Garlic extract diallyl sulfide (DAS) activates nuclear receptor CAR to induce the Sult1e1 gene in mouse liver

Constituent chemicals in garlic extract are known to induce phase I and phase II enzymes in rodent livers. Here we have utilized Car(+/+) and Car(-/-) mice to demonstrate that the nuclear xenobiotic receptor CAR regulated the induction of the estrogen sulfotransferase Sult1e1 gene by diallyl sulfide (DAS) treatment in mouse liver. DAS treatment caused CAR accumulation in the nucleus, resulting in a remarkable increase of SULT1E1 mRNA (3,200 fold) and protein in the livers of Car(+/+) females but not of Car(-/-) female mice. DAS also induced other CAR-regulated genes such as Cyp2b10, Cyp3a11 and Gadd45β. Compared with the rapid increase of these mRNA levels, which began as early as 6 hours after DAS treatment, the levels of SULT1E1 mRNA began increasing after 24 hours. This slow response to DAS suggested that CAR required an additional factor to activate the Sult1e1 gene or that this activation was indirect. Despite the remarkable induction of SULT1E1, there was no decrease in the serum levels of endogenous E2 or increase of estrone sulfate while the clearance of exogenously administrated E2 was accelerated in DAS treated mice.     

The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene

The endogenous CYP2B6 gene becomes phenobarbital (PB) inducible in androstenol-treated HepG2 cells either transiently or stably transfected with a nuclear receptor CAR expression vector. The PB induction mediated by CAR is regulated by a conserved 51-base pair element called PB-responsive enhancer module (PBREM) that has now been located between -1733 and -1683 bp in the gene's 5'-flanking region. An in vitro translated CAR acting as a retinoid X receptor alpha heterodimer binds directly to the two nuclear receptor sites NR1 and NR2 within PBREM. In a stably transfected HepG2 cell line, both PBREM and NR1 are activated by PB and PB-type compounds such as chlorinated pesticides, polychlorinated biphenyls and chlorpromazine. In addition to PBREM, CAR also transactivates the steroid/rifampicin-response element of the human CYP3A4 gene in HepG2 cells. Thus, activation of the repressed nuclear receptor CAR appears to be a versatile mediator that regulates PB induction of the CYP2B and other genes.     

The Nuclear Orphan Receptor CAR-Retinoid X Receptor Heterodimer Activates the Phenobarbital-Responsive Enhancer Module of the CYP2B Gene

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.     

Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids

The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.     

Cytoplasmic localization of pregnane X receptor and ligand-dependent nuclear translocation in mouse liver

The pregnane X receptor (PXR) plays an important role in the response to xenobiotics and endogenous toxins. We have used a specific anti-PXR antibody in the Western blotting of mouse liver nuclear extracts to show that PXR is accumulated in the nucleus after treatment with 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), followed by an increase in Cyp3a11 mRNA. Expression of wild type PXR and various mutants as green fluorescent fusion proteins in mouse livers showed that PXR was retained in the cytoplasm from where PCN treatment translocated PXR into the nucleus. Furthermore, the xenochemical response signal, the nuclear translocation signal, and the activation function 2 domain were all required for the nuclear translocation to occur. Immunoprecipitation experiments using the hsp90 antibody demonstrated the presence of PXR in a complex with the endogenous cytoplasmic constitutive active/androstane receptor retention protein (CCRP) in HepG2 cells. Fluorescence resonance energy transfer analysis of mouse liver sections after co-expression of cyan fluorescent protein-CCRP and yellow fluorescent protein-PXR also indicated that CCRP and PXR were closely associated in vivo. Overexpression of exogenous CCRP increased the cytoplasmic level of the PXR.CCRP.hsp90 complex, whereas a decrease in endogenous CCRP by treatment with small interfering RNA for CCRP repressed the PXR-mediated reporter activity in HepG2 cells. We conclude that the CCRP mediates the retention of PXR in the cytosol and modulates the activation of PXR in response to PCN treatment.     

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.     

Different regulation of the expression of mouse hepatic cytochrome P450 2B enzymes by glucocorticoid and phenobarbital.

The effect of the synthetic glucocorticoid, dexamethasone, and phenobarbital upon the expression of Cyp2b9 and Cyp2b10, major CYP2B subfamilies in the mouse, was differentiated in C57BL/6 mouse liver and hepatocytes in primary culture. Overall expression was higher in the untreated female liver than in the male liver. More Cyp2b9 than Cyp2b10 mRNA was present in the female liver, whereas the level of Cyp2b10 was higher in the male. Phenobarbital increased Cyp2b10 expression more than did Cyp2b9 in both sexes. Treatment with dexamethasone markedly induced Cyp2b10 expression dose dependently, but simultaneously suppressed Cyp2b9 in both sexes. Evidence of this was obtained both in vivo and in hepatocyte culture. Furthermore, the existence of at least two unknown species of CYP2B, whose expressions were either increased or decreased by dexamethasone was suggested. Adrenalectomy increased the expression of Cyp2b9 and Cyp2b10 mRNAs, especially that of Cyp2b9 in the male liver. In addition, the expression of one unknown species which was constitutively suppressed increased in adrenalectomized male mice. That the treatment of dexamethasone or adrenalectomy altered the expression of CYP2B subfamilies suggests that endogenous glucocorticoid hormone plays a basic role in the constitutive expression of cytochrome P450. Furthermore, the sex-related difference in the expression of Cyp2b9 and Cyp2b10 suggests that sex-dependent secretion of endogeneous modulating factors is involved in the regulatory pathway.     

Functional inhibitory cross-talk between constitutive androstane receptor and hepatic nuclear factor-4 in hepatic lipid/glucose metabolism is mediated by competition for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha

The role of the constitutive androstane receptor (CAR) in xenobiotic metabolism by inducing expression of cytochromes P450 is well known, but CAR has also been implicated in the down-regulation of key genes involved in bile acid synthesis, gluconeogenesis, and fatty acid beta-oxidation by largely unknown mechanisms. Because a key hepatic factor, hepatic nuclear factor-4 (HNF-4), is crucial for the expression of many of these genes, we examined whether CAR could suppress HNF-4 transactivation. Expression of CAR inhibited HNF-4 transactivation of CYP7A1, a key gene in bile acid synthesis, in HepG2 cells, and mutation of the DNA binding domain of CAR impaired this inhibition. Gel shift assays revealed that CAR competes with HNF-4 for binding to the DR1 motif in the CYP7A1 promoter. TCPOBOP, a CAR agonist that increases the interaction of CAR with coactivators, potentiated CAR inhibition of HNF-4 transactivation. Furthermore, inhibition by CAR was reversed by expression of increasing amounts of GRIP-1 or PGC-1alpha, indicating that CAR competes with HNF-4 for these coactivators. Treatment of mice with phenobarbital or TCPOBOP resulted in decreased hepatic mRNA levels of the reported genes down-regulated by CAR, including Cyp7a1 and Pepck. In vivo recruitment of endogenous CAR to the promoters of Cyp7a1 and Pepck was detected in mouse liver after phenobarbital treatment, whereas association of HNF-4 and coactivators, GRIP-1, p300, and PGC-1alpha, with these promoters was significantly decreased. Our data suggest that CAR inhibits HNF-4 activity by competing with HNF-4 for binding to the DR1 motif and to the common coactivators, GRIP-1 and PGC-1alpha, which may be a general mechanism by which CAR down-regulates key genes in hepatic lipid and glucose metabolism.     

Estrogen receptor 1 modulates circadian rhythms in adult female mice

Estradiol influences the level and distribution of daily activity, the duration of the free-running period, and the behavioral phase response to light pulses. However, the mechanisms by which estradiol regulates daily and circadian rhythms are not fully understood. We tested the hypothesis that estrogens modulate daily activity patterns via both classical and “non-classical” actions at the estrogen receptor subtype 1 (ESR1). We used female transgenic mice with mutations in their estrogen response pathways; ESR1 knock-out (ERKO) mice and “non-classical” estrogen receptor knock-in (NERKI) mice. NERKI mice have an ESR1 receptor with a mutation in the estrogen-response-element binding domain, allowing only actions via “non-classical” genomic and second messenger pathways. Ovariectomized female NERKI, ERKO, and wildtype (WT) mice were given a subcutaneous capsule with low- or high-dose estradiol and compared with counterparts with no hormone replacement. We measured wheel-running activity in a light:dark cycle and constant darkness, and the behavioral phase response to light pulses given at different points during the subjective day and night. Estradiol increased average daily wheel-running, consolidated activity to the dark phase, and shortened the endogenous period in WT, but not NERKI and ERKO mice. The timing of activity onset during entrainment was advanced in all estradiol-treated animals regardless of genotype suggesting an ESR1-independent mechanism. We propose that estradiol modifies period, activity level, and distribution of activity via classical actions of ESR1 whereas an ESR1 independent mechanism regulates the phase of rhythms.     

Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response

Expression of P-450 (Cyp) enzymes is reduced in liver during the acute phase response, contributing to the decrease in bile acid levels and drug metabolism during infection. Nuclear hormone receptors CAR and PXR are key transactivators of Cyp2b and Cyp3a genes, respectively. Injection of bacterial lipopolysaccharide (LPS) induced the expected reduction in Cyp2b10 and Cyp3a mRNA levels in mouse liver. These decreases were associated with a marked reduction in CAR and PXR mRNA levels within 4 h following treatment. LPS-induced CAR and PXR repression were dose-dependent and sustained for at least 16 h. LPS treatment also reversed the up-regulation of Cyp3a in mice pre-treated with PXR ligand RU486. In addition, we observed a concomitant decrease in RXR (retinoid X receptor) mRNA levels, the obligatory partner of both CAR and PXR for high affinity binding to DNA. These findings represent one possible molecular mechanism underlying sepsis-induced repression of Cyp enzymes.     

Repression of CFTR activity in human MMNK-1 cholangiocytes induces sulfotransferase 1E1 expression in co-cultured HepG2 hepatocytes

Mouse models of cystic fibrosis (CF) indicate that sulfotransferase (SULT) 1E1 is significantly induced in livers of many mice lacking cystic fibrosis transmembrane receptor (CFTR) activity. Increased SULT1E1 activity results in the alteration of estrogen-regulated protein expression in the livers of these mice. In this study, human MMNK-1 cholangiocytes with repressed CFTR function were used to induce SULT1E1 expression in human HepG2 hepatocytes to investigate whether SULT1E1 can be increased in human CF liver. CFTR expression was inhibited in MMNK-1 cholangiocytes using CFTR-siRNA, then the MMNK-1 and HepG2 cells were co-cultured in a membrane-separated Transwell system. Expression of SULT1E1 and selected estrogen-regulated proteins were then assayed in the HepG2 cells. Results demonstrate that inhibition of CFTR expression in MMNK-1 cells results in the induction of SULT1E1 message and activity in HepG2 cells in the Transwell system. The expression of estrogen-regulated proteins including insulin-like growth factor (IGF)-1, glutathione-S-transferase (GST) P1 and carbonic anhydrase (CA) II expression are repressed in the HepG2 cells cultured with the CFTR-siRNA-MMNK-1 cells apparently in response to the increased sulfation of beta-estradiol. Thus, we have shown that co-culture of HepG2 hepatocytes with MMNK-1 cholangiocytes with siRNA repressed CFTR expression results in the selective induction of SULT1E1 in the HepG2 cells. Loss of CFTR function in cholangiocytes may have a paracrine regulatory effect on hepatocytes via the induction of SULT1E1 and the increased sulfation of beta-estradiol. Experiments are presently underway in our laboratory to elucidate the identity of these paracrine regulatory factors.     

Generation of Cyp17iCre transgenic mice and their application to conditionally delete estrogen receptor alpha (Esr1) from the ovary and testis

A transgenic mouse line that expresses iCre under regulation of the Cytochrome P(450) 17alpha-hydroxylase/17, 20-lyase (Cyp17) promoter was developed as a novel transgenic mouse model for the conditional deletion of genes specifically in the theca/interstitial cells of the ovary and Leydig cells of the testis. In this report, we describe the development of Cyp17iCre mice and the application of these mice for conditional deletion of the estrogen receptor alpha (Esr1) gene in the theca/interstitial and Leydig cells of the female and male gonad, respectively. These mice will prove a powerful tool to inactivate genes in the gonad in a cell-specific manner.     

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).