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Autophagy is a process for the turnover of intracellular organelles and molecules during stress responses. microRNAs (miRNAs) are small, non-coding endogenous RNAs that may regulate almost every cellular process. However, the roles of miRNAs in autophagy are still poorly understood. In this study, we show that miR-26a enhances autophagy in both culture cells and the mouse liver. Hepatic overexpression of miR-26a in mice alleviated ethanol-induced hepatic steatosis and liver injury. Overexpression of miR-26a increased the expression of the autophagy mediator Beclin-1, which is regulated by mitogen-activated protein kinases (MAPKs). We identified DUSP4 and DUSP5, two MAPKs inhibitors, as direct targets of miR-26a. We further demonstrated that miR-26a targeted the 3′-UTRs of several other negative regulators of autophagy. Our results thus identify a novel miRNA-mediated mechanism that enhances cytoprotective autophagy in the liver. Key messages • miR-26a enhances autophagy in liver cells. • Hepatic overexpression of miR-26a in mice alleviates ethanol-induced liver injury. • Overexpression of miR-26a increases the expression of autophagy mediator Beclin-1. • DUSP4 and DUSP5, two MAPKs inhibitors, were identified as direct targets of miR-26a. Electronic supplementary material The online version of this article (doi:10.1007/s00109-015-1282-2) contains supplementary material, which is available to authorized users.

As phospho-eIF4E (p-eIF4E), unlike total eIF4E (t-eIF4E) essential for normal cells, is specifically required by cancer cells, it is an attractive, yet unrealized, target for anti-tumor intervention. Here we identify a small molecule, homoharringtonine (HHT), that antagonizes p-eIF4E function and eradicates acute myeloid leukemia (AML) expressing high level of p-eIF4E in vitro and in vivo. HHT selectively reduces p-eIF4E levels of leukemia cells without affecting t-eIF4E. HHT targets the phosphorylated serine 209 residue of p-eIF4E and induces p-eIF4E oligomerization, which enhances its interaction with the small ubiquitin-like protein modifier (SUMO)-conjugating enzyme UBC9, resulting in proteasome-dependent degradation of p-eIF4E via SUMO2/3-mediated SUMOylation. These results suggest that the phosphorylated serine 209 residue of p-eIF4E might be a potential target for developing small molecule-based new therapies for leukemia.

The liver is able to regenerate itself in response to partial hepatectomy or liver injury. This is accomplished by a complex network of different cell types and signals both inside and outside the liver. Bile acids (BAs) are recently identified as liver-specific metabolic signals and promote liver regeneration by activating their receptors: Farnesoid X Receptor (FXR) and G-protein-coupled BA receptor 1 (GPBAR1, or TGR5). FXR is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. FXR promotes liver regeneration after 70% partial hepatectomy (PHx) or liver injury. Moreover, activation of FXR is able to alleviate age-related liver regeneration defects. Both liver- and intestine-FXR are activated by BAs after liver resection or injury and promote liver regeneration through distinct mechanism. TGR5 is a membrane-bound BA receptor and it is also activated during liver regeneration. TGR5 regulates BA hydrophobicity and stimulates BA excretion in urine during liver regeneration. BA signaling thus represents a novel metabolic pathway during liver regeneration. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Vertical sleeve gastrectomy (VSG) produces sustainable weight loss, remission of type 2 diabetes (T2D), and improvement of nonalcoholic fatty liver disease (NAFLD). However, the molecular mechanisms underlying the metabolic benefits of VSG have remained elusive. According to our previous results, diet-induced obesity induces epigenetic modifications to chromatin in mouse liver. We demonstrate here that VSG in C57BL/6J wild-type male mice can reverse these chromatin modifications and thereby impact the expression of key metabolic genes. Genes involved in lipid metabolism, especially omega-6 fatty acid metabolism, are up-regulated in livers of mice after VSG while genes in inflammatory pathways are down-regulated after VSG. Consistent with gene expression changes, regulatory regions near genes involved in inflammatory response displayed decreased chromatin accessibility after VSG. Our results indicate that VSG induces global regulatory changes that impact hepatic inflammatory and lipid metabolic pathways, providing new insight into the mechanisms underlying the beneficial metabolic effects induced by VSG.

Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and thymine DNA glycosylase (TDG) play crucial roles in early embryonic and germ cell development by mediating DNA demethylation. However, the molecular mechanisms that regulate TETs/TDG expression and their role in cellular differentiation, including that of the pancreas, are not known. Here, we report that (i) TET1/2/3 and TDG can be direct targets of the microRNA miR-26a, (ii) murine TETs, especially TET2 and TDG, are down-regulated in islets during postnatal differentiation, whereas miR-26a is up-regulated, (iii) changes in 5-hydroxymethylcytosine accompany changes in TET mRNA levels, (iv) these changes in mRNA and 5-hydroxymethylcytosine are also seen in an in vitro differentiation system initiated with FACS-sorted adult ductal progenitor-like cells, and (v) overexpression of miR-26a in mice increases postnatal islet cell number in vivo and endocrine/acinar colonies in vitro. These results establish a previously unknown link between miRNAs and TET expression levels, and suggest a potential role for miR-26a and TET family proteins in pancreatic cell differentiation.

Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here, we report that berbamine and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca2+/calmodulin-dependent protein kinase II (CAMKII). Furthermore, berbamine inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice and downregulated the self-renewal abilities of liver cancer–initiating cells. Chemical inhibition or short hairpin RNA–mediated knockdown of CAMKII recapitulated the effects of berbamine, whereas overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to berbamine treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peritumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of berbamine for liver cancer therapies. Our data suggest that berbamine and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII. Mol Cancer Ther; 12(10); 2067–77. ©2013 AACR.

The current study was designed to investigate the effect of ginsenoside Rb1 (Rb1) on apoptosis induced by hypoxia and oxidative stress in a retinal ganglion cell line (RGC-5). The underlying mechanism was also investigated. RGC-5 cells were pretreated with 10 µmol/l Rb1 for 24 h and exposed to 400 µmol/l cobalt chloride (CoCl2) for 48 h or 600 µmol/l H2O2 for 24 h. The percentage of cells actively undergoing apoptosis was determined by flow cytometry with Annexin V/propidium iodide (PI) double staining. The expression of caspases was determined using western blot analysis. CoCl2 and H2O2 treatments significantly increased the apoptotic percentages to 24.5 and 21.63%, respectively. Pretreatment of Rb1 reduced the total apoptotic percentages to 15.12 and 12.03%, respectively. The expression of cleaved caspase-3, -9 and -8 was increased in the CoCl2-treated group, however, caspase-3 was not increased in the H2O2-treated group. Pretreatment of Rb1 reduced the expression of cleaved caspase-3 and -9 in the CoCl2-treated group, but reduced only cleaved caspase-9 in the H2O2-treated group. These results suggest that Rb1 may prevent RGC-5 cells from apoptosis against hypoxia and oxidative stress via the mitochondrial pathway.

HERV-K (human endogenous retrovirus type K) type 1-encoded Np9 is a tumor-specific biomarker, but its oncogenic role and targets in human leukemia remain elusive. We first identified Np9 as a potent viral oncogene in human leukemia. Silencing of Np9 inhibited the growth of myeloid and lymphoblastic leukemic cells, whereas expression of Np9 significantly promoted the growth of leukemia cells in vitro and in vivo. Np9 not only activated ERK, AKT and Notch1 pathways but also upregulated β-catenin essential for survival of leukemia stem cells. In human leukemia, Np9 protein level in leukemia patients was substantially higher than that in normal donors (56% vs 4.5%). Moreover, Np9 protein level was correlated with the number of leukemia stem/progenitor cells but not detected in normal CD34(+) hematopoietic stem cells. In addition, Np9-positive samples highly expressed leukemia-specific pol-env polyprotein, env and transmembrane proteins as well as viral particles. Thus, the viral oncogene Np9 is a critical molecular switch of multiple signaling pathways regulating the growth of leukemia stem/progenitor cells. These findings open a new perspective to understand the etiology of human common leukemia and provide a novel target for treating leukemia.

Transgenerational effects on health and development of early-life nutrition have gained increased attention recently. However, the underlying mechanisms of transgenerational transmission are only starting to emerge, with epigenetics as perhaps the most important mechanism. We recently reported the first animal model to study transgenerational programming of longevity after early-life dietary manipulations, enabling investigations to identify underlying epigenetic mechanisms. We report here that post-eclosion dietary manipulation (PDM) with a low-protein (LP) diet upregulates the protein level of E(z), an H3K27 specific methyltransferase, leading to higher levels of H3K27 trimethylation (H3K27me3). This PDM-mediated change in H3K27me3 corresponded with a shortened longevity of F0 flies as well as their F2 offspring. Specific RNAi-mediated post-eclosion knockdown of E(z) or pharmacological inhibition of its enzymatic function with EPZ-6438 in the F0 parents improved longevity while rendering H3K27me3 low across generations. Importantly, addition of EPZ-6438 to the LP diet fully alleviated the longevity-reducing effect of the LP PDM, supporting the increased level of E(z)-dependent H3K27me3 as the primary cause and immediate early-life period as the critical time to program longevity through epigenetic regulation. These observations establish E(z)-mediated H3K27me3 as one epigenetic mechanism underlying nutritional programming of longevity and support the use of EPZ-6438 to extend lifespan.

Zicao is being highlighted as a promising Chinese medicine due to all the beneficial effects that have been associated with its use. Unfortunately, studies on the toxicity of Zicao in different species are still missing and should be carried out. In this study, we investigated whether Acetylshikonin (AS) from Zicao has an anti-fertility effect through mating experiments and explored its underling mechanism. Sprague-Dawley rats received no treatment or were treated with 120, 360 or 1080 mg/kg AS extract by intragastric administration for 2 weeks. The rat pregnancy rate of the 1080 mg/kg dose group was significantly decreased relative to control group, while it recovered after a month of drug withdrawal, which indicated that the effect of antifertility is reversible. Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in rat were significantly decreased by AS. The secretion of FSH in rat anterior pituitary cells was decreased but the synthesis was not affected. AS reduced the number of developing follicle and mature follicle in rat ovarian cortical. Maybe all of these resulted from AS decreased the expression of synaptotagmin-1 and SNAP-25 which were the critical proteins of exocytosis. Our data suggested that AS at high dose can suppress the ability of pregnancy of the rats through decreasing serum FSH and LH levels by affecting exocytosis process of gonadotropic hormone (GTH).

Metabolic disorders such as diabetes are known risk factors for developing cholesterol gallstone disease (CGD). Cholesterol gallstone disease is one of the most prevalent digestive diseases, leading to considerable financial and social burden worldwide. Ursodeoxycholic acid (UDCA) is the only bile acid drug approved by FDA for the non-surgical treatment of gallstones. However, the molecular link between UDCA and CGD is unclear. Previous data suggest that the farnesoid X receptor (FXR), a bile acid nuclear receptor, may protect against the development of CGD. In studies aimed at identifying the role of FXR, we recently identify a novel chemical tool, 6EUDCA (6-αethyl-ursodeoxycholic acid), a synthetic derivative of UDCA, for studying FXR. We found that 6EUDCA binds FXR stronger than UDCA in a TR-FRET binding assay. This result was supported by computational docking models that suggest 6EUDCA forms a more extensive hydrogen bound network with FXR. Interestingly, neither compound could activate FXR target genes in human nor mouse liver cells, suggesting UDCA and 6EUDCA activate non-genomic signals in an FXR-dependent manner. Overall these studies may lead to the identification of a novel mechanism by which bile acids regulate cell function, and 6EUDCA may be an effective targeted CGD therapeutic.

Vertical sleeve gastrectomy (VSG) is one of the most commonly performed clinical bariatric surgeries used for the remission of obesity and diabetes. However, the precise molecular mechanism by which VSG exerts its beneficial effects remains elusive. We report that the membrane-bound G protein-coupled bile acid receptor, GPBAR-1 (also known as TGR5), is required to mediate the effects of anti-obesity, anti-hyperglycemia, and improvements of fatty liver of VSG in mice. In the absence of TGR5, the beneficial metabolic effects of VSG in mice are lost. Moreover, we found that the expression of TGR5 increased significantly after VSG, and VSG alters both BA levels and composition in mice, resulting in enhancement of TGR5 signaling in the ileum and brown adipose tissues, concomitant with improved glucose control and increased energy expenditure. Conclusion: Our study elucidates a novel underlying mechanism by which VSG achieves its postoperative therapeutic effects through enhanced TGR5 signaling. (Hepatology 2016;64:760-773)

Obesity and its major co-morbidity, type 2 diabetes, have reached an alarming epidemic prevalence without an effective treatment available. It has been demonstrated that inhibition of SREBP pathway may be a useful strategy to treat obesity with type 2 diabetes. Sterol regulatory element-binding proteins (SREBPs) are major transcription factors regulating the expression of genes involved in biosynthesis of cholesterol, fatty acid and triglyceride. In current study, we identified a small molecule, curcumin, inhibited the SREBP expression in vitro. The inhibition of SREBP by curcumin decreased the biosynthesis of cholesterol and fatty acid. In vivo, curcumin ameliorated HFD-induced body weight gain and fat accumulation in liver or adipose tissues, and improved serum lipid levels and insulin sensitivity in HFD-induced obese mice. Consistently, curcumin regulates SREBPs target genes and metabolism associated genes in liver or adipose tissues, which may directly contribute to the lower lipid level and improvement of insulin resistance. Take together, curcumin, a major active component of Curcuma longa could be a potential leading compound for development of drugs for the prevention of obesity and insulin resistance.

The nuclear receptor farnesoid X receptor (FXR) acts as a liver protector by regulating normal liver homeostasis. Spontaneously developed liver tumors have been found in FXR-null mice. However, the role of FXR in the tumorigenesis of human hepatocellular carcinoma (HCC) is still poorly understood. In this study, we measured the expression of FXR and its primary target gene, small heterodimer partner, and analyzed the clinical significance of FXR expression in HCC patients. A lentiviral vector that selectively overexpresses FXR was used to investigate the function of FXR in HCC cell proliferation both in vitro and in vivo . Our data showed that in human HCC, FXR expression was significantly reduced and was positively correlated with multiple malignant clinicopathological characteristics. Lentivirus-mediated exogenous FXR expression resulted in a marked increase of small heterodimer partner expression, significant repression of liver cancer cell proliferation, and tumor growth in nude mice. These results suggest that FXR may be of clinical and pharmacological importance as a promising biomarker of HCC.

Background & Aims Liver tumor, especially hepatocellular carcinoma (HCC), is closely associated with chronic inflammation. We previously showed that farnesoid X receptor knockout ( FXR − / − ) mice displayed chronic inflammation and developed spontaneous liver tumors when they aged. However, the mechanism by which inflammation leads to HCC in the absence of FXR is unclear. Because IFNγ is one of the most upregulated pro-inflammatory cytokines in FXR − / − livers, we generated IFNγ − / − FXR − / − double knockout mice to determine IFNγ's roles in hepatocarcinogenesis. Methods IFNγ − / − mice were crossed with an FXR − / − C57BL/6 background or injected i.p. with the hepatocarcinogen diethylnitrosamine (DEN). Hepatocarcinogenesis was analyzed with biochemical and histological methods. Results IFNγ deletion accelerated spontaneous hepatocarcinogenesis in FXR − / − mice and increased the susceptibility to DEN-induced hepatocarcinogenesis. IFNγ deletion enhanced activation of HCC promoters STAT3 and JNK/c-Jun, but abolished induction of p53 in IFNγ − / − livers after acute DEN-induced injury. Furthermore, hepatic p53 expression increased in aged wild type mice but not in aged IFNγ − / − and IFNγ − / − FXR − / − mice, while activation of STAT3 and JNK/c-Jun was enhanced in aged IFNγ − / − and IFNγ − / − FXR − / − mice. In addition, IFNγ inhibited liver cancer xenograft growth and impaired IL-6-induced STAT3 phosphorylation by inducing SOCS1/3 expression. Conclusions Increased IFNγ expression in FXR − / − livers represents a protective response of the liver against chronic injury and tumorigenesis. IFNγ suppresses hepatocarcinogenesis by inducing p53 expression and preventing STAT3 activation.

Aberrant epigenetic alterations during development may result in long-term epigenetic memory and have a permanent effect on the health of subjects. Constitutive androstane receptor (CAR) is a central regulator of drug/xenobiotic metabolism. Here, we report that transient neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism. CAR activation by neonatal exposure to the CAR-specific ligand 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) led to persistently induced expression of the CAR target genes Cyp2B10 and Cyp2C37 throughout the life of exposed mice. These mice showed a permanent reduction in sensitivity to zoxazolamine treatment as adults. Compared with control groups, the induction of Cyp2B10 and Cyp2C37 in hepatocytes isolated from these mice was more sensitive to low concentrations of the CAR agonist TCPOBOP. Accordingly, neonatal activation of CAR led to a permanent increase of histone 3 lysine 4 mono-, di-, and trimethylation and decrease of H3K9 trimethylation within the Cyp2B10 locus. Transcriptional coactivator activating signal cointegrator-2 and histone demethylase JMJD2d participated in this CAR-dependent epigenetic switch. Conclusion: Neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism. (HEPATOLOGY 2012)

Hypertrophic cardiomyopathy is a primary disorder characterized by asymmetric thickening of the septum and left ventricular wall, which affects 1 in 500 individuals in the general population. Mutations in mitochondrial DNA have been found to be one of the most important causes of hypertrophic cardiomyopathy. Here we report the clinical, genetic and molecular characterization of a Han Chinese family with a likely maternally transmitted hypertrophic cardiomyopathy. Four (2 men/2 women) of 5 matrilineal relatives in this 3-generation family exhibited the variable severity and age at onset of 44 to 79 years old. Sequence analysis of the entire mitochondrial DNA in this pedigree identified the known homoplasmic ND5 12338T>C variant. This mitochondrial DNA haplogroup belongs to the Eastern Asian F2a. The 12338T>C variant, highly evolutionarily conserved, resulted in the replacement of the translation initiating methionine with a threonine, shortening the ND5 polypeptide by 2 amino acids. The occurrence of ND5 12338T>C variant exclusively in maternal members of this Chinese family suggested that the 12338T>C variant is associated with maternally inherited hypertrophic cardiomyopathy. Our findings will provide theoretical basis for genetic counseling of maternally inherited hypertrophic cardiomyopathy.

Persistent Jak/Stat3 signal transduction plays a crucial role in tumorigenesis and immune development. Activated Jak/Stat3 signaling has been validated as a promising molecular target for cancer therapeutics discovery and development. Berbamine (BBM), a natural bis-benzylisoquinoline alkaloid, was identified from the traditional Chinese herbal medicine Berberis amurensis used for treatment of cancer patients. While BBM has been shown to have potent antitumor activities with low toxicity in various cancer types, the molecular mechanism of action of BBM remains largely unknown. Here, we determine the antitumor activities of 13 synthetic berbamine derivatives (BBMDs) against human solid tumor cells. BBMD3, which is the most potent in this series of novel BBMDs, exhibits over 6-fold increase in biological activity compared to natural BBM. Moreover, BBMD3, directly inhibits Jak2 autophosphorylation kinase activity in vitro with IC(50)0.69 μM. Autophosphorylation of Jak2 kinase at Tyr1007/1008 sites also was strongly inhibited in the range of 15 μM of BBMD3 in human melanoma cells at 4h after treatment. Following inhibition of autophosphorylation of Jak2, BBMD3 blocked constitutive activation of downstream Stat3 signaling in melanoma cells. BBMD3 also down-regulated expression of the Stat3 target proteins Mcl-1and Bcl-x(L), associated with induction of apoptosis. In sum, our findings demonstrate that the novel berbamine derivative BBMD3 is an inhibitor of the Jak2/Stat3 signaling pathway, providing evidence for a molecular mechanism whereby BBMD3 exerts at least in part the apoptosis of human melanoma cells. In addition, BBMD3 represents a promising lead compound for development of new therapeutics for cancer treatment.

Previous experimental studies have shown that acute graft-versus-host disease (GVHD) is associated with two waves of donor CD8+ T cell expansion. In the current studies, we used in vivo bioluminescent imaging, in vivo BrdU labeling, and three different experimental GVHD systems to show that B7-H1 expression by recipient parenchymal cells controls the second wave of alloreactive donor CD8+ T cell expansion and the associated second phase of GVHD. Loss of B7-H1 expression by parenchymal cells during the course of GVHD was associated with persistent proliferation of donor CD8+ T cells in GVHD target tissues and continued tissue injury, whereas persistent expression of B7-H1 expression by parenchymal cells led to reduced proliferation of donor CD8+ T cells in GVHD target tissues and resolution of GVHD. These studies demonstrate that parenchymal cell expression of B7-H1 is required for tolerizing infiltrating T cells and preventing the persistence of GVHD. Our results suggest that therapies designed to preserve or restore expression of B7-H1 expression by parenchymal tissues in the recipient could prevent or ameliorate GVHD in humans.

Acute lung injury and its more severe form, acute respiratory distress syndrome, are characterized by an acute inflammatory response in the airspaces and lung parenchyma. The nuclear receptor farnesoid X receptor (FXR) is expressed in pulmonary artery endothelial cells. Here, we report a protective role of FXR in a lipopolysaccharide-induced mouse model of acute lung injury. Upon intratracheal injection of lipopolysaccharide, FXR-/- mice showed higher lung endothelial permeability, released more bronchoalveolar lavage cells to the alveoli, and developed acute pneumonia. Cell adhesion molecules were expressed at higher levels in FXR-/- mice as compared with control mice. Furthermore, lung regeneration was much slower in FXR-/- mice. In vitro experiments showed that FXR activation blocked TNFα-induced expression of P-selectin but stimulated proliferation of lung microvascular endothelial cells through up-regulation of Foxm1b. In addition, expression of a constitutively active FXR repressed the expression of proinflammatory genes and improved lung permeability and lung regeneration in FXR-/- mice. This study demonstrates a critical role of FXR in suppressing the inflammatory response in lung and promoting lung repair after injury.

Background & Aims Previous studies indicate that bile acids (BAs) promote normal liver regeneration and repair after injury. However, the impact of insufficient BA signaling, which is observed in patients with BA sequestrant medication or cerebrotendinous xanthomatosis (CTX) disease, on liver injury is still unknown. Our aim is to determine the outcomes of reduced BA levels upon liver injury. Methods Seventy percent partial hepatectomy (PH) and carbon tetrachloride (CCl 4 ) treatment were performed using CYP27 −/− mice, a genetic animal model with low BA levels. The liver repair of CYP27 −/− mice after the treatments was characterized by histological staining, chemical analysis, and quantitative real-time PCR. Results CYP27 −/− mice exhibited enhanced CCl 4 -induce liver injury, and defective liver regeneration and prolonged steatosis after 70% PH. Due to the insufficient BA signaling, farnesoid X receptor (FXR) activities were significantly reduced in CYP27 −/− livers after 70% PH. Activation of FXR by either 0.2% cholic acid feeding or oral infusion of an FXR agonist greatly promoted liver regeneration in CYP27 −/− mice. Conclusions Normal physiological levels of BAs are required for liver repair. Patients with BA sequestrant medications or CTX disease due to CYP27 gene mutations may have an increased risk of liver failure, and treatment with FXR ligands can promote liver regeneration of patients with low BA levels.

Gpbar1 (TGR5), a membrane-bound bile acid receptor, is well known for its roles in regulation of energy homeostasis and glucose metabolism. TGR5 also displays strong attenuation of macrophage reactivity in vitro, but the physiological roles of TGR5 in inflammatory response, and its mechanism, is unknown. Here, we demonstrate that TGR5 is a negative modulator of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB)-mediated inflammation. TGR5 activation suppresses the phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), the translocation of p65, NF-κB DNA-binding activity, and its transcription activity. Furthermore, TGR5 activation enhances the interaction of IκBα and β-arrestin2. Suppression of NF-κB transcription activity and its target gene expression by TGR5 agonist are specifically abolished by the expression of anti-β-arrestin2 small interfering RNA. These results show that TGR5 suppresses the NF-κB pathway by mediation of the interaction between IκBα and β-arrestin2. In a lipopolysaccharide (LPS)-induced inflammation model, TGR5−/− mice show more severe liver necroses and inflammation, compared with wild-type (WT) mice. Activation of TGR5 by its agonist ligand inhibits the expression of inflammatory mediators in response to NF-κB activation induced by LPS in WT, but not TGR5−/−, mouse liver. Conclusion: These findings identify TGR5 as a negative mediator of inflammation that may serve as an attractive therapeutic tool for immune and inflammatory liver diseases. (HEPATOLOGY 2011;)

The liver can fully regenerate itself by a compensatory regrowth in response to partial hepatectomy or injury. This process consists of a variety of well-orchestrated phases and is mediated by many signals. Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. Bile acids are FXR physiological ligands. As a metabolic regulator, FXR plays key roles in regulating metabolism of bile acids, lipids and glucose. Recently, bile acid/FXR signaling pathway is shown to be required for normal liver regeneration. Furthermore, FXR promotes liver repair after injury and activation of FXR is able to alleviate age-related defective liver regeneration. These novel findings suggest that FXR-mediated bile acid signaling is an integrated component of normal liver regeneration machinery, and also highlight the potential use of FXR ligands to promote liver regeneration after segmental liver transplantation or resection of liver tumors. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Liver cancer, hepatocellular carcinoma (HCC) in particular, is one of the most deadly cancers worldwide, and the incidence of HCC is increasing rapidly in the United States and other developed countries. Epidemiological studies have identified major risk factors for HCC, including infection with hepatitis B and C virus (HBV and HCV), exposure to certain chemicals, high intake of alcohol, as well as metabolic diseases such as obesity and diabetes that are rapidly rising in the US. Although the etiologies for liver carcinogenesis are relatively well defined, the exact mechanism and pathways leading to cancer development are still unclear. Chronic liver injury, followed by inflammation and irregular liver regeneration has been suggested as an important step in hepatocarcinogenesis. Therefore, the identification of key factors that protect the liver from injury and inflammation could provide valuable insight into the development of HCC. In this review, we will summarize the recent findings in HCC studies, mainly focusing on the new molecular link among inflammation, liver repair and HCC.

Introduction. We examined the murine hepatectomy model of liver regeneration (LR) in the setting of neoadjuvant chemotherapy. Methods. C57BL/6 mice were randomized to receive neoadjuvant intraperitoneal (IP) injections of a control, oxaliplatin (15 mg/kg), or irinotecan (100 mg/Kg or 250 mg/Kg) solution. Hepatectomy (70%) was performed 14 days after the final IP treatment. Animals were sacrificed at postoperative day (D) 0, 1, 2, 3, and 7. Liver remnants and serum were collected for analysis. 𝑇 -tests for independent samples were used for statistical comparisons. Results. For oxaliplatin, percent LR did not differ at D1 or D2 but was significantly less at D3 (89.0% versus 70.0%, 𝑃 = 0 . 0 4 8 ) with no difference on D7 ( 𝑃 = 0 . 2 1 ). Irinotecan-treated mice at both dose levels (100 mg/Kg and 250 mg/Kg) showed no significant differences in LR. BrdU incorporation was significantly decreased in oxaliplatin-treated animals (D1,2,3). Conclusions. Neoadjuvant oxaliplatin but not irinotecan impairs early LR in a posthepatectomy murine model which correlates with decreased DNA synthesis.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by interacting with the 3′ untranslated region (3′-UTR) of multiple mRNAs. Recent studies have linked miRNAs to the development of cancer metastasis. In this study, we show that miR-194 is specifically expressed in the human gastrointestinal tract and kidney. Moreover, miR-194 is highly expressed in hepatic epithelial cells, but not in Kupffer cells or hepatic stellate cells, two types of mesenchymal cells in the liver. miR-194 expression was decreased in hepatocytes cultured in vitro, which had undergone a dedifferentiation process. Furthermore, expression of miR-194 was low in liver mesenchymal-like cancer cell lines. The overexpression of miR-194 in liver mesenchymal-like cancer cells reduced the expression of the mesenchymal cell marker N-cadherin and suppressed invasion and migration of the mesenchymal-like cancer cells both in vitro and in vivo. We further demonstrated that miR-194 targeted the 3′-UTRs of several genes that were involved in epithelial-mesenchymal transition and cancer metastasis. Conclusion: These results support a role of miR-194, which is specifically expressed in liver parenchymal cells, in preventing liver cancer cell metastasis. (HEPATOLOGY 2010;.)

Constitutive androstane receptor CAR (NR1I3) has been identified as a central mediator of coordinate responses to xenobiotic and endobiotic stress. Here we use leptin-deficient mice (ob/ob) and ob/ob, CAR −/− double mutant mice to identify a metabolic role of CAR in type 2 diabetes. Activation of CAR significantly reduces serum glucose levels and improves glucose tolerance and insulin sensitivity. Gene expression analyses and hyperinsulinemic euglycemic clamp results suggest that CAR activation ameliorates hyperglycemia by suppressing glucose production and stimulating glucose uptake and usage in the liver. In addition, CAR activation dramatically improves fatty liver by both inhibition of hepatic lipogenesis and induction of β-oxidation. We conclude that CAR activation improves type 2 diabetes, and that these actions of CAR suggest therapeutic approaches to the disease.

In comparison with the relatively slow progress of battery technology, semiconductor memory has improved much more rapidly, making storage a less critical limiting factor in designing low power embedded systems such as PDAs. To exploit such technology trends, we present a novel framework, a joint encoder–decoder framework (JEDF), which allows the decoder to tradeoff energy and memory consumption without sacrificing playback quality. We employ sum-of-powers-of-two (SOPOT) technique, an approximate signal processing (ASP) technique, in an MPEG AAC decoder to reduce the computational workload. The SOPOT introduces additional ASP noise (in the decoder) on top of the quantization noise introduced in the process of lossy compression (in the encoder). The sum of these two kinds of noise may become audible when it exceeds the masking threshold. We tackle this problem from a new perspective: the proposed JEDF allows the ASP and quantization noises to be shaped jointly to match the masking threshold. In the case that the perceptual room between the masking threshold and the quantization noise is insufficient for the ASP noise, the JEDF can reduce the quantization noise level which results in an increase in bitrate. To implement the proposed scheme, we have developed two new techniques: (1) SOPOT truncation noise shaping; (2) truncation noise allocation based on a perceptual model. Experimental results show the effectiveness of our approach.

The constitutive androstane receptor (CAR) is a xenosensing nuclear receptor and regulator of cytochrome P450s (CYPs). However, the role of CAR as a basal regulator of CYP expression nor its role in sexually dimorphic responses have been thoroughly studied. We investigated basal regulation and sexually dimorphic regulation and induction by the potent CAR activator TCPOBOP and the moderate CAR activator Nonylphenol (NP). NP is an environmental estrogen and one of the most commonly found environmental toxicants in Europe and the United States. Previous studies have demonstrated that NP induces several CYPs in a sexually dimorphic manner, however the role of CAR in regulating NP-mediated sexually dimorphic P450 expression and induction has not been elucidated. Therefore, wild-type and CAR-null male and female mice were treated with honey as a carrier, NP, or TCPOBOP and CYP expression monitored by QPCR and Western blotting. CAR basally regulates the expression of Cyp2c29, Cyp2b13, and potentially Cyp2b10 as demonstrated by QPCR. Furthermore, we observed a shift in the testosterone 6alpha/15alpha-hydroxylase ratio in untreated CAR-null female mice to the male pattern, which indicates an alteration in androgen status and suggests a role for androgens as CAR inverse agonists. Xenobiotic-treatments with NP and TCPOBOP induced Cyp2b10, Cyp2c29, and Cyp3a11 in a CAR-mediated fashion; however NP only induced these CYPs in females and TCPOBOP induced these CYPs in both males and females. Interestingly, Cyp2a4, was only induced in wild-type male mice by TCPOBOP suggesting Cyp2a4 induction is not sensitive to CAR-mediated induction in females. Overall, TCPOBOP and NP show similar CYP induction profiles in females, but widely different profiles in males potentially related to lower sensitivity of males to either indirect or moderate CAR activators such as NP. In summary, CAR regulates the basal and chemically inducible expression of several sexually dimorphic xenobiotic metabolizing P450s in a manner that varies depending on the ligand.

Cholesterol 7alpha-hydroxylase (CYP7a1) is the rate-limiting enzyme in the classic pathway of bile acid synthesis. Expression of CYP7a1 is regulated by a negative feedback pathway of bile acid signaling. Previous studies have suggested that bile acid signaling is also required for normal liver regeneration, and CYP7a1 expression is strongly repressed after 70% partial hepatectomy (PH). Both the effect of CYP7a1 suppression on liver regrowth and the mechanism by which 70% PH suppresses CYP7a1 expression are unknown. Here we show that liver-specific overexpression of an exogenous CYP7a1 gene impaired liver regeneration after 70% PH, which was accompanied by increased hepatocyte apoptosis and liver injury. CYP7a1 expression was initially suppressed after 70% PH in an farnesoid X receptor/ small heterodimer partner-independent manner; however, both farnesoid X receptor and small heterodimer partner were required to regulate CYP7a1 expression at the later stage of liver regeneration. c-Jun N-terminus kinase and hepatocyte growth factor signaling pathways are activated during the acute phase of liver regeneration. We determined that hepatocyte growth factor and c-Jun N-terminus kinase pathways were involved in the suppressing of the CYP7a1 expression in the acute phase of live regeneration. Taken together, our results provide the significance that CYP7a1 suppression is required for liver protection after 70% PH and there are two distinct phases of CYP7a1 gene regulation during liver regeneration.

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. As a metabolic regulator, FXR plays key roles in bile acid, cholesterol, lipid, and glucose metabolism. Therefore, FXR is a potential drug target for a number of metabolic disorders, especially those related to the metabolic syndrome. More recently, our group and others have extended the functions of FXR to more than metabolic regulation, which include anti-bacterial growth in intestine, liver regeneration, and hepatocarcinogenesis. These new findings suggest that FXR has much broader roles than previously thought, and also highlight FXR as a drug target for multiple diseases. This review summarizes the basic information of FXR but focuses on its new functions.

The farnesoid X receptor (FXR) is a nuclear receptor that plays key roles in hepatoprotection by maintaining the homeostasis of liver metabolism. FXR null mice display strong hepatic inflammation and develop spontaneous liver tumors. In this report, we demonstrate that FXR is a negative modulator of nuclear factor kappaB (NF-kappaB)-mediated hepatic inflammation. Activation of FXR by its agonist ligands inhibited the expression of inflammatory mediators in response to NF-kappaB activation in both HepG2 cells and primary hepatocytes cultured in vitro. In vivo, compared with wild-type controls, FXR(-/-) mice displayed elevated messenger RNA (mRNA) levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interferon-inducible protein 10, and interferon-gamma in response to lipopolysaccharide (LPS). Examination of FXR(-/-) livers showed massive necroses and inflammation after treatment with LPS at a dose that does not induce significant liver damage or inflammation in wild-type mice. Moreover, transfection of a constitutively active FXR expression construct repressed the iNOS, COX-2, interferon-inducible protein 10 and interferon-gamma mRNA levels induced by LPS administration. FXR activation had no negative effects on NF-kappaB-activated antiapoptotic genes, suggesting that FXR selectively inhibits the NF-kappaB-mediated hepatic inflammatory response but maintains or even enhances the cell survival response. On the other hand, NF-kappaB activation suppressed FXR-mediated gene expression both in vitro and in vivo, indicating a negative crosstalk between the FXR and NF-kappaB signaling pathways. Our findings reveal that FXR is a negative mediator of hepatic inflammation, which may contribute to the critical roles of FXR in hepatoprotection and suppression of hepatocarcinogenesis.

Oltipraz (OPZ) is a well known inducer of NAD(P)H:quinone oxidoreductase (NQO1) along with other enzymes that comprise the nuclear factor E2-related factor 2 (Nrf2) battery of detoxification genes. However, OPZ treatment also induces expression of CYP2B, a gene regulated by the constitutive androstane receptor (CAR). Therefore, this study was designed to determine whether OPZ induces gene expression in the mouse liver through activation of CAR in addition to Nrf2. OPZ increased the mRNA expression of both Cyp2b10 and Nqo1 in C57BL/6 mouse livers. As expected, in livers from Nrf2-/- mice, OPZ induction of Nqo1 was reduced, indicating Nqo1 induction is dependent on Nrf2 activation, whereas Cyp2b10 induction was unchanged. The robust induction of Cyp2b10 by OPZ in wild-type mice was completely absent in CAR-/- mice, revealing a CAR-dependent induction by OPZ. OPZ also induced transcription of the human CYP2B6 promoter-reporter containing the phenobarbital (PB) responsive element in mouse liver using an in vivo transcription assay. Additionally, OPZ induced in vivo nuclear accumulation of CAR at 3 h but, as with PB, was unable to reverse androstanol repression of mouse CAR constitutive activity in transiently transfected HepG2 cells. In summary, OPZ induces expression of Cyp2b10 and Nqo1 via the activation of CAR and Nrf2, respectively.

Activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) by xenobiotic inducers of cytochromes P450 is part of a pleiotropic response that includes liver hypertrophy, tumor promotion, effects on lipid homeostasis, and energy metabolism. Here, we describe an acute response to CAR and PXR activators that is associated with induction of Insig-1, a protein with antilipogenic properties. We first observed that activation of CAR and PXR in mouse liver results in activation of Insig-1 along with reduced protein levels of the active form of sterol regulatory element binding protein 1 (Srebp-1). Studies in mice deficient in CAR and PXR revealed that the effect on triglycerides involves these two nuclear receptors. Finally, we identified a functional binding site for CAR and PXR in the Insig-1 gene by in vivo, in vitro, and in silico genomic analysis. Our experiments suggest that activation Insig-1 by drugs leads to reduced levels of active Srebp-1 and consequently to reduced target gene expression including the genes responsible for triglyceride synthesis. The reduction nuclear Srebp-1 by drugs is not observed when Insig-1 expression is repressed by small interfering RNA. In addition, observed that Insig-1 is also a target of AMP-activated kinase, the hepatic activity of which is increased by activators of CAR and PXR and is known to cause a reduction of triglycerides. The fact that drugs that serve as CAR or PXR ligands induce Insig-1 might have clinical consequences and explains alterations lipid levels after drug therapy.

Our previous microarray expression analysis of the long-lived Little mice (Ghrhr(lit/lit)) showed a concerted up-regulation of xenobiotic detoxification genes. Here, we show that this up-regulation is associated with a potent increase in resistance against the adverse effects of a variety of xenobiotics, including the hepatotoxins acetaminophen and bromobenzene and the paralyzing agent zoxazolamine. The classic xenobiotic receptors Car (Constitutive Androstane Receptor) and Pxr (Pregnane X Receptor) are considered key regulators of xenobiotic metabolism. Using double and triple knockout/mutant mouse models we found, however, that Car and Pxr are not required for the up-regulation of xenobiotic genes in Little mice. Our results suggest instead that bile acids and the primary bile acid receptor Fxr (farnesoid X receptor) are likely mediators of the up-regulation of xenobiotic detoxification genes in Little mice. Bile acid levels are considerably elevated in the bile, serum, and liver of Little mice. We found that treatment of wild-type animals with cholic acid, one of the major bile acids elevated in Little mice, mimics in large part the up-regulation of xenobiotic detoxification genes observed in Little mice. Additionally, the loss of Fxr had a major effect on the expression of the xenobiotic detoxification genes up-regulated in Little mice. A large fraction of these genes lost or decreased their high expression levels in double mutant mice for Fxr and Ghrhr. The alterations in xenobiotic metabolism in Little mice constitute a form of increased stress resistance and may contribute to the extended longevity of these mice.

Gpbar1 (TGR5), a membrane-bound bile acid receptor, is well-known for its roles in regulation of energy homeostasis and glucose metabolism. Here, we show that mice lacking TGR5 were much more susceptible to lipopolysaccharide (LPS)-induced acute gastric inflammation than wild-type (WT) mice and TGR5 is a negative regulator of gastric inflammation through antagonizing NF-κB signaling pathway. We found that the treatment of TGR5 ligands 23(S)-mCDCA and GPBARA (3-(2-Chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethylisoxazole-4-carboxamide) suppressed gene and protein expression mediated by NF-κB signaling. TGR5 overexpression with ligand treatment inhibited gene expression of interferon-inducible protein 10 (IP-10), TNF-α, and chemoattractant protein-1 (MCP-1) induced by LPS. Furthermore, we revealed that TGR5 activation antagonized NF-κB signaling pathway through suppressing its transcription activity, the phosphorylation of IκBα and p65 translocation, which suggests that TGR5 antagonizes gastric inflammation at least in part by inhibiting NF-κB signaling. These findings identify TGR5 as a negative mediator of gastric inflammation that may serve as an attractive therapeutic tool for human gastric inflammation and cancer.

Salvia miltiorrhiza and ligustrazine are traditional Chinese medicines that have been used in combination for treatment of cardiovascular disease, including coronary heart disease, cardiac angina and atherosclerosis in Asia, in particular, China. The present study aimed to determine the effect of S. miltiorrhiza and ligustrazine injection (SLI) on myocardial ischemia/reperfusion (I/R) and hypoxia/reoxygenation (H/R) injuries via the Akt serine/threonine kinase (Akt)‑endothelial nitric oxide synthase (eNOS) signaling pathway. Male Sprague‑Dawley rats were randomly assigned into six groups: i) Sham group; ii) I/R group; iii) Low‑SLI group (6.8 mg/kg/day, i.p.); iv) Medium‑SLI group (20.4 mg/kg/day, i.p.); v) High‑SLI group (61.2 mg/kg/day, i.p.); vi) verapamil group (6 mg/kg/day, i.p.). Prior to surgery, the aforementioned groups were pretreated with a homologous drug once per day for 3 days. The effect of SLI following 35 min coronary artery occlusion and 2 h reperfusion was evaluated by determining infarct size, hemodynamics, biochemical values and histological observations. Additionally, cell viability, caspase‑3 expression, B cell leukemia/lymphoma‑2 (Bcl‑2)/Bcl‑2‑associated X protein (Bax) ratio, phosphorylated (p‑)Akt and p‑eNOS were also investigated following 2 h simulated ischemia and 2 h simulated reperfusion in H9C2 cardiomyocyte cells. Pretreatment with SLI significantly improved cardiac function in a dose‑dependent manner and reduced myocardial infarct size, creatine kinase, lactate dehydrogenase and malondialdehyde levels in blood serum. Additionally, myocardial histopathology changes in the rat model were also alleviated in SLI treatment groups. The present in vitro study revealed that treatment with SLI reduced the apoptotic rate of H9C2 cells by inhibiting the activation of caspase‑3 and increasing the Bcl‑2/Bax ratio. The effect of SLI was associated with increased phosphorylation of the survival kinase Akt at Ser473 and its downstream target eNOS following H/R. The present study determined that SLI may alleviate I/R injury in cardiomyocytes and inhibit apoptosis in rats by the activation of the Akt‑eNOS signaling pathway, and downregulation of the expression levels of proapoptotic factors, including caspase-3.

Aberrant β-catenin activation contributes to a third or more of human hepatocellular carcinoma (HCC), but β-catenin activation alone is not sufficient to induce liver cancer in mice. Differentiated hepatocytes proliferate upon acute activation of either β-catenin or the nuclear xenobiotic receptor CAR. These responses are strictly limited and are tightly linked, since β-catenin is activated in nearly all of the CAR-dependent tumours generated by the tumour promoter phenobarbital. Here, we show that full activation of β-catenin in the liver induces senescence and growth arrest, which is overcome by combined CAR activation, resulting in uncontrolled hepatocyte proliferation, hepatomegaly and rapid lethality despite maintenance of normal liver function. Combining CAR activation with limited β-catenin activation induces tumorigenesis, and the tumours share a conserved gene expression signature with β-catenin-positive human HCC. These results reveal an unexpected route for hepatocyte proliferation and define a murine model of hepatocarcinogenesis with direct relevance to human HCC.

The liver can rapidly regenerate itself in response to partial hepatectomy or liver injury, which is accomplished by a complex network of different cell types and signals both inside and outside the liver. Bile acids (BAs) are liver-specific metabolic signals and promote liver regeneration by activating two BA receptors: farnesoid X receptor (FXR) and G-protein-coupled BA receptor 1 (GPBAR1 or TGR5). FXR is a member of the nuclear hormone receptor superfamily. Both liver- and intestine-FXR are activated after liver resection or injury and promote liver regeneration through distinct mechanism. Moreover, activation of FXR is able to alleviate age-related liver regeneration defects. TGR5 is a membrane-bound BA receptor, and it regulates BA hydrophobicity and stimulates BA excretion in urine during liver regeneration. FXR and TGR5 thus constitute a novel metabolic pathway during liver regeneration. Aberrant regulation of this pathway is linked to liver carcinogenesis and other liver diseases.

The CYP3As are broad-spectrum drug-metabolizing enzymes that are collectively responsible for more than 50% of xenobiotic metabolism. Unlike other CYP3As, murine CYP3A44 is expressed predominantly in the female liver, with much lower levels in male livers and no detectable expression in brain or kidney in either gender. In this study, we examined the role of nuclear hormone receptors in the regulation of Cyp3a44 gene expression. Interestingly, we observed differential effects of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) -mediated activation of Cyp3a44 gene expression, which was gender-specific. For example, activation of PXR by pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone (DEX) induced CYP3A44 mRNA levels in a PXR-dependent fashion in male mice, whereas no induction was detected in female mice. In contrast, PCN and DEX down-regulated CYP3A44 expression in female PXR null animals. Similar to PXR, CAR activation also showed a male-specific induction with no effect on CYP3A44 levels in females. When PXR knockout mice were challenged with the CAR activator phenobarbital, a significant up-regulation of male CYP3A44 levels was observed, whereas levels in females remained unchanged. We conclude that gender has a critical impact on PXR- and CAR-mediated effects of CYP3A44 expression.

Liver mass depends on one or more unidentified humoral signals that drive regeneration when liver functional capacity is diminished. Bile acids are important liver products, and their levels are tightly regulated. Here, we identify a role for nuclear receptor–dependent bile acid signaling in normal liver regeneration. Elevated bile acid levels accelerate regeneration, and decreased levels inhibit liver regrowth, as does the absence of the primary nuclear bile acid receptor FXR. We propose that FXR activation by increased bile acid flux is a signal of decreased functional capacity of the liver. FXR, and possibly other nuclear receptors, may promote homeostasis not only by regulating expression of appropriate metabolic target genes but also by driving homeotrophic liver growth.

The constitutive androstane receptor (CAR) modulates xeno- and endobiotic hepatotoxicity by regulating detoxification pathways. Whether activation of CAR may also protect against liver injury by directly blocking apoptosis is unknown. To address this question, CAR wild-type (CAR+/+) and CAR knockout (CAR-/-) mice were treated with the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and then with the Fas agonist Jo2 or with concanavalin A (ConA). Following the administration of Jo2, hepatocyte apoptosis, liver injury, and animal fatalities were abated in TCPOBOP-treated CAR+/+ but not in CAR-/- mice. Likewise, acute and chronic ConA-mediated liver injury and fibrosis were also reduced in wild-type versus CAR(-/-) TCPOBOP-treated mice. The proapoptotic proteins Bak (Bcl-2 antagonistic killer) and Bax (Bcl-2-associated X protein) were depleted in livers from TCPOBOP-treated CAR+/+ mice. In contrast, mRNA expression of the antiapoptotic effector myeloid cell leukemia factor-1 (Mcl-1) was increased fourfold. Mcl-1 promoter activity was increased by transfection with CAR and administration of TCPOBOP in hepatoma cells, consistent with a direct CAR effect on Mcl-1 transcription. Indeed, site-directed mutagenesis of a putative CAR consensus binding sequence on the Mcl-1 promoter decreased Mcl-1 promoter activity. Mcl-1 transgenic animals demonstrated little to no acute liver injury after administration of Jo2, signifying Mcl-1 cytoprotection. In conclusion, these observations support a prominent role for CAR cytoprotection against Fas-mediated hepatocyte injury via a mechanism involving upregulation of Mcl-1 and, likely, downregulation of Bax and Bak.

A double null mouse line (2XENKO) lacking the xenobiotic receptors CAR (constitutive androstane receptor) (NR1I3) and PXR (pregnane X receptor) (NR1I2) was generated to study their functions in response to potentially toxic xenobiotic and endobiotic stimuli. Like the single knockouts, the 2XENKO mice are viable and fertile and show no overt phenotypes under normal conditions. As expected, they are completely insensitive to broad range xenobiotic inducers able to activate both receptors, such as clotrimazole and dieldrin. Comparisons of the single and double knockouts reveal specific roles for the two receptors. Thus, PXR does not contribute to the process of acetaminophen hepatotoxicity mediated by CAR, but both receptors contribute to the protective response to the hydrophobic bile acid lithocholic acid (LCA). As previously observed with PXR (Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C. M., Nelson, M. C., Ong, E. S., Waxman, D. J., and Evans, R. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3375-3380), pharmacologic activation of CAR induces multiple LCA detoxifying enzymes and provides strong protection against LCA toxicity. Comparison of their responses to LCA treatment demonstrates that CAR predominantly mediates induction of the cytochrome p450 CYP3A11 and the multidrug resistance-associated protein 3 transporter, whereas PXR is the major regulator of the Na+-dependent organic anion transporter 2. These differential responses may account for the significant sensitivity of the CAR knockouts, but not the PXR knockouts, to an acute LCA dose. Because this sensitivity is not further increased in the 2XENKO mice, CAR may play a primary role in acute responses to this toxic endobiotic. These results define a central role for CAR in LCA detoxification and show that CAR and PXR function coordinately to regulate both xenobiotic and bile acid metabolism.