The small heterodimer partner (SHP; NROB2), a member of the nuclear receptor superfamily, contributes to the biological regulation of several major functions of the liver. However, the role of SHP in cellular proliferation and tumorigenesis has not been investigated before. Here we report that SHP negatively regulates tumorigenesis both in vivo and in vitro. SHP−/− mice aged 12 to 15 months old developed spontaneous hepatocellular carcinoma, which was found to be strongly associated with enhanced hepatocyte proliferation and increased cyclin D1 expression. In contrast, overexpressing SHP in hepatocytes of SHP-transgenic mice reversed this effect. Embryonic fibroblasts lacking SHP showed enhanced proliferation and produced increased cyclin D1 messenger RNA and protein, and SHP was shown to be a direct negative regulator of cyclin D1 gene transcription. The immortal SHP−/− fibroblasts displayed characteristics of malignant transformed cells and formed tumors in nude mice.
These results provide first evidence that SHP plays tumor suppressor function by negatively regulating cellular growth.
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; NR1I3) 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 a 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 (H3K4) mono-, di- and trimethylation and decrease of H3K9 trimethylation within the Cyp2B10 locus. Transcriptional coactivator ASC-2 and histone demethylase JMJD2d participated in this CAR-dependent epigenetic switch.
Neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism.
CAR; nuclear receptor; epigenetic memory; drug metabolism; histone methylation
Several pathways and pathologies have been suggested as connections between obesity and diabetes, including inflammation of adipose and other tissues, toxic lipids, endoplasmic reticulum stress, and fatty liver. One specific proposal is that insulin resistance induces a vicious cycle in which hyperinsulinemia increases hepatic lipogenesis and exacerbates fatty liver, in turn further increasing insulin resistance. Here I suggest that reversing this cycle via suppression of the lipogenic transcription factor SREBP-1c is a common thread that connects the antidiabetic effects of a surprising number of nuclear hormone receptors, including CAR, LRH-1, TRβ, ERα and FXR/SHP.
Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR-/- mice, but not in hypothyroid PXR-/- mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR-/- hypothyroid mice, and this induction was abolished in CAR-/- mice and in and CAR-/- PXR-/- double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR-/- mice and lowest in WT and PXR-/- mice. Hypothyroid WT or PXR-/- mice survived chronic CBZ treatment, but all hypothyroid CAR-/- and CAR-/- PXR-/- mice died, with CAR-/-PXR-/- mice surviving longer than CAR-/- mice (12.3 ±3.3 days vs. 6.3 ±2.1 days, p=0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge.
thyroid hormone; cytochrome P450; xenobiotic receptor
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.
The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.
Bmal1; circadian clock; diabetes; insulin secretion; mitochondria; β-cells
Multiple nuclear receptors, including hepatocyte nuclear factor 4α (HNF4α), retinoid X receptor α (RXRα) plus peroxisome proliferator-activated receptor α (PPARα), RXRα plus farnesoid X receptor α (FXRα), liver receptor homolog 1 (LRH1), and estrogen-related receptors (ERRs), have been shown to support efficient viral biosynthesis in nonhepatoma cells in the absence of additional liver-enriched transcription factors. Although HNF4α has been shown to be critical for the developmental expression of hepatitis B virus (HBV) biosynthesis in the liver, the relative importance of the various nuclear receptors capable of supporting viral transcription and replication in the adult in vivo has not been clearly established. To investigate the role of the nuclear receptor FXR and the corepressor small heterodimer partner (SHP) in viral biosynthesis in vivo, SHP-expressing and SHP-null HBV transgenic mice were fed a bile acid-supplemented diet. The increased FXR activity and SHP expression levels resulting from bile acid treatment did not greatly modulate HBV RNA and DNA synthesis. Therefore, FXR and SHP appear to play a limited role in modulating HBV biosynthesis, suggesting that alternative nuclear receptors are more critical determinants of viral transcription in the HBV transgenic mouse model of chronic viral infection. These observations suggest that hepatic bile acid levels or therapeutic agents targeting FXR may not greatly modulate viremia during natural infection.
2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car−/−) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car−/− livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor.
Ortho-Aminoazotoluene (OAT); Constitutive Androstane Receptor (CAR); CYP450s; c-Myc; hepatocyte proliferation
Bile acid homeostasis is tightly regulated via a feedback loop operated by the nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP). Contrary to current models, which place FXR upstream of SHP in a linear regulatory pathway, here we show that the phenotypic consequences in mice of the combined loss of both receptors are much more severe than the relatively modest impact of the loss of either Fxr or Shp alone. Fxr–/–Shp–/– mice exhibited cholestasis and liver injury as early as 3 weeks of age, and this was linked to the dysregulation of bile acid homeostatic genes, particularly cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1). In addition, double-knockout mice showed misregulation of genes in the C21 steroid biosynthesis pathway, with strong induction of cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1), resulting in elevated serum levels of its enzymatic product 17-hydroxyprogesterone (17-OHP). Treatment of WT mice with 17-OHP was sufficient to induce liver injury that reproduced many of the histopathological features observed in the double-knockout mice. Therefore, our data indicate a pathologic role for increased production of 17-hydroxy steroid metabolites in liver injury and suggest that Fxr–/–Shp–/– mice could provide a model for juvenile onset cholestasis.
DamIP is a new method for studying DNA-protein interaction in vivo. A mutant form of DNA adenine methyltransferase (DamK9A) from E. coli is fused to the protein of interest and expressed. The fusion protein will bind to target binding sites and introduce N-6-adenine methylation in nearby sites in the genomic DNA. Methylated DNA fragments are enriched with an antibody against N-6-methyladenine and used for further analysis, e.g. real-time PCR, microarray or high-throughput sequencing. This method is simple and does not require either protein-DNA crosslinking or a specific antibody to the protein of interest. This unit describes the application of this method for the identification of DNA binding sites in vivo.
DNA adenine methyltransferase; transcription factor binding sites; DamIP; chromatin immunoprecipitation
All organisms have devised strategies to counteract energy depletion in order to promote fitness for survival. We show here that cellular energy depletion puts into play a surprising strategy that leads to absorption of exogenous fuel for energy repletion. We found that the energy depletion sensing kinase AMPK, binds, phosphorylates, and activates the transcriptional coactivator SRC-2, which in a liver-specific manner, promotes absorption of dietary fat from the gut. Hepatocyte-specific deletion of SRC-2 results in intestinal fat malabsorption and attenuated entry of fat into the blood stream. This defect can be attributed to AMPK and SRC-2 mediated transcriptional regulation of hepatic bile-acid secretion into the gut, as it can be completely rescued by replenishing intestinal BA, or by genetically restoring the levels of hepatic Bile Salt Export Pump (BSEP). Our results position the hepatic AMPK-SRC-2 axis as an energy rheostat which upon cellular energy depletion resets whole-body energy by promoting absorption of dietary fuel.
Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (NR5A2) regulates bile acid biosynthesis1,2. Structural studies have identified phospholipids as potential LRH-1 ligands3–5, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine, DLPC) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signaling pathway that regulates bile acid metabolism and glucose homeostasis.
Nonalcoholic fatty liver disease (NAFLD) is a common complication of obesity that can progress to nonalcoholic steatohepatitis (NASH), a serious liver pathology that can advance to cirrhosis. The mechanisms responsible for NAFLD progression to NASH remain unclear. Lack of a suitable animal model that faithfully recapitulates the pathophysiology of human NASH is a major obstacle in delineating mechanisms responsible for progression of NAFLD to NASH and, thus, development of better treatment strategies. We identified and characterized a novel mouse model, middle-aged male LDLR−/− mice fed high-fat diet (HFD), which developed NASH associated with 4 of 5 metabolic syndrome (MS) components. In MS mice, as observed in humans, liver steatosis and oxidative stress promoted NASH development. Aging exacerbated the HFD-induced NASH such that liver steatosis, inflammation, fibrosis, oxidative stress and liver injury markers were greatly enhanced in middle-aged versus young LDLR−/− mice. While expression of genes mediating fatty acid oxidation and antioxidant responses were upregulated in young LDLR−/− mice fed HFD, they were drastically reduced in MS mice. However, similar to recent human trials, NASH was partially attenuated by an insulin-sensitizing peroxisome proliferator-activated receptor-gamma (PPARγ) ligand, rosiglitazone. In addition to expected improvements in MS, newly identified mechanisms of PPARγ ligand effects included stimulation of antioxidant gene expression and mitochondrial β-oxidation, and suppression of inflammation and fibrosis. LDLR-deficiency promoted NASH, since middle-aged C57BL/6 mice fed HFD did not develop severe inflammation and fibrosis, despite increased steatosis.
MS mice represent an ideal model to investigate NASH in the context of MS, as commonly occurs in human disease, and NASH development can be substantially attenuated by PPARγ activation, which enhances β-oxidation.
NASH; aging; oxidative stress; Nrf2; Rosiglitazone; chronic liver disease; mitochondrial dysfunction; LDLR−/−
The bile acid receptor farnesoid X receptor (FXR; NR1H4) is a central regulator of bile acid and lipid metabolism. We show here that FXR plays a key regulatory role in glucose homeostasis. FXR-null mice developed severe fatty liver and elevated circulating FFAs, which was associated with elevated serum glucose and impaired glucose and insulin tolerance. Their insulin resistance was confirmed by the hyperinsulinemic euglycemic clamp, which showed attenuated inhibition of hepatic glucose production by insulin and reduced peripheral glucose disposal. In FXR–/– skeletal muscle and liver, multiple steps in the insulin signaling pathway were markedly blunted. In skeletal muscle, which does not express FXR, triglyceride and FFA levels were increased, and we propose that their inhibitory effects account for insulin resistance in that tissue. In contrast to the results in FXR–/– mice, bile acid activation of FXR in WT mice repressed expression of gluconeogenic genes and decreased serum glucose. The absence of this repression in both FXR–/– and small heterodimer partner–null (SHP–/–) mice demonstrated that the previously described FXR-SHP nuclear receptor cascade also targets glucose metabolism. Taken together, our results identify a link between lipid and glucose metabolism mediated by the FXR-SHP cascade.
Yin Zhi Huang, a decoction of Yin Chin (Artemisia capillaris) and three other herbs, is widely used in Asia to prevent and treat neonatal jaundice. We recently identified the constitutive androstane receptor (CAR, NR1I3) as a key regulator of bilirubin clearance in the liver. Here we show that treatment of WT and humanized CAR transgenic mice with Yin Zhi Huang for 3 days accelerates the clearance of intravenously infused bilirubin. This effect is absent in CAR knockout animals. Expression of bilirubin glucuronyl transferase and other components of the bilirubin metabolism pathway is induced by Yin Zhi Huang treatment of WT mice or mice expressing only human CAR, but not CAR knockout animals. 6,7-Dimethylesculetin, a compound present in Yin Chin, activates CAR in primary hepatocytes from both WT and humanized CAR mice and accelerates bilirubin clearance in vivo. We conclude that CAR mediates the effects of Yin Zhi Huang on bilirubin clearance and that 6,7-dimethylesculetin is an active component of this herbal medicine. CAR is a potential target for the development of new drugs to treat neonatal, genetic, or acquired forms of jaundice.
The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7α-hydroxylase (CYP7A1) gene expression in primary human hepatocytes. Adenovirusmediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a ~ 6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increased hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.
bile acid synthesis; insulin; gene expression; nuclear receptor; metabolic diseases
Untreated type 1 diabetes increases hepatic drug metabolism in both human patients and rodent models. We used mouse knockouts to test the role of the nuclear xenobiotic receptors CAR and PXR in this process. Streptozotocin induced diabetes resulted in increased expression of drug metabolizing cytochrome P450's and also increased the clearance of the cytochrome P450 substrate zoxazolamine. This induction was completely absent in Car-/- mice, but was not affected by the loss of PXR. Among the many effects of diabetes on the liver, we identified elevations in bile acids and activated AMP kinase as potential CAR activating stimuli. Expression of the CAR coactivator PGC-1α was also increased in mouse models of type 1 diabetes. The CAR-dependent induction of drug metabolism in newly diagnosed or poorly managed type 1 diabetes has the potential for significant impact on the efficacy or toxicity of therapeutic agents.
Bile acids; CAR; cytochrome P450; streptozotocin; drug metabolism
Identifying binding sites and target genes of transcription factors is a major biologic problem. The most commonly used current technique, chromatin immunoprecipitation (ChIP), is dependent on a high quality antibody for each protein of interest, which is not always available, and is also cumbersome, involving sequential cross-linking and reversal of cross-linking. We have developed a novel strategy to study protein DNA binding sites in vivo, which we term DamIP. By tethering a mutant form of E. coli DNA adenine methyltransferase to the target protein, the fusion protein introduces N-6-adenosine methylation to sequences proximal to the protein binding sites. DNA fragments with this modification, which is absent in eukaryotes, are detected using an antibody directed against methylated adenosine. For an initial test of the method we used human estrogen receptor α (hERα), one of the best studied transcription factors. We found that expression of Dam-hERα fusion proteins in MCF-7 cells introduces adenosine methylation near a series of known direct hERα binding sites. Specific methylation tags are also found at indirect hERα binding sites, including both primary binding sites for the ER interactors AP-1 and SP1, and promoters that are activated by upstream ER bound enhancers. DamIP provides a new tool for the study of DNA interacting protein function in vivo.
6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (CD437/AHPN) and 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC/MM002) are inducers of apoptosis of malignant cells both in vitro and in vivo. Numerous mechanisms have been proposed for how these compounds exert this effect. This report shows that AHPN/3-Cl-AHPC binds specifically to the orphan nuclear receptor small heterodimer partner (SHP; NR0B2), and this binding promotes interaction of the receptor with a corepressor complex that minimally contains Sin3A, N-CoR, histone deacetylase 4, and HSP90. Formation of the SHP-Sin3A complex is essential for the ability of AHPN and 3-Cl-AHPC to induce apoptosis, as both knockout SHP and knockdown of Sin3A compromise the proapoptotic activity of these compounds but not other apoptosis inducers. These results suggest that AHPN/3-Cl-AHPC and their analogues are SHP ligands and their induction of apoptosis is mediated by their binding to the SHP receptor.
Cytochromes P450 (P450s) and glutathione S-transferases (GSTs) constitute two important enzyme families involved in carcinogen metabolism. Generally, P450s play activation or detoxifying roles while GSTs act primarily as detoxifying enzymes. We previously demonstrated that oral administration of the linear furanocoumarins, isopimpinellin and imperatorin, modulated P450 and GST activities in various tissues of mice. The purpose of the present study was to compare a broader range of naturally occurring coumarins (simple coumarins, and furanocoumarins of the linear and angular type) for their abilities to modulate hepatic drug metabolizing enzymes when administered orally to mice. We now report that all of the different coumarins tested (coumarin, limettin, auraptene, angelicin, bergamottin, imperatorin and isopimpinellin) induced hepatic GST activities, whereas the linear furanocoumarins possessed the greatest abilities to induce hepatic P450 activities, in particular P450 2B and 3A. In both cases, this corresponded to an increase in protein expression of the enzymes. Induction of P4502B10, 3A11, and 2C9 by xenobiotics often are a result of activation of the pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Using a pregnane X receptor reporter system, our results demonstrated that isopimpinellin activated both PXR and its human ortholog SXR by recruiting coactivator SRC-1 in transfected cells. In CAR transfection assays, isopimpinellin counteracted the inhibitory effect of androstanol on full length mCAR, a Gal4-mCAR ligand binding domain fusion, and restored coactivator binding. Orally administered isopimpinellin induced hepatic mRNA expression of Cyp2b10,Cyp3a1, GSTa in CAR(+/+) wild-type mice. In contrast, the induction of Cyp2b10 mRNA by isopimpinellin was attenuated in the CAR(−/−) mice, suggesting that isopimpinellin induces Cyp2b10 via the CAR receptor. Overall, the current data indicate that naturally occurring coumarins have diverse activities in terms of inducing various xenobiotic metabolizing enzymes based on their chemical structure.
coumarins; furanocoumarins; P450s; pregnane X-receptor; constitutive androstane receptor
Bile acids are potentially toxic compounds and their levels of hepatic production, uptake and export are tightly regulated by many inputs, including circadian rhythm. We tested the impact of disrupting the peripheral circadian clock on integral steps of bile acid homeostasis.
Both restricted feeding, which phase shifts peripheral clocks, and genetic ablation in Per1−/−/Per2−/− (PERDKO) mice disrupted normal bile acid control and resulted in hepatic cholestasis. Restricted feeding caused a dramatic, transient elevation in hepatic bile acid levels that was associated with activation of the xenobiotic receptors CAR and PXR and elevated serum aspartate aminotransferase (AST), indicative of liver damage. In the PERDKO mice, serum bile acid levels were elevated and the circadian expression of key bile acid synthesis and transport genes, including Cyp7A1 and NTCP, was lost. This was associated with blunted expression of a primary clock output, the transcription factor DBP, which transactivates the promoters of both genes.
We conclude that disruption of the circadian clock results in dysregulation of bile acid homeostasis that mimics cholestatic disease.
Nonylphenol (NP) and its parent compounds, the nonylphenol ethoxylates are some of the most prevalent chemicals found in U.S. waterways. NP is also resistant to biodegradation and is a known environmental estrogen, which makes NP a chemical of concern. Our data show that NP also activates the constitutive androstane receptor (CAR), an orphan nuclear receptor important in the induction of detoxification enzymes, including the P450s. Transactivation assays demonstrate that NP increases murine CAR (mCAR) transcriptional activity, and NP treatment can overcome the inhibitory effects of the inverse agonist, androstanol, on mCAR activation. Treatment of wild-type (CAR +/+) mice with NP at 50 or 75 mg/kg/day increases Cyp2b protein expression in a dose-dependent manner as demonstrated by Western blotting, and was confirmed by quantitative reverse transcription–PCR of Cyp2b10 transcript levels. CAR-null (CAR −/−) mice show no increased expression of Cyp2b following NP treatment, indicating that CAR is required for NP-mediated Cyp2b induction. In addition, NP increases the translocation of CAR into the nucleus, which is the key step in the commencement of CAR's transcriptional activity. NP also induced CYP2B6 in primary human hepatocytes, and increased Cyp2b10 messenger RNA and protein expression in humanized CAR mice, indicating that NP is an activator of human CAR as well. In conclusion, NP is a CAR activator, and this was demonstrated in vitro with transactivation assays and in vivo with transgenic CAR mouse models.
Nonylphenol; CAR; PXR; Cyp2b10; P450
We explored the effects of bile acids on triglyceride (TG) homeostasis using a combination of molecular, cellular, and animal models. Cholic acid (CA) prevents hepatic TG accumulation, VLDL secretion, and elevated serum TG in mouse models of hypertriglyceridemia. At the molecular level, CA decreases hepatic expression of SREBP-1c and its lipogenic target genes. Through the use of mouse mutants for the short heterodimer partner (SHP) and liver X receptor (LXR) α and β, we demonstrate the critical dependence of the reduction of SREBP-1c expression by either natural or synthetic farnesoid X receptor (FXR) agonists on both SHP and LXRα and LXRβ. These results suggest that strategies aimed at increasing FXR activity and the repressive effects of SHP should be explored to correct hypertriglyceridemia.
CAR/RXR heterodimers bind a variety of hormone response elements and activate transcription in the absence of added ligands. This constitutive activity of murine CAR can be inhibited by the inverse agonist ligand androstanol or increased by the agonist TCPOBOP. RXR agonists activate some RXR heterodimer complexes, which are termed permissive, while other non-permissive complexes are not responsive to such ligands.
Direct protein-protein interaction studies demonstrate that the RXR agonist 9-cis-RA increases interaction of CAR/RXR heterodimers with the coactivator SRC-3, but also inhibits the ability of TCPOBOP to increase and androstanol to decrease coactivator binding. CAR transactivation of a response element with a five nucleotide spacer (DR-5) is unaffected by 9-cis-RA or the synthetic RXR agonist LG1069. In agreement with the inhibitory effect observed in vitro, these rexinoids block both the TCPOBOP mediated transactivation of this element and the androstanol dependent inhibition. In contrast, CAR transactivation of other response elements is increased by rexinoids. Stable expression of CAR in a HepG2 derived cell line increases expression of the endogenous CAR target CYP2B6. This expression is further increased by TCPOBOP but decreased by either androstanol or LG1069, and LG1069 blocks the stimulatory effect of TCPOBOP but not the inhibitory effect of androstanol.
We conclude that CAR/RXR heterodimers are neither strictly permissive nor non-permissive for RXR signaling. Instead, rexinoids have distinct effects in different contexts. These results expand the potential regulatory mechanisms of rexinoids and suggest that such compounds may have complex and variable effects on xenobiotic responses.