Regulation of constitutive androstane receptor and its target genes by fasting, cAMP, hepatocyte nuclear factor alpha, and the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha

Animal studies reveal that fasting and caloric restriction produce increased activity of specific metabolic pathways involved in resistance to weight loss in liver. Evidence suggests that this phenomenon may in part occur through the action of the constitutive androstane receptor (CAR, NR1I3). Currently, the precise molecular mechanisms that activate CAR during fasting are unknown. We show that fasting coordinately induces expression of genes encoding peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), CAR, cytochrome P-450 2b10 (Cyp2b10), UDP-glucuronosyltransferase 1a1 (Ugt1a1), sulfotransferase 2a1 (Sult2a1), and organic anion-transporting polypeptide 2 (Oatp2) in liver in mice. Treatments that elevate intracellular cAMP levels also produce increased expression of these genes in cultured hepatocytes. Our data show that PGC-1alpha interaction with hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1) directly regulates CAR gene expression through a novel and evolutionarily conserved HNF4-response element (HNF4-RE) located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Genetic studies reveal that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Taken together, our data show that fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver. Given the recent finding that mice lacking CAR exhibit a profound decrease in resistance to weight loss during extended periods of caloric restriction, our findings have important implications in the development of drugs for the treatment of obesity and related diseases.     

The nuclear receptor CAR (NR1I3) regulates serum triglyceride levels under conditions of metabolic stress

The nuclear receptor constitutive androstane receptor (CAR) (NR1I3) regulates hepatic genes involved in xenobiotic detoxification as well as genes involved in energy homeostasis. We provide data that extend the role of CAR to regulation of serum triglyceride levels under conditions of metabolic/nutritional stress. The typically high serum triglyceride levels of ob/ob mice were completely normalized when crossed onto a Car(-/-) (mice deficient for the Car gene) genetic background. Moreover, increases in serum triglycerides observed after a high-fat diet (HFD) regime were not observed in Car(-/-) animals. Conversely, pharmacological induction of CAR activity using the selective mouse CAR agonist TCPOBOP during HFD feeding resulted in a CAR-dependent increase in serum triglyceride levels. A major regulator of hepatic fatty oxidation is the nuclear receptor PPARalpha (NR1C1). The expression of peroxisome proliferator-activated receptor alpha (PPARalpha) target genes was inversely related to the activity of CAR. Consistent with these observations, Car(-/-) animals exhibited increased hepatic fatty acid oxidation. Treatment of mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) significantly decreased expression of PPARalpha mRNA as well as Cyp4a14, CPT1alpha, and cytosolic Acyl-CoA thioesterase (CTE) in the liver. These data have implications in disease therapy such as for diabetes and nonalcoholic steatohepatitis (NASH).     

Hepatocyte nuclear factor-4alpha regulates the human apolipoprotein AV gene: identification of a novel response element and involvement in the control by peroxisome proliferator-activated receptor-gamma coactivator-1alpha, AMP-activated protein kinase, and mitogen-activated protein kinase pathway

The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4alpha (HNF-4alpha) as a novel regulator of human apoAV gene. Inhibition of HNF-4alpha expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4alpha directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4alpha consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor-gamma coactivator-1alpha was capable of stimulating the HNF-4alpha-dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4alpha. Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4alpha gene revealed a species-distinct regulation of apoAV by HNF-4alpha, which resembles that of a subset of HNF-4alpha target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4alpha and underscore the role of HNF-4alpha in regulating triglyceride metabolism.     

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha activation of CYP7A1 during food restriction and diabetes is still inhibited by small heterodimer partner

Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic pathway of hepatic bile acid biosynthesis from cholesterol. During fasting and in type I diabetes, elevated levels of peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) induce expression of the Cyp7A1 gene and overexpression of PGC-1alpha in hepatoma cells stimulates bile acid synthesis. Using Ad-PGC-1alpha-RNA interference to induce acute disruption of PGC-1alpha in mice, here we show that PGC-1alpha is necessary for fasting-mediated induction of CYP7A1. Co-immunoprecipitation and promoter activation studies reveal that the induction of CYP7A1 is mediated by direct interaction between PGC-1alpha and the AF2 domain of liver receptor homolog-1 (LRH-1). In contrast, the very similar PGC-1beta could not substitute for PGC-1alpha. We also show that transactivation of PGC-1alpha and LRH-1 is repressed by the small heterodimer partner (SHP). Treatment of mice with GW4064, a synthetic agonist for farnesoid X receptor, induced SHP expression and decreased both the recruitment of PGC-1alpha to the Cyp7A1 promoter and the fasting-induced expression of CYP7A1 mRNA. These data suggest that PGC-1alpha is an important co-activator for LRH-1 and that SHP targets the interaction between LRH-1 and PGC-1alpha to inhibit CYP7A1 expression. Overall, these studies provide further evidence for the important role of PGC-1alpha in bile acid homeostasis and suggest that pharmacological targeting of farnesoid X receptor in vivo can be used to reverse the increase in CYP7A1 associated with adverse metabolic conditions.     

Complementary roles of farnesoid X receptor,pregnane X receptor,and constitutive androstane receptor in protection against bile acid toxicity

The nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR), are important in maintaining bile acid homeostasis. Deletion of both FXR and PXR in vivo by cross-breeding B6;129-Fxrtm1Gonz (FXR-null) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice revealed a more severe disruption of bile acid, cholesterol, and lipid homeostasis in B6;129-Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice fed a 1% cholic acid (CA) diet. Hepatic expression of the constitutive androstane receptor (CAR) and its target genes was induced in FXR- and FPXR-null mice fed the CA diet. To test whether up-regulation of CAR represents a means of protection against bile acid toxicity to compensate for the loss of FXR and PXR, animals were pretreated with CAR activators, phenobarbital or 1,4-bis[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), followed by the CA diet. A role for CAR in protection against bile acid toxicity was confirmed by a marked reduction of serum bile acid and bilirubin concentrations, with an elevation of the expression of the hepatic genes involved in bile acid and/or bilirubin metabolism and excretion (CYP2B, CYP3A, MRP2, MRP3, UGT1A, and glutathione S-transferase alpha), following pretreatment with phenobarbital or TCPOBOP. In summary, the current study demonstrates a critical and combined role of FXR and PXR in maintaining not only bile acid but also cholesterol and lipid homeostasis in vivo. Furthermore, FXR, PXR, and CAR protect against hepatic bile acid toxicity in a complementary manner, suggesting that they serve as redundant but distinct layers of defense to prevent overt hepatic damage by bile acids during cholestasis.     

The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR

A wide range of xenobiotic compounds are metabolized by cytochrome P450 (CYP) enzymes, and the genes that encode these enzymes are often induced in the presence of such compounds. Here, we show that the nuclear receptor CAR can recognize response elements present in the promoters of xenobiotic-responsive CYP genes, as well as other novel sites. CAR has previously been shown to be an apparently constitutive transactivator, and this constitutive activity is inhibited by androstanes acting as inverse agonists. As expected, the ability of CAR to transactivate the CYP promoter elements is blocked by the inhibitory inverse agonists. However, CAR transactivation is increased in the presence of 1,4-bis[2-(3, 5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent known member of the phenobarbital-like class of CYP-inducing agents. Three independent lines of evidence demonstrate that TCPOBOP is an agonist ligand for CAR. The first is that TCPOBOP acts in a dose-dependent manner as a direct agonist to compete with the inhibitory effect of the inverse agonists. The second is that TCPOBOP acts directly to stimulate coactivator interaction with the CAR ligand binding domain, both in vitro and in vivo. The third is that mutations designed to block ligand binding block not only the inhibitory effect of the androstanes but also the stimulatory effect of TCPOBOP. Importantly, these mutations do not block the apparently constitutive transactivation by CAR, suggesting that this activity is truly ligand independent. Both its ability to target CYP genes and its activation by TCPOBOP demonstrate that CAR is a novel xenobiotic receptor that may contribute to the metabolic response to such compounds.     

Regulation of gene expression of hepatic drug metabolizing enzymes and transporters by the Toll-like receptor 2 ligand, lipoteichoic acid

Expression of hepatic drug metabolizing enzymes (DMEs) is altered in infection and inflammation. However, the role of Gram+ve bacterial components and their receptor, Toll-like receptor (TLR) 2 in regulation of hepatic DMEs is unknown. Gene expression of DMEs is regulated by members of the nuclear receptor superfamily (PXR, CAR and RXRα). The TLR2 ligand, lipoteichoic acid (LTA) reduced RNA levels of CAR and its target genes, Cyp2b10, Cyp2a4 and Sultn in mouse liver (∼60-80% reduction). Hepatic genes regulated by PXR and CAR, Cyp3a11 and Mrp2 were moderately reduced by LTA, along with ∼50% reduction of PXR RNA and nuclear protein levels of RXRα. The effects of LTA were significantly attenuated by pre-treatment with the Kupffer cell inhibitor, gadolinium chloride, indicating that Kupffer cells contribute to LTA-mediated down-regulation of hepatic genes. These results indicate that treatment with Gram+ve bacterial components preferentially down-regulate CAR and its target genes in the liver.     

Chromatin assembly enhances binding to the CYP2B1 phenobarbital-responsive unit (PBRU) of nuclear factor-1, which binds simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor (RXR) and enhances CAR/RXR-mediated activation of the PBRU

Phenobarbital induction of CYP2B genes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivo footprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR.     

Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo

Phenobarbital (PB) induction of CYP2B genes is mediated by translocation of the constitutively active androstane receptor (CAR) to the nucleus. Interaction of CAR with p160 coactivators and enhancement of CAR transactivation by the coactivators have been shown in cultured cells. In the present studies, the interaction of CAR with the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was examined in vitro and in vivo. Binding of GRIP1 to CAR was shown by glutathione S-transferase (GST) pull-down and affinity DNA binding. N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of only the fragments containing the C-terminal region. In gel shift analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a CAR agonist and decreased by a CAR antagonist. Expression of GRIP1 enhanced CAR-mediated transactivation in cultured hepatic-derived cells 2-3-fold. In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the CYP2B1 PB-dependent enhancer 15-fold, whereas CAR expression alone resulted in only a 3-fold enhancement in untreated mice. Remarkably, CAR and GRIP1 together synergistically transactivated the enhancer about 150-fold, which is approximately equal to activation by PB treatment. In PB-treated mice, expression of exogenous CAR alone had little effect, expression of GRIP1 increased transactivation about 2-fold, and with CAR and GRIP, a 4-fold activation was observed. In untreated mice, expression of GRIP resulted in nuclear translocation of green fluorescent protein-CAR. These results strongly suggest that a p160 coactivator functions in CAR-mediated transactivation in vivo in response to PB treatment and that the synergistic activation of CAR by GRIP in untreated animals results from both nuclear translocation and activation of CAR.