One Sentence Summary:Inhibiting injury-induced senescence mediated by TGFβ signaling in regenerative epithelium improves liver regeneration. Accessible Summary:The liver is a paradigm of organ regeneration, however regeneration may fail in a previously normal liver following acute severe injury such as acetaminophen poisoning. We show that, a process with prevents proliferation termed senescence, which is classically associated with aging and carcinogenesis, stops the liver's regenerative cells. This senescence can be spread from cell to cell by the signaling molecule TGFβ. When TGFβ signaling is inhibited during acetaminophen poisoning in mice, senescence is impeded, regeneration accelerates, and survival is improved. Therefore targeting senescence induced by acute tissue injury is an attractive therapeutic approach to improve regeneration. Abstract:Liver injury results in rapid regeneration through hepatocyte proliferation and hypertrophy.However, after acute severe injury, such as acetaminophen poisoning, effective regeneration may fail. We investigated how senescence may underlie this regenerative failure. In human acute liver disease, and murine models, p21-dependent hepatocellular senescence was proportionate to disease severity and was associated with impaired regeneration. In an acetaminophen injury mouse model, a transcriptional signature associated with the induction 4 of paracrine senescence was observed within twenty four hours, and was followed by one of impaired proliferation. In mouse genetic models of hepatocyte injury and senescence we observed transmission of senescence to local uninjured hepatocytes. Spread of senescence depended upon macrophage-derived TGFβ1 ligand. In acetaminophen poisoning, inhibition of TGFβ receptor 1 (TGFβR1) improved mouse survival. TGFβR1 inhibition reduced senescence and enhanced liver regeneration even when delivered after the current therapeutic window for acetaminophen poisoning. This mechanism, in which injury-induced senescence impairs liver regeneration, is an attractive therapeutic target for developing treatments for acute liver failure.

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked -acetylglucosamine (-GlcNAcylation) via overexpression of the GlcNAc-regulating enzymesGlcNAc transferase (OGT) or GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations inGlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevatedGlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevatedGlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), chemicals present in a multitude of consumer products, are persistent organic pollutants. Both compounds induce hepatotoxic effects in rodents, including steatosis, hepatomegaly and liver cancer. The mechanisms of PFOA- and PFOS-induced hepatic dysfunction are not completely understood. We present evidence that PFOA and PFOS induce their hepatic effects via targeting hepatocyte nuclear factor 4-alpha (HNF4α). Human hepatocytes treated with PFOA and PFOS at a concentration relevant to occupational exposure caused a decrease in HNF4α protein without affecting HNF4α mRNA or causing cell death. RNA sequencing analysis combined with Ingenuity Pathway Analysis of global gene expression changes in human hepatocytes treated with PFOA or PFOS indicated alterations in the expression of genes involved in lipid metabolism and tumorigenesis, several of which are regulated by HNF4α. Further investigation of specific HNF4α target gene expression revealed that PFOA and PFOS could promote cellular dedifferentiation and increase cell proliferation by down regulating positive targets (differentiation genes such as CYP7A1) and inducing negative targets of HNF4α (pro-mitogenic genes such as CCND1). Furthermore, in silico docking simulations indicated that PFOA and PFOS could directly interact with HNF4α in a similar manner to endogenous fatty acids. Collectively, these results highlight HNF4α degradation as novel mechanism of PFOA and PFOS-mediated steatosis and tumorigenesis in human livers.

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Macroautophagy is a major cellular degradation pathway for long-lived proteins and cellular organelles to maintain cellular homeostasis. Reduced autophagy has been implicated in neurodegenerative diseases, metabolic syndrome, and tumorigenesis. In contrast, increased autophagy has been shown to protect against tissue injury and aging. Here we employed a cell-based quantitative high-throughput image screening (qHTS) for autophagy modulators using mouse embryonic fibroblasts (MEFs) that are stably expressing GFP-LC3. The library of pharmacologically active compounds (LOPAC) was used to screen for the autophagy modulators in compounds alone or in combination with the lysosome inhibitor chloroquine (CQ). The GFP-LC3 puncta were then quantified to measure autophagic flux. The primary screening revealed 173 compounds with efficacy more than 40%. These compounds were cherry-picked and re-tested at multiple different concentrations using the same assay. A number of novel autophagy inducers, inhibitors, and modulators with dual-effects on autophagy were identified from the cherry-pick screening. Interestingly, we found a group of compounds that induce autophagy are related to dopamine receptors and are commonly used as clinical psychiatric drugs. Among them, indatraline hydrochloride (IND), a dopamine inhibitor, and chlorpromazine hydrochloride (CPZ) and fluphenazine dihydrochloride (FPZ), two dopamine receptor antagonists, were further evaluated. We found that FPZ-induced autophagy through mTOR inhibition but IND and CPZ induced autophagy in an mTOR-independent manner. Our data suggest that image-based autophagic flux qHTS can efficiently identify autophagy inducers and inhibitors.

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single β-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

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 posttranslational modification (PTM) regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a critical termination signal for liver regeneration (LR) following partial hepatectomy (PHX). 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. 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-Seq 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 IHC 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. These studies show that O-GlcNAcylation plays a critical role in the termination of LR via regulation of HNF4α in hepatocytes.

Estrogen receptor alpha (ESR1) is one of the two intracellular receptors for estrogen and is expressed by hepatocytes in the liver. The role of ESR1 in the regulation of toxicant-induced liver injury and compensatory regeneration is not completely clear. We investigated the role of ESR1 in liver regeneration after carbon tetrachloride (CCl4)-induced liver injury using wild type (WT) and ESR1 knockout rats (ESR1-KO). Adult female WT and ESR1-KO rats were treated with 1ml/kg CCl4 and euthanized over a time course of 0–48 hr. Liver Injury measured by serum alanine amino transaminase (ALT) and histopathological analysis showed significantly higher liver injury in ESR1-KO as compared to WT rats. Hematoxylin and eosin (H&E) staining revealed two-fold higher necrosis and significant inflammatory cell infiltration in ESR1-KO rats. Chloracetate esterase staining revealed higher neutrophil infiltration in ESR1-KO rat livers. Interestingly, Proliferating cell nuclear antigen (PCNA) immunohistochemistry showed that in spite of two-fold higher liver injury, the ESR1KO rats had equal liver regeneration as compared to WT rats. Western blot analysis of cyclin D1 and phosphorylated Rb, proteins involved in the initiation of the cell cycle, were significantly higher at all time points in ESR1KO rats. Further analysis revealed faster activation of canonical Wnt/β-catenin and NF-κB signaling in ESR1-KO rats characterized by higher activated β-catenin and phosphorylated p65 at 12 hr after CCl4 treatment. Taken together, these data indicate that ESR1-mediated signaling inhibits liver regeneration by down regulation of Wnt signaling resulting in lower cyclin D1 activation after chemical-induced liver injury.