[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal Of Pathology 09/2014; 184(11). DOI:10.1016/j.ajpath.2014.07.019 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bile acids play a critical role in liver injury and regeneration, but their role in acetaminophen (APAP)-induced liver injury is not known. We tested the effect of bile acid modulation on APAP hepatotoxicity using C57BL/6 mice, which were fed a normal diet, a 2% cholestyramine (CSA)-containing diet for bile acid depletion, or a 0.2% cholic acid (CA)-containing diet for 1 week before treatment with 400 mg/kg APAP. CSA-mediated bile acid depletion resulted in significantly higher liver injury and delayed regeneration after APAP treatment. In contrast, 0.2% CA supplementation in the diet resulted in a moderate delay in progression of liver injury and significantly higher liver regeneration after APAP treatment. Either CSA-mediated bile acid depletion or CA supplementation did not affect hepatic CYP2E1 levels or GSH depletion after APAP treatment. CSA-fed mice exhibited significantly higher activation of c-Jun N-terminal protein kinases and a significant decrease in intestinal fibroblast growth factor 15 mRNA after APAP treatment. In contrast, mice fed a 0.2% CA diet had significantly lower c-Jun N-terminal protein kinase activation and 12-fold higher fibroblast growth factor 15 mRNA in the intestines. Liver regeneration after APAP treatment was significantly faster in CA diet-fed mice after APAP administration secondary to rapid cyclin D1 induction. Taken together, these data indicate that bile acids play a critical role in both initiation and recovery of APAP-induced liver injury.
American Journal Of Pathology 09/2013; 50(5). DOI:10.1016/j.ajpath.2013.07.012 · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 HNF4αin adult mice resulted in increased hepatocyte proliferation with a significant increase in liver to body weight ratio. We determined global gene expression changes usingIlluminaHiSeq-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 HNF4αsignificantly 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 HNF4αknockout mice showed significant induction in c-Mycexpression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Mycactivation. (HEPATOLOGY 2013.).
[Show abstract][Hide abstract] ABSTRACT: 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. (Hepatology 2012.).
[Show abstract][Hide abstract] ABSTRACT: HNF4α is known as the master regulator of hepatocyte differentiation. Recent studies indicate that HNF4α may inhibit hepatocyte proliferation via yet to be identified mechanisms. We investigated the role of HNF4α in the regulation of hepatocyte proliferation using a novel HNF4α knockdown mouse model based on delivery of inducible Cre recombinase using an AAV8 viral vector. Hepatocyte specific deletion of HNF4α resulted in increased hepatocyte proliferation. Global gene expression analysis showed that a majority of the down regulated genes were previously known HNF4α target genes involved in hepatic differentiation. Interestingly, 500+ up regulated genes were associated with cell proliferation and cancer. Further, we identified potential negative target genes of HNF4α, many of which are involved in the stimulation of proliferation. We confirmed binding of HNF4α at three of these genes using ChIP analysis. Furthermore, in vitro over expression of HNF4α in mouse HCC cells resulted in a decrease in pro-mitogenic gene expression and cell cycle arrest. Taken together, these data indicate that, apart from its role in hepatocyte differentiation, HNF4α actively inhibits hepatocyte proliferation by repression of specific pro-mitogenic genes.
[Show abstract][Hide abstract] ABSTRACT: 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.