CAR2 displays unique ligand binding and RXRalpha heterodimerization characteristics.
ABSTRACT: The constitutive androstane receptor (CAR; NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional four amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4, and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Overexpression of the RXRalpha ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXRalpha. Mutagenesis of Ser233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating Ser233 to an aspartate residue drastically reduced the transactivation capacity of CAR2. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR-4 x 3 reporter element correlated with their ability to interact with RxRalpha and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXRalpha-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol compared with the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the four-amino-acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.
Project description:Phthalates and other endocrine-disruptive chemicals are manufactured in large quantities for use as plasticizers and other commercial applications, resulting in ubiquitous human exposure and thus, concern regarding their toxicity. Innate defense against small molecule exposures is controlled in large part by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). The human CAR gene undergoes multiple alternative splicing events resulting in the CAR2 and CAR3 variant receptors. Recent studies from our laboratory show that CAR2 is potently and specifically activated by di(2-ethylhexyl) phthalate (DEHP). We hypothesized that alternative splicing is a mechanism for increasing CAR's functional diversity, broadening the human receptors' repertoire of response to environmental xenobiotics. In these studies, we examine the interaction of alternatively spliced CARs and PXR with a range of suspected endocrine disruptors, including phthalates, bisphenol A (BPA), and 4-N-nonylphenol (NP). Transactivation and two-hybrid studies in COS-1 cells revealed differential selectivity of endocrine-disrupting chemicals for the variant CAR and PXR. Ex vivo studies showed DEHP and di-isononyl phthalate potently induced CYP2B6 and CYP3A4 expression in human hepatocytes. Mutation analysis of CAR2, in silico modeling, and ligand docking studies suggested that the SPTV amino acid insertion of CAR2 creates a unique ligand-binding pocket. Alternative gene splicing results in variant CAR receptors that selectively recognize phthalates and BPA. The interaction of phthalates with CAR and PXR suggests a xenobiotic response that is complex and biologically redundant.
Project description:Downstream in-frame start codons produce amino-terminal-truncated human constitutive androstane receptor protein isoforms (DeltaNCARs). The DeltaNCARs are expressed in liver and in vitro cell systems following translation from in-frame methionine AUG start codons at positions 76, 80, 125, 128, 168 and 265 within the full-length CAR mRNA. The resulting CAR proteins lack the N-terminal DNA-binding domain (DBD) of the receptor, yielding DeltaNCAR variants with unique biological function. Although the DeltaNCARs maintain full retinoid X receptor alpha (RXRalpha) heterodimerization capacity, the DeltaNCARs are inactive on classical CAR-inducible direct repeat (DR)-4 elements, yet efficiently transactivate a DR-1 element derived from the endogenous PPAR-inducible acyl-CoA oxidase gene promoter. RXRalpha heterodimerization with CAR1, CAR76 and CAR80 isoforms is necessary for the DR-1 PPRE activation, a function that exhibits absolute dependence on both the respective RXRalpha DBD and CAR activation (AF)-2 domains, but not the AF-1 or AF-2 domain of RXRalpha, nor CAR's DBD. A new model of CAR DBD-independent transactivation is proposed, such that in the context of a DR-1 peroxisome proliferator-activated response element, only the RXRalpha portion of the CAR-RXRalpha heterodimer binds directly to DNA, with the AF-2 domain of tethered CAR mediating transcriptional activation of the receptor complex.
Project description:Control of oestradiol-responsive gene regulation by oestrogen receptors (ERs) may involve complex cross-talk with retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Recently, we have shown that ERalpha directly interacts with RARalpha and RXRalpha through their ligand binding domains (LBDs). In the present work, we extend these results by showing that ERbeta binds similarly to RARalpha and RXRalpha but not to the glucocorticoid receptor, as demonstrated by the yeast two-hybrid tests and glutathione S-transferase pull-down assays. These direct interactions were also demonstrated in gel-shift assays, in which the oestrogen response element (ERE) binding by ERalpha was enhanced by the RXRalpha LBD but was abolished by the RARalpha LBD. In addition, we showed that RARalpha and RXRalpha bound the ERE as efficiently as ERalpha, suggesting that competition for DNA binding may affect the transactivation function of the ER. In transient transfection experiments, co-expression of RARalpha or RXRalpha, along with ERalpha or ERbeta, revealed differential modulation of the ERE-dependent transactivation, which was distinct from the results when each receptor alone was co-transfected. Importantly, when the LBD of RARalpha was co-expressed with ERalpha, transactivation of ERalpha on the ERE was repressed as efficiently as when wild-type RARalpha was co-expressed. Furthermore, liganded RARalpha or unliganded RXRalpha enhanced the ERalpha transactivation, suggesting the formation of transcriptionally active heterodimer complexes between the ER and retinoid receptors. Taken together, these results suggest that direct protein-protein interactions may play major roles in the determination of the biological consequences of cross-talk between ERs and RARalpha or RXRalpha.
Project description:The effects of contaminants are typically studied in individual exposures; however, environmental exposures are rarely from a single contaminant. Therefore, the study of chemical mixtures is important in determining the effects of xenobiotics. The constitutive androstane receptor (CAR) responds to endobiotics and xenobiotics, and in turn induces detoxification enzymes involved in their elimination. First, we compared several androgens as inverse agonists, including androgens allegedly used by Bay Area Laboratory Co-operative to enhance athletic performance. CAR inverse agonists ranked in order of potency were dihydroandrosterone (DHA) > tetrahydrogestrinone (THG) > androstanol > norbolethone. Therefore, we used DHA as an inverse agonist during transactivation assays. Next, we examined the effects of several pesticides, plasticizers, steroids, and bile acids on CAR activation. Our data demonstrates that several pesticides and plasticizers, including diethylhexylphthalate, nonylphenol, cypermethrin, and chlorpyrifos activate CAR. Both full and partial CAR activators were discovered, and EC(50) values and Hillslopes were determined for use in the concentration addition models. Concentration addition models with and without restraint values to account for partial activators were developed. Measured results from transactivation assays with a mixture of two to five chemicals indicate that the concentration addition model without restraints correctly predicts activity unless all of the chemicals in the mixture are partial activators, and then restraint values be considered. Overall, our data indicates that it is important to consider that we are exposed to a milieu of chemicals, and the efficacy of each individual chemical is not the sole factor in determining CAR's activity in mixture modeling.
Project description:We have shown previously that the hepatic control region 1 (HCR-1) enhances the activity of the human apolipoprotein C-II (apoC-II) promoter in HepG2 cells via two hormone response elements (HREs) present in the apoC-II promoter. In the present paper, we report that the HCR-1 selectively mediates the transactivation of the apoC-II promoter by chenodeoxycholic acid (CDCA) and 9- cis -retinoic acid. CDCA, which is a natural ligand of farnesoid X receptor alpha (FXRalpha), increases the steady-state apoC-II mRNA levels in HepG2 cells. This increase in transcription requires the binding of retinoid X receptor alpha (RXRalpha)-FXRalpha heterodimers to a novel inverted repeat with one nucleotide spacing (IR-1) present in the HCR-1. This element also binds hepatocyte nuclear factor 4 and apoA-I regulatory protein-1. Transactivation of the HCR-1/apoC-II promoter cluster by RXRalpha-FXRalpha heterodimers in the presence of CDCA was abolished by mutations either in the IR-1 HRE of the HCR-1 or in the thyroid HRE of the proximal apoC-II promoter, which binds RXRalpha-thyroid hormone receptor beta (T3Rbeta) heterodimers. The same mutations also abolished transactivation of the HCR-1/apoC-II promoter cluster by RXRalpha-T3Rbeta heterodimers in the presence of tri-iodothyronine. The findings establish synergism between nuclear receptors bound to specific HREs of the proximal apoC-II promoter and the HCR-1, and suggest that this synergism mediates the induction of the HCR-1/apoC-II promoter cluster by bile acids and retinoids.
Project description:Daphnia pulex is the first crustacean to have its genome sequenced. The genome project provides new insight and data into how an aquatic crustacean may respond to environmental stressors, including toxicants. We cloned Daphnia pulex HR96 (DappuHR96), a nuclear receptor orthologous to the CAR/PXR/VDR group of nuclear receptors. In Drosophila melanogaster, (hormone receptor 96) HR96 responds to phenobarbital exposure and has been hypothesized as a toxicant receptor. Therefore, we set up a transactivation assay to test whether DappuHR96 is a promiscuous receptor activated by xenobiotics and endobiotics similar to the constitutive androstane receptor (CAR) and the pregnane X-receptor (PXR). Transactivation assays performed with a GAL4-HR96 chimera demonstrate that HR96 is a promiscuous toxicant receptor activated by a diverse set of chemicals such as pesticides, hormones, and fatty acids. Several environmental toxicants activate HR96 including estradiol, pyriproxyfen, chlorpyrifos, atrazine, and methane arsonate. We also observed repression of HR96 activity by chemicals such as triclosan, androstanol, and fluoxetine. Nearly 50% of the chemicals tested activated or inhibited HR96. Interestingly, unsaturated fatty acids were common activators or inhibitors of HR96 activity, indicating a link between diet and toxicant response. The omega-6 and omega-9 unsaturated fatty acids linoleic and oleic acid activated HR96, but the omega-3 unsaturated fatty acids alpha-linolenic acid and docosahexaenoic acid inhibited HR96, suggesting that these two distinct sets of lipids perform opposing roles in Daphnia physiology. This also provides a putative mechanism by which the ratio of dietary unsaturated fats may affect the ability of an organism to respond to a toxic insult. In summary, HR96 is a promiscuous nuclear receptor activated by numerous endo- and xenobiotics.
Project description:Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (?3.5-fold) in primary hepatocytes isolated from both CAR-wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism.
Project description:Unlike classical nuclear receptors that require ligand for transcriptional activity, the constitutive androstane receptor (CAR) is active in the absence of ligand. To determine the molecular contacts that underlie this constitutive activity, we created a three-dimensional model of CAR and verified critical structural features by mutational analysis. We found that the same motifs that facilitate ligand-dependent activity in classical receptors also mediated constitutive activity in CAR. This raises a critical question: how are these motifs maintained in an active conformation in unliganded CAR? The model identified several novel interactions that account for this activity. First, CAR possesses a short loop between helix 11 and the transactivation domain (helix 12), as well as a short carboxy-terminal helix. Together, these features favor ligand-independent docking of the transactivation domain in a position that is characteristic of ligand-activated receptors. Second, this active conformation is further stabilized by a charge-charge interaction that anchors the carboxy-terminal activation domain to helix 4. Mutational analysis of these interactions provides direct experimental support for this model. We also show that ligand-mediated repression of constitutive activity reflects both a displacement of coactivator and a recruitment of corepressor. Our data demonstrate that CAR utilizes the same conserved structural motifs and coregulator proteins as originally defined for classical nuclear receptors. Despite these remarkable similarities, our model demonstrates how a few critical changes in CAR can dramatically reverse the transcriptional activity of this protein.
Project description:Nuclear receptors (NRs) can function as ligandinducible transregulators in both mammalian and yeast cells, indicating that important features of transcriptional control have been conserved throughout evolution. We report here the isolation and characterization of an essential yeast protein of unknown function, PSU1, which exhibits properties expected for a co-activator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of NRs. PSU1 interacts in a ligand-dependent manner with the LBD of several NRs, including retinoic acid (RARalpha), retinoid X (RXRalpha), thyroid hormone (TRalpha), vitamin D3 (VDR) and oestrogen (ERalpha) receptors. Importantly, both in yeast and in vitro, these interactions require the integrity of the AF-2 activating domain. When tethered to a heterologous DNA-binding domain, PSU1 can activate transcription on its own. By using yeast reporter cells that express PSU1 conditionally, we show that PSU1 is required for transactivation by the AF-2 of ERalpha. Taken together these data suggest that in yeast, PSU1 is involved in ligand-dependent transactivation by NRs. Sequence analysis revealed that in addition to a highly conserved motif found in a family of MutT-related proteins, PSU1 contains several alpha-helical leucine-rich motifs sharing the consensus sequence LLxPhiL (x, any amino acid; Phi, hydrophobic amino acid) in regions that elicit either transactivation or NR-binding activity.
Project description:Prostate cancer is often slowly progressive, and it can be difficult to treat with conventional cytotoxic drugs. Nonsteroidal antiinflammatory drugs inhibit the development of prostate cancer, but the mechanism of chemoprevention is unknown. Here, we show that the R-enantiomer of the nonsteroidal antiinflammatory drug etodolac inhibited tumor development and metastasis in the transgenic mouse adenocarcinoma of the prostate (TRAMP) model, by selective induction of apoptosis in the tumor cells. This proapoptotic effect was associated with loss of the retinoid X receptor (RXRalpha) protein in the adenocarcinoma cells, but not in normal prostatic epithelium. R-etodolac specifically bound recombinant RXRalpha, inhibited RXRalpha transcriptional activity, and induced its degradation by a ubiquitin and proteasome-dependent pathway. The apoptotic effect of R-etodolac could be controlled by manipulating cellular RXRalpha levels. These results document that pharmacologic antagonism of RXRalpha transactivation is achievable and can have profound inhibitory effects in cancer development.