific functions. Loss of HNF4α expression and function is associated with the progression of chronic liver disease. Further, HNF4α is a target of chemical-induced liver injury. In this review, we discuss the role of HNF4α in liver pathophysiology and highlight its potential use as a therapeutic target for liver diseases.

Human Wharton's Jelly-derived mesenchymal stem cells prevent acetaminophen-induced liver injury in a mouse model unlike human dermal fibroblasts.

The persistence of hepatotoxicity induced by N-acetyl-para-aminophenol (Acetaminophen or Paracetamol, abbreviated as APAP) as the most common cause of acute liver failure in the United States, despite the availability of N-acetylcysteine, illustrates the clinical relevance of additional therapeutic approaches. While human mesenchymal stem cells (MSCs) have shown protection in mouse models of liver injury, the MSCs used are generally not cleared for human use and it is unclear whether these effects are due to xenotransplantation. Here we evaluated GMP manufactured clinical grade human Wharton's Jelly mesenchymal stem cells (WJMSCs), which are currently being investigated in human clinical trials, in a mouse model of APAP hepatotoxicity in comparison to human dermal fibroblasts (HDFs) to address these issues. C57BL6J mice were treated with a moderate APAP overdose (300 mg/kg) and WJMSCs were administered 90 min later. Liver injury was evaluated at 6 and 24 h after APAP. WJMSCs treatment reduced APAP-induced liver injury at both time points unlike HDFs, which showed no protection. APAP-induced JNK activation as well as AIF and Smac release from mitochondria were prevented by WJMSCs treatment without influencing APAP bioactivation. Mechanistically, WJMSCs treatment upregulated expression of Gclc and Gclm to enhance recovery of liver GSH levels to attenuate mitochondrial dysfunction and accelerated recovery of pericentral hepatocytes to re-establish liver zonation and promote liver homeostasis. Notably, preventing GSH resynthesis with buthionine sulfoximine prevented the protective effects of WJMSCs. These data indicate that these GMP-manufactured WJMCs could be a clinically relevant therapeutic approach in the management of APAP hepatotoxicity in humans.

Regulation of hepatic xenosensor function by HNF4alpha.

Nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as aryl hydrocarbon receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these 4 major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.

Multi-Omics after O-GlcNAc Alteration Identified Cellular Processes Promoting Aneuploidy after Loss of O-GlcNAc Transferase.

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address this issue, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. Moreover, these data show how a multi-Omics platform can discern how OGT can fine-tune multiple cellular pathways involved in in aneuploidy.

Multi-Omics after O-GlcNAc Alteration Identifies Cellular Processes Working Synergistically to Promote Aneuploidy.

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address this issue, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. Moreover, these data show how a multi-Omics platform can discern how OGT can synergistically fine-tune multiple cellular pathways.

Hepatic Transcriptome Comparative In Silico Analysis Reveals Similar Pathways and Targets Altered by Legacy and Alternative Per- and Polyfluoroalkyl Substances in Mice.

Per- and poly-fluoroalkyl substances (PFAS) are a large class of fluorinated carbon chains that include legacy PFAS, such as perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorohexane sulfonate (PFHxS). These compounds induce adverse health effects, including hepatotoxicity. Potential alternatives to the legacy PFAS (HFPO-DA (GenX), HFPO4, HFPO-TA, F-53B, 6:2 FTSA, and 6:2 FTCA), as well as a byproduct of PFAS manufacturing (Nafion BP2), are increasingly being found in the environment. The potential hazards of these new alternatives are less well known. To better understand the diversity of molecular targets of the PFAS, we performed a comparative toxicogenomics analysis of the gene expression changes in the livers of mice exposed to these PFAS, and compared these to five activators of PPARα, a common target of many PFAS. Using hierarchical clustering, pathway analysis, and predictive biomarkers, we found that most of the alternative PFAS modulate molecular targets that overlap with legacy PFAS. Only three of the 11 PFAS tested did not appreciably activate PPARα (Nafion BP2, 6:2 FTSA, and 6:2 FTCA). Predictive biomarkers showed that most PFAS (PFHxS, PFOA, PFOS, PFNA, HFPO-TA, F-53B, HFPO4, Nafion BP2) activated CAR. PFNA, PFHxS, PFOA, PFOS, HFPO4, HFPO-TA, F-53B, Nafion BP2, and 6:2 FTSA suppressed STAT5b, activated NRF2, and activated SREBP. There was no apparent relationship between the length of the carbon chain, type of head group, or number of ether linkages and the transcriptomic changes. This work highlights the similarities in molecular targets between the legacy and alternative PFAS.

Leukocyte cell-derived chemotaxin 2 correlates with pediatric non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), newly renamed metabolic dysfunction-associated liver disease (MASLD), is a leading cause of liver disease in children and adults. There is a paucity of data surrounding potential biomarkers and therapeutic targets, especially in pediatric NAFLD. Leukocyte cell-derived chemotaxin 2 (LECT2) is a chemokine associated with both liver disease and skeletal muscle insulin resistance. Our aim was to determine associations between LECT2 and common clinical findings of NAFLD in pediatric patients. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum LECT2 concentrations in children (aged 2-17 years) with and without NAFLD. LECT2 concentrations were then correlated to clinical parameters in NAFLD. Mean LECT2 was significantly elevated in children with NAFLD versus healthy controls (n = 63 vs. 42, 5.83 ± 1.98 vs. 4.02 ± 2.02 ng/mL, p < 0.005). Additionally, LECT2 had strong correlations with body mass index (BMI) (Pearson r = 0.301, p = 0.002). A LECT2 concentration of 3.76 mg/mL predicts NAFLD with a sensitivity of 90.5% and specificity of 54.8%. Principal component analysis and logistic regression models further confirmed associations between LECT2 and NAFLD status. This study demonstrates increased serum LECT2 concentrations in pediatric NAFLD, which correlates with BMI and shows strong predictive value within these patients. Our data indicate that LECT2 is a potential diagnostic biomarker of disease and should be further investigated in pediatric as well as adult NAFLD.

The Essential Role of O-GlcNAcylation in Hepatic Differentiation.

Background & Aims: O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase (OGT), which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and hepatocellular carcinoma. Methods: Single-cell RNA-sequencing was used to investigate hepatocyte differentiation in hepatocyte-specific OGT-KO mice with increased hepatic O-GlcNAcylation and in OGA-KO mice with decreased O-GlcNAcylation in hepatocytes. HCC patient samples and the DEN-induced hepatocellular carcinoma (HCC) model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. Results: Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation characterized by dysregulation of glycogen storage and glucose production. OGT-KO mice exacerbated DEN-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, OGA-KO mice with increased hepatic O-GlcNAcylation inhibited DEN-induced HCC. A progressive loss of O-GlcNAcylation was observed in HCC patients. Conclusions: Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC.

Identifying Human Specific Adverse Outcome Pathways of Per- and Polyfluoroalkyl Substances Using Liver-Chimeric Humanized Mice.

Background: Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants with myriad adverse effects. While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are the most common contaminants, levels of replacement PFAS, such as perfluoro-2-methyl-3-oxahexanoic acid (GenX), are increasing. In rodents, PFOA, PFOS, and GenX have several adverse effects on the liver, including nonalcoholic fatty liver disease. Objective: We aimed to determine human-relevant mechanisms of PFAS induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes. Methods: Male humanized mice were treated with 0.067 mg/L of PFOA, 0.145 mg/L of PFOS, or 1 mg/L of GenX in drinking water for 28 days. Liver and serum were collected for pathology and clinical chemistry, respectively. RNA-sequencing coupled with pathway analysis was used to determine molecular mechanisms. Results: PFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. PFOA had no significant changes in serum LDL/VLDL and total cholesterol. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed inhibition of NR1D1, a transcriptional repressor important in circadian rhythm, as the major common molecular change in all PFAS treatments. PFAS treated mice had significant nuclear localization of NR1D1. In silico modeling showed PFOA, PFOS, and GenX potentially interact with the DNA-binding domain of NR1D1. Discussion: These data implicate PFAS in circadian rhythm disruption via inhibition of NR1D1. These studies show that FRG humanized mice are a useful tool for studying the adverse outcome pathways of environmental pollutants on human hepatocytes in situ.

The essential role of O-GlcNAcylation in hepatic differentiation.

BACKGROUND: O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase, which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and HCC. METHODS: Single-cell RNA-sequencing (RNA-seq) was used to investigate hepatocyte differentiation in hepatocyte-specific O-GlcNAc transferase-knockout (OGT-KO) mice with decreased hepatic O-GlcNAcylation and in O-GlcNAcase-KO mice with increased O-GlcNAcylation in hepatocytes. Patients HCC samples and the diethylnitrosamine-induced HCC model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. RESULTS: Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation, characterized by dysregulation of glycogen storage and glucose production. O-GlcNAc transferase-KO mice exacerbated diethylnitrosamine-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, O-GlcNAcase -KO mice with increased hepatic O-GlcNAcylation inhibited diethylnitrosamine-induced HCC. A progressive loss of O-GlcNAcylation was observed in patients with HCC. CONCLUSIONS: Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC.

Hyaluronan synthesis inhibition normalizes ethanol-enhanced hepatic stellate cell activation.

BACKGROUND: Chronic ethanol overconsumption promotes alcohol-associated liver disease (ALD), characterized by hepatocyte injury, inflammation, hepatic stellate cell (HSC) activation, and fibrosis. Hyaluronan (HA) concentration is greater in livers and blood from advanced ALD patients than patients with advanced non-ALD. In the liver, HSCs are the major HA producers. The relationship between ethanol, HA, and HSC activation is incompletely understood. Thus, here, we tested the hypothesis that ethanol enhances HSC activation in a HA-dependent manner. METHODS: Liver tissue microarrays (TMAs) containing steatotic livers from donors with or without a history of alcohol consumption were used to measure HA and collagen content. Mice were fed a moderate (2%, v/v) ethanol-containing diet or pair-fed control diet for 2 days, after which they were given a single carbon tetrachloride (CCl4 ) injection. To inhibit HA synthesis, we provided 4-methylumbelliferone (4MU) daily. We used LX2 cells, a human HSC cell line, to determine the impact ethanol had on LPS responses, with or without concurrent 4MU exposure. RESULTS: CCl4 induced liver injury, but it did not differ between ethanol or control diet fed mice with or without 4MU treatment. Ethanol feeding enhanced CCl4 -induced hepatic HA content, which was paralleled by HA synthase (Has)2 transcript abundance; 4MU treatment normalized both. Consistently, HSC activation, assessed by measuring αSMA mRNA and protein, was induced by CCl4 exposure, enhanced by ethanol feeding, and normalized by 4MU. Hepatic transcripts, but not protein, for Ccl2 were enhanced by ethanol feeding and normalized by 4MU exposure. Finally, ethanol-exposed LX2 cells made more LPS-stimulated CCL2 mRNA and protein than cells not exposed to ethanol; 4MU prevented this. CONCLUSION: These data show that ethanol augments HSC activation through HA synthesis and enhances hepatic profibrogenic features. Therefore, targeting HSC HA production could potentially attenuate liver disease in ALD patients.

GenX induces fibroinflammatory gene expression in primary human hepatocytes.

The toxicity induced by the persistent organic pollutants per- and polyfluoroalkyl substances (PFAS) is dependent on the length of their polyfluorinated tail. Long-chain PFASs have significantly longer half-lives and profound toxic effects compared to their short-chain counterparts. Recently, production of a short-chain PFAS substitute called ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoate, also known as GenX, has significantly increased. However, the adverse health effects of GenX are not completely known. In this study, we investigated the dose-dependent effects of GenX on primary human hepatocytes (PHH). Freshly isolated PHH were treated with either 0.1, 10, or 100 µM of GenX for 48 and 96 h; then, global transcriptomic changes were determined using Human Clariom™ D arrays. GenX-induced transcriptional changes were similar at 0.1 and 10 µM doses but were significantly different at the 100 µM dose. Genes involved in lipid, monocarboxylic acid, and ketone metabolism were significantly altered following exposure of PHH at all doses. However, at the 100 µM dose, GenX caused changes in genes involved in cell proliferation, inflammation and fibrosis. A correlation analysis of concentration and differential gene expression revealed that 576 genes positively (R > 0.99) and 375 genes negatively (R < -0.99) correlated with GenX concentration. The upstream regulator analysis indicated HIF1α was inhibited at the lower doses but were activated at the higher dose. Additionally, VEGF, PPARα, STAT3, and SMAD4 signaling was induced at the 100 µM dose. These data indicate that at lower doses GenX can interfere with metabolic pathways and at higher doses can induce fibroinflammatory changes in human hepatocytes.

Regulation of Liver Regeneration by Hepatocyte O-GlcNAcylation in Mice.

BACKGROUND & AIMS: The liver has a unique capacity to regenerate after injury in a highly orchestrated and regulated manner. Here, we report that O-GlcNAcylation, an intracellular post-translational modification regulated by 2 enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration following partial hepatectomy (PHX). METHODS: We studied liver regeneration after PHX on hepatocyte specific OGT and OGA knockout mice (OGT-KO and OGA-KO), which caused a significant decrease (OGT-KO) and increase (OGA-KO) in hepatic O-GlcNAcylation, respectively. RESULTS: OGA-KO mice had normal regeneration, but the OGT-KO mice exhibited substantial defects in termination of liver regeneration with increased liver injury, sustained cell proliferation resulting in significant hepatomegaly, hepatic dysplasia, and appearance of small nodules at 28 days after PHX. This was accompanied by a sustained increase in expression of cyclins along with significant induction in pro-inflammatory and pro-fibrotic gene expression in the OGT-KO livers. RNA-sequencing studies revealed inactivation of hepatocyte nuclear 4 alpha (HNF4α), the master regulator of hepatic differentiation and a known termination signal, in OGT-KO mice at 28 days after PHX, which was confirmed by both Western blot and immunohistochemistry analysis. Furthermore, a significant decrease in HNFα target genes was observed in OGT-KO mice, indicating a lack of hepatocyte differentiation following decreased hepatic O-GlcNAcylation. Immunoprecipitation experiments revealed HNF4α is O-GlcNAcylated in normal differentiated hepatocytes. CONCLUSIONS: These studies show that O-GlcNAcylation plays a critical role in the termination of liver regeneration via regulation of HNF4α in hepatocytes.