Antagonist- and inverse agonist-driven interactions of the vitamin D receptor and the constitutive androstane receptor with corepressor protein

Ligand-dependent signal transduction by nuclear receptors (NRs) includes dynamic exchanges of coactivator (CoA) and corepressor (CoR) proteins. Here we focused on the structural determinants of the antagonist- and inverse agonist-enhanced interaction of the endocrine NR vitamin D receptor (VDR) and the adopted orphan NR constitutive androstane receptor (CAR) from two species with the CoR NR corepressor. We found that the pure VDR antagonist ZK168281 and the human CAR inverse agonist clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors, whereas ZK168281 resembled more the mouse CAR inverse agonist androstanol in its ability to recruit CoR proteins. Molecular dynamics simulations resulted in comparable models for the CoR receptor interaction domain peptide bound to VDR/antagonist or CAR/inverse agonist complexes. A salt bridge between the CoR and a conserved lysine in helix 4 of the NR is central to this interaction, but also helix 12 was stabilized by direct contacts with residues of the CoR. Fixation of helix 12 in the antagonistic/inverse agonistic conformation prevents an energetically unfavorable free floatation of the C terminus. The comparable molecular mechanisms that explain the similar functional profile of antagonist and inverse agonists are likely to be extended from VDR and CAR to other members of the NR superfamily and may lead to the design of even more effective ligands.     

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.     

Meclizine is an agonist ligand for mouse constitutive androstane receptor (CAR) and an inverse agonist for human CAR

The constitutive androstane receptor (CAR, NR1I3) is a key regulator of xenobiotic and endobiotic metabolism. The ligand-binding domains of murine (m) and human (h) CAR are divergent relative to other nuclear hormone receptors, resulting in species-specific differences in xenobiotic responses. Here we identify the widely used antiemetic meclizine (Antivert; Bonine) as both an agonist ligand for mCAR and an inverse agonist for hCAR. Meclizine increases mCAR transactivation in a dose-dependent manner. Like the mCAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, meclizine stimulates binding of steroid receptor coactivator 1 to the murine receptor in vitro. Meclizine administration to mice increases expression of CAR target genes in a CAR-dependent manner. In contrast, meclizine suppresses hCAR transactivation and inhibits the phenobarbital-induced expression of the CAR target genes, cytochrome p450 monooxygenase (CYP)2B10, CYP3A11, and CYP1A2, in primary hepatocytes derived from mice expressing hCAR, but not mCAR. The inhibitory effect of meclizine also suppresses acetaminophen-induced liver toxicity in humanized CAR mice. These results demonstrate that a single compound can induce opposite xenobiotic responses via orthologous receptors in rodents and humans.     

Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR(-/-)) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR(-/-) mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR(-/-) mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.     

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).     

Agonist-dependent and agonist-independent transactivations of the human constitutive androstane receptor are modulated by specific amino acid pairs

The constitutive androstane receptor (CAR) is an interesting member of the nuclear receptor superfamily because of its exceptionally high constitutive activity due to ligand-independent interaction of the ligand-binding domain with co-activator proteins. This study compares the agonist-dependent and agonist-independent activities of human CAR with those of mouse CAR and the vitamin D receptor and demonstrates that the constitutive activity of CAR is mediated by at least three contacts between the amino acids of helix 12, partner amino acids in helices 4 and 11, and a charge clamp between helices 12 and 3. The stabilization of helix 12 by a contact between its C terminus and the lysine of helix 4 has the same impact in human and mouse CARs. In addition, the charge clamp between the glutamate in helix 12 and the lysine in helix 3 is also important for the constitutive activity of both receptor orthologs but less critical for the agonist-dependent stabilization of their respective helices 12. Interestingly, Cys-357 in mouse CAR has significantly more impact on the stabilization of helix 12 than does the orthologous position Cys-347 in human CAR. This deficit appears to be compensated by a more dominant role of Ile-330 in human CAR over Leu-340 in mouse CAR because it is more efficient than Cys-347 in controlling the flexibility of helix 12 in the presence of an agonist. The constitutive activity of other members of the nuclear receptor superfamily could be explained by a homologous hydrophobic interaction between large, non-polar amino acids of helices 11 and 12.     

The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction

The orphan nuclear receptor CAR (NR1I3) has been characterized as a central component in the coordinate response to xenobiotic and endobiotic stress. In this study, we demonstrate that CAR plays a pivotal function in energy homeostasis and establish an unanticipated metabolic role for this nuclear receptor. Wild-type mice treated with the synthetic CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) exhibited decreased serum concentration of the thyroid hormone (TH) thyroxine (T(4)). However, treatment of Car(-/-) mice with TCPOBOP failed to elicit these changes. To examine whether CAR played a role in the regulation of TH levels under physiological conditions, wild-type and Car(-/-) mice were fasted for 24 h, a process known to alter TH metabolism in mammals. As expected, the serum triiodothyronine and T(4) concentrations decreased in wild-type mice. However, triiodothyronine and T(4) levels in fasted Car(-/-) mice remained significantly higher than those in fasted wild-type animals. Concomitant with the changes in serum TH levels, both CAR agonist treatment and fasting induced the expression of CAR target genes (notably, Cyp2b10, Ugt1a1, Sultn, Sult1a1, and Sult2a1) in a receptor-dependent manner. Importantly, the Ugt1a1, Sultn, Sult1a1, and Sult2a1 genes encode enzymes that are capable of metabolizing TH. An attenuated reduction in TH levels during fasting, as observed in Car(-/-) mice, would be predicted to increase weight loss during caloric restriction. Indeed, when Car(-/-) animals were placed on a 40% caloric restriction diet for 12 weeks, Car(-/-) animals lost over twice as much weight as their wild-type littermates. Thus, CAR participates in the molecular mechanisms contributing to homeostatic resistance to weight loss. These data imply that CAR represents a novel therapeutic target to uncouple metabolic rate from food intake and has implications in obesity and its associated disorders.     

Ligand-mediated conformational changes of the VDR are required for gene transactivation

The central element of the molecular switch of nuclear 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) signaling is the ligand-binding domain (LBD) of the Vitamin D receptor (VDR), which can be stabilized by 1alpha,25(OH)(2)D(3) or its analogues in to agonistic, antagonistic or inverse agonistic conformations. The positioning of helix 12 of the LBD is of most critical importance for these conformations, because it determines the distance between the charge clamp amino acids K246 and E420 that are essential for VDR-coactivator (CoA) interaction. Most VDR ligands have been identified as agonists and only a few (e.g., ZK168281 and TEI-9647) as pure or partial antagonists. Antagonists induce corepressor (CoR) dissociation from the VDR but prevent completely or partially CoA interaction and thus transactivation. Gemini is a 1alpha,25(OH)(2)D(3) analogue with two identical side chains that despite its significantly increased volume binds to the VDR and acts under most conditions as an agonist. Interestingly, supramolar CoR concentrations shift Gemini from an agonist to an inverse agonist, which actively recruits CoR to the VDR and thus mediates repression of 1alpha,25(OH)(2)D(3) target genes. Gemini is the first described (conditional) inverse agonist to an endocrine nuclear receptor (NR) and may function as a sensor for cell-specific CoA/CoR ratios.     

Xenobiotic-Induced Hepatocyte Proliferation Associated with Constitutive Active/Androstane Receptor (CAR) or Peroxisome Proliferator-Activated Receptor α (PPARα) Is Enhanced by Pregnane X Receptor (PXR) Activation in Mice

Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR), constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPARα activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital, or PPARα activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16α-carbonitrile (PCN) alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPARα is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.     

Characterization of transactivational property and coactivator mediation of rat mineralocorticoid receptor activation function-1 (AF-1)

The autonomous activation function-2 (AF-2) in the mineralocorticoid receptor (MR) E/F domain is known to play a major role in the ligand-induced transactivation function of MR; however, it remained unclear about the transactivation function of its A/B domain. We therefore tried to characterize the MR A/B domain as the AF-1 and further studied the actions of known coactivators for AF-2 in the E/F ligand-binding domain in the function of the MR A/B domain. Deletion analyses of rat and human MRs revealed that the A/B domains harbor a transactivation function acting as AF-1. The MR mutant (E959Q) with a point mutation in helix 12, which causes a complete loss of MR AF-2 activity, still retained ligand-induced transactivation function, indicating a significant role for AF-1 in the full activity of the ligand-induced MR function. Among the coactivators tested to potentiate the MR AF-2, TIF2 and p300 potentiated the MR AF-1 through two different core regions [amino acids (a.a.) 1-169, a.a. 451-603] and exhibited functional interactions with the MR A/B domain in the cultured cells. However, such interactions were undetectable in a yeast and in an in vitro glutathione-S-transferase pull-down assay, indicating that the functional interaction of TIF2 and p300 with the MR A/B domain to support the MR AF-1 activity require some unknown nuclear factor(s) or a proper modification of the A/B domain in the cells.