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.     

Structure basis of bigelovin as a selective RXR agonist with a distinct binding mode

The nuclear receptor retinoid X receptor (RXR) functions potently in the regulation of homeostasis and cell development, while rexinoids as RXR agonists have proved their therapeutic potential in the treatment of metabolic diseases and cancer. Here, the natural product bigelovin was identified as a selective RXRα agonist. Interestingly, this compound could not transactivate RXRα:RXRα homodimer but could enhance the transactivation of RXRα:peroxisome proliferator-activated receptor γ heterodimer and repress that of RXRα:liver X receptor (LXR) α heterodimer, while it had no effects on RXRα:farnesoid X receptor heterodimer. Considering that the effective role of LXR response element involved transactivation of sterol regulatory element-binding protein-1c mediated by RXRα:LXRα in triglyceride elevation, such LXR response element repressing by bigelovin has obviously addressed its potency for further research. Moreover, our determined crystal structure of the bigelovin-activated RXRα ligand-binding domain with the coactivator human steroid receptor coactivator-1 peptide revealed that bigelovin adopted a distinct binding mode. Compared with the known RXR ligands, bigelovin lacks the acidic moiety in structure, which indicated that the acidic moiety rendered little effects on RXR activation. Our results have thereby provided new insights into the structure-based selective rexinoids design with bigelovin as a potential lead compound.     

Helix 11 dynamics is critical for constitutive androstane receptor activity

The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339-345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.     

The rexinoid LG100754 is a novel RXR:PPARgamma agonist and decreases glucose levels in vivo.

The RXR serves as a heterodimer partner for the PPARgamma and the dimer is a molecular target for insulin sensitizers such as the thiazolidinediones. Ligands for either receptor can activate PPAR-dependent pathways via PPAR response elements. Unlike PPARgamma agonists, however, RXR agonists like LG100268 are promiscuous and activate multiple RXR heterodimers. Here, we demonstrate that LG100754, a RXR:RXR antagonist and RXR:PPARalpha agonist, also functions as a RXR:PPARgamma agonist. It does not activate other LG100268 responsive heterodimers like RXR:liver X receptoralpha, RXR:liver X receptorbeta, RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor induced gene B. This unique RXR ligand triggers cellular RXR:PPARgamma-dependent pathways including adipocyte differentiation and inhibition of TNFalpha-mediated hypophosphorylation of the insulin receptor, but does not activate key farnesoid X receptor and liver X receptor target genes. Also, LG100754 treatment of db/db animals leads to an improvement in insulin resistance in vivo. Interestingly, activation of RXR:PPARgamma by LG100268 and LG100754 occurs through different mechanisms. Therefore, LG100754 represents a novel class of insulin sensitizers that functions through RXR but exhibits greater heterodimer selectivity compared with LG100268. These results establish an approach to the design of novel RXR-based insulin sensitizers with greater specificity.     

Retinoid signalling and gene expression in neuroblastoma cells: RXR agonist and antagonist effects on CRABP-II and RARbeta expression

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.     

Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer

Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand.     

Retinoid X receptor (RXR) agonist-induced antagonism of farnesoid X receptor (FXR) activity due to absence of coactivator recruitment and decreased DNA binding

The bile salt export pump (BSEP) plays an integral role in lipid homeostasis by regulating the canalicular excretion of bile acids. Induction of BSEP gene expression is mediated by the farnesoid X receptor (FXR), which binds as a heterodimer with the retinoid X receptor (RXR) to the FXR response element (FXRE) located upstream of the BSEP gene. RXR ligands mimic several partner ligands and show additive effects upon coadministration. Using real-time quantitative PCR and cotransfection reporter assays, we demonstrate that the RXR agonist LG100268 antagonizes induction of BSEP expression mediated by endogenous and synthetic FXR ligands, CDCA and GW4064, respectively. Moreover, this antagonism is a general feature of RXR agonists and is attributed to a decrease in binding of FXR/RXR heterodimers to the BSEP-FXRE coupled with the inability of RXR agonists to recruit coactivators to FXR/RXR. Our data suggest that FXR/RXR is a conditionally permissive heterodimer and is the first example of RXR ligand-mediated antagonism of FXR activity. Because FXR agonists lower triglyceride levels, our results suggest a novel role for RXR-mediated antagonism of FXR activity in the development of hypertriglyceridemia observed with RXR agonists in rodents and humans.     

Discrete roles for peroxisome proliferator-activated receptor gamma and retinoid X receptor in recruiting nuclear receptor coactivators

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in adipogenesis. PPARgamma binds to DNA as a heterodimer with retinoid X receptor (RXR), and PPARgamma-RXR can be activated by ligands specific for either receptor; the presence of both ligands can result in a cooperative effect on the transactivation of target genes. How these ligands mediate transactivation, however, remains unclear. PPARgamma is known to interact with both the p160/SRC-1 family of coactivators and the distinct, multisubunit coactivator complex called DRIP. A single DRIP subunit, DRIP205 (TRAP220, PBP), binds directly to PPARgamma. Here we report that PPARgamma and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner. At physiological concentrations, RXR-specific ligands only induced p160 binding to RXR, and PPARgamma-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARgamma. This selectivity was not observed in interaction assays off DNA, implying that the specificity of coactivator binding in response to ligand is strongly influenced by the allosteric effects of DNA-bound heterodimers. These coactivator-selective effects were also observed in transient-transfection assays in the presence of overexpressed p160 or DRIP coactivators. The results suggest that the cooperative effects of PPARgamma- and RXR-specific ligands may occur at the level of selective coactivator recruitment.     

The critical role of carboxy-terminal amino acids in ligand-dependent and -independent transactivation of the constitutive androstane receptor

The mouse constitutive androstane receptor (CAR) is a unique member of the nuclear receptor superfamily, for which an inverse agonist, the testosterone metabolite 5alpha-androstan-3alpha-ol (androstanol), and an agonist, the xenobiotic 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene, are known. In this study the role of the transactivation domain 2 (AF-2) of CAR was investigated, which is formed by the seven most carboxy-terminal amino acids of the receptor. The AF-2 domain was shown to be critical for the constitutive activity by mediating a ligand-independent interaction of CAR with coactivator (CoA) proteins. In addition this domain increased and decreased contact with CoAs in the presence of agonist and inverse agonist, respectively. In analogy to classical endocrine nuclear receptors, in CAR the charge clamp between K187 (in helix 3) and E355 (within the AF-2 domain) was expected to be critical for its interaction with CoAs. However, the hydrophobic amino acids L352, L353, and I356 on the surface of the AF-2 domain were found to be more important for this protein-protein interaction. Moreover, these amino acids and C357 were shown to be involved in the response of CAR to androstanol. Interestingly, the cysteine at position 357 appears to block classical endocrine responsiveness of CAR to agonists, since mutagenesis of this amino acid both reduced CoA interaction in the absence of ligand and drastically increased inducibility by 1,4-bis[2-(3, 5-dichloropyridyloxy)] benzene. We showed that this blockade is not due to an intramolecular disulfide bridge, but is probably caused by an interaction between C357 and Y336.