Acetaminophen (APAP) overdose results in acute liver failure and has limited treatment options. Previous studies show that stimulating liver regeneration is critical for survival after APAP overdose, but the mechanisms remain unclear. In this study, we identified major signaling pathways involved in liver regeneration after APAP-induced acute liver injury using a novel incremental dose model. Liver injury and regeneration were studied in C57BL/6 mice treated with either 300 mg/kg (APAP300) or 600 mg/kg (APAP600) APAP. Mice treated with APAP300 developed extensive liver injury and robust liver regeneration. In contrast, APAP600-treated mice exhibited significant liver injury but substantial inhibition of liver regeneration, resulting in sustained injury and decreased survival. The inhibition of liver regeneration in the APAP600 group was associated with cell cycle arrest and decreased cyclin D1 expression. Several known regenerative pathways, including the IL-6/STAT-3 and epidermal growth factor receptor/c-Met/mitogen-activated protein kinase pathways, were activated, even at APAP600, where regeneration was inhibited. However, canonical Wnt/β-catenin and NF-κB pathways were activated only in APAP300-treated mice, where liver regeneration was stimulated. Furthermore, overexpression of a stable form of β-catenin, where serine 45 is mutated to aspartic acid, in mice resulted in improved liver regeneration after APAP overdose. Taken together, our incremental dose model has identified a differential role of several signaling pathways in liver regeneration after APAP overdose and highlighted canonical Wnt signaling as a potential target for regenerative therapies for APAP-induced acute liver failure.

HNF4α, the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation via unknown mechanisms. We investigated the mechanisms of HNF4α-induced inhibition of hepatocyte proliferation using a novel TAM-inducible, hepatocyte specific HNF4α knockdown mouse model. Hepatocyte specific deletion of HNF4α in adult mice resulted in increased hepatocyte proliferation with a significant increase in liver to body weight ratio. We determined global gene expression changes using Illumina HiSeq-based RNA sequencing, which revealed that, a significant number of up-regulated genes following deletion of HNF4α were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c-Myc-regulated gene expression network was highly activated following HNF4α deletion. To determine whether deletion of HNF4α affects cancer pathogenesis, HNF4α knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4α significantly increased the number and size of DEN-induced hepatic tumors. Pathological analysis revealed that tumors in HNF4α deleted mice were well-differentiated hepatocellular carcinoma (HCC) and mixed HCC-cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN-treated HNF4α knockout mice showed significant induction in c-Myc expression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Myc activation.

Autophagy is an evolutionarily conserved biological process that degrades intracellular proteins and organelles including damaged mitochondria through the formation of autophagosome. We have previously demonstrated that pharmacological induction of autophagy by rapamycin protects against acetaminophen (APAP)-induced liver injury in mice. In contrast, in the present study, we found that mice with the liver-specific loss of Atg5, an essential autophagy gene, were resistant to APAP-induced liver injury. Hepatocyte-specific deletion of Atg5 resulted in mild liver injury characterized by increased apoptosis and compensatory hepatocyte proliferation. The lack of autophagy in the Atg5-deficient mouse livers was confirmed by increased p62 protein levels and the absence of LC3-lipidation as well as autophagosome formation. Analysis of histological and clinical chemistry parameters indicated that the Atg5 liver-specific knockout mice are resistant to APAP overdose (500 mg/kg). Further investigations revealed that the bioactivation of APAP is normal in Atg5 liver-specific knockout mice although they had lower CYP2E1 expression. There was an increased basal hepatic glutathione (GSH) content and a faster recovery of GSH after APAP treatment due to persistent activation of Nrf2, a transcriptional factor regulating drug detoxification and GSH synthesis gene expression. In addition, we found significantly higher hepatocyte proliferation in the livers of Atg5 liver-specific knockout mice. Taken together, our data suggest that persistent activation of Nrf2 and increased hepatocyte proliferation protect against APAP-induced liver injury in Atg5 liver-specific knockout mice.

Hepatocyte nuclear factor-4␣ (HNF4␣) is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4␣ may inhibit hepatocyte proliferation via mechanisms that have yet to be identified. Using a HNF4␣ knockdown mouse model based on delivery of inducible Cre recombinase via an adeno-associated virus 8 viral vector, we investigated the role of HNF4␣ in the regulation of hepatocyte proliferation. Hepatocyte-specific deletion of HNF4␣ resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the downregulated genes were previously known HNF4␣ target genes involved in hepatic differentiation. Interestingly, Ն500 upregulated genes were associated with cell proliferation and cancer. Furthermore, we identified potential negative target genes of HNF4␣, many of which are involved in the stimulation of proliferation. Using chromatin immunoprecipitation analysis, we confirmed binding of HNF4␣ at three of these genes. Furthermore, overexpression of HNF4␣ in mouse hepatocellular carcinoma cells resulted in a decrease in promitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4␣ actively inhibits hepatocyte proliferation by repression of specific promitogenic genes. adeno-associated virus; Ect2; gene repression; hepatocyte proliferation; liver regeneration THE LIVER IS KNOWN for its exceptional capability to regenerate in response to surgical removal, as well as following druginduced liver injury. Liver regeneration is critical in maintaining liver health, because the liver is the major site of xenobiotic detoxification and is exposed to a variety of drugs and chemicals. The mechanisms of initiation and progression of liver regeneration are well studied, but the mechanisms of termination of regeneration are not completely understood (27). It is important to understand the mechanisms of termination of liver regeneration, because many of the pathways involved in stimulation of liver regeneration can be carcinogenic if left unchecked. It is known that the mechanisms of termination of regeneration are essential to maintain controlled liver growth and inhibit progression to hepatocellular carcinoma (HCC). However, the detailed mechanisms of termination of regeneration and their link to prevention of HCC are not completely clear.Hepatocyte nuclear factor-4␣ (HNF4␣, representative public ID NR2A1) is considered the master regulator of hepatocyte differentiation. It plays an important role in the regulation of many hepatocyte-specific genes, including those involved in glycolysis, gluconeogenesis, ureagenesis, fatty acid metabolism, bile acid synthesis, drug metabolism, apolipoprotein synthesis, and blood coagulation (11, 15, 18 -21). Recent studies suggest a novel role of HNF4␣ in the regulation of cell proliferation (8,9,11,12,15,23,26,29,36). Data indicate that HNF4␣ may have antiproliferative activity in human embryonic kidney cells and rat pancreatic beta cell...

Farnesoid X Receptor (FXR), the primary bile acid-sensing nuclear receptor, also plays a role in stimulation of liver regeneration. Whole body deletion of FXR results in significant inhibition of liver regeneration after partial hepatectomy (PHX). FXR is expressed in liver and intestine and recent ChIP-seq analysis indicates that FXR regulates distinct set of genes in a tissue-specific manner. These data raise the question about relative contribution of hepatic and intestinal FXR in regulation of liver regeneration. We studied liver regeneration after PHX in hepatocyte-specific FXR knockout (hepFXR-KO) mice over a time course of 0 to 14 days. Whereas the overall kinetics of liver regrowth in hepFXR-KO mice was unaffected, a delay in peak hepatocyte proliferation from day 2 to day 3 after PHX was observed in the hepFXR-KO mice as compared to Cre- control mice. Real Time PCR, Western blot and co-IP studies revealed decreased Cyclin D1 expression and decreased association of Cyclin D1 with CDK4 in hepFXR-KO mice after PHX, correlating with decreased phosphorylation of pRb and delayed cell proliferation in the hepFXR-KO livers. The hepFXR-KO mice also exhibited delay in acute hepatic fat accumulation following PHX, which is associated with regulation of cell cycle. Further, a significant delay in HGF-initiated signaling, including AKT, c-myc and ERK-1/2 pathways, was observed in hepFXR-KO mice. UPLC-mass spectroscopy analysis of hepatic bile acids indicated no difference in levels of bile acids in hepFXR-KO and control mice. In Conclusion, deletion of hepatic FXR did not completely inhibit but delays liver regeneration after PHX secondary to delayed Cyclin D1 activation.