Nuclear Receptor Pregnane Research Articles

Pregnane X receptor mediates dyslipidemia induced by the HIV protease inhibitor amprenavir in mice.

Human immunodeficiency virus (HIV) protease inhibitors (PIs) have been used successfully in extending the life span of people infected with HIV. The use of PIs has also been associated with dyslipidemia and an increased risk of cardiovascular disease, but the underlying mechanisms remain elusive. Several PIs have been implicated in activating the nuclear receptor pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. Recent studies indicate that PXR may also play an important role in the regulation of lipid homeostasis. In the present study, we identified amprenavir, a widely used HIV PI, as a potent PXR-selective agonist. Computational docking studies combined with site-direct mutagenesis identified several key residues within the ligand-binding pocket of PXR that constitute points of interaction with amprenavir. Amprenavir efficiently activated PXR and induced PXR target gene expression in vitro and in vivo. Short-term exposure to amprenavir significantly increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. Amprenavir-mediated PXR activation stimulated the expression of several key intestinal genes involved in lipid homeostasis. These findings provide critical mechanistic insight for understanding the impact of PIs on cardiovascular disease and demonstrate a potential role of PXR in mediating the adverse effects of HIV PIs in humans.

Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood–brain barrier after CITCO activation

ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood–brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity.

Role of pregnane X receptor (PXR) in diet‐induced obesity

Clinical obesity is a complex metabolic disorder that affects 1 in 3 adults. While a high‐fat diet (HFD) causes obesity and insulin resistance, the specific genes that determine sensitivity to obesity are not known. Recent reports suggest that the nuclear receptor pregnane X receptor (PXR), a xenobiotic receptor that defends against toxic agents, may also play a role in obesity: PXR ligands alter patient plasma lipid levels and affect lipid homeostatic and energy metabolic gene expression. Because of differences in ligand specificity between human and mouse PXRs, we examined the role of PXR in diet‐induced obesity using male PXR‐humanized (hPXR) transgenic and PXR‐knockout (PXR‐KO) vs. wild type (WT) mice. After 16 weeks of high (45%) or normal (12%) fat diets, WT mice had a higher susceptibility to obesity compared to hPXR and PXR‐KO mice. PXR‐KO mice showed the smallest gain in white adipose tissue mass. In contrast, only HFD‐fed hPXR mice had elevated serum glucose and triglycerides and reduced hepatic adiponectin, even though all three genotypes on a HFD displayed insulin resistance, hyperleptinemia and hepatotoxicity. Taken together, our data indicate that while the mouse PXR promoted HFD‐induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and can be used to explore the role of human PXR in metabolic syndrome. [Supported by NIH grants RO1 HL064761, R25HL059868, P20 MD000175, and SC1 HL099139]

Expression, localization and polymorphisms of the nuclear receptor PXR in Barrett's esophagus and esophageal adenocarcinoma

BackgroundThe continuous exposure of esophageal epithelium to refluxate may induce ectopic expression of bile-responsive genes and contribute to the development of Barrett's esophagus (BE) and esophageal adenocarcinoma. In normal physiology of the gut and liver, the nuclear receptor Pregnane × Receptor (PXR) is an important factor in the detoxification of xenobiotics and bile acid homeostasis. This study aimed to investigate the expression and genetic variation of PXR in reflux esophagitis (RE), Barrett's esophagus (BE) and esophageal adenocarcinoma.MethodsPXR mRNA levels and protein expression were determined in biopsies from patients with adenocarcinoma, BE, or RE, and healthy controls. Esophageal cell lines were stimulated with lithocholic acid and rifampicin. PXR polymorphisms 25385C/T, 7635A/G, and 8055C/T were genotyped in 249 BE patients, 233 RE patients, and 201 controls matched for age and gender.ResultsPXR mRNA levels were significantly higher in adenocarcinoma tissue and columnar Barrett's epithelium, compared to squamous epithelium of these BE patients (P < 0.001), and RE patients (P = 0.003). Immunohistochemical staining of PXR showed predominantly cytoplasmic expression in BE tissue, whereas nuclear expression was found in adenocarcinoma tissue. In cell lines, stimulation with lithocholic acid did not increase PXR mRNA levels, but did induce nuclear translocation of PXR protein. Genotyping of the PXR 7635A/G polymorphism revealed that the G allele was significantly more prevalent in BE than in RE or controls (P = 0.037).ConclusionsPXR expresses in BE and adenocarcinoma tissue, and showed nuclear localization in adenocarcinoma tissue. Upon stimulation with lithocholic acid, PXR translocates to the nuclei of OE19 adenocarcinoma cells. Together with the observed association of a PXR polymorphism and BE, this data implies that PXR may have a function in prediction and treatment of esophageal disease.

Pregnane X receptor activation induces FGF19-dependent tumor aggressiveness in humans and mice

The nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required FGF19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and "normal" intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.

Abstract 1703: Importance of pregnane X receptor (PXR) and P-glycoprotein (MDR1) to tamoxifen response and endoxifen disposition

Abstract Tamoxifen, a widely prescribed anti-estrogen for treating estrogen receptor positive breast cancer, is a prodrug which requires bioactivation by cytochrome P450 (CYP) enzymes 2D6 and 3A4 to generate the active metabolite, endoxifen. One obstacle to optimal tamoxifen therapy is the emergence of acquired endocrine resistance during therapy. Although resistance may be multifactorial, studies have shown reduced intra-tumour tamoxifen concentrations in patients with acquired resistance, suggesting the involvement of drug transporters. Tamoxifen is known to induce the expression of the nuclear hormone receptor pregnane X receptor (PXR), which we have shown to be upregulated in breast tumour cells compared with normal adjacent tissue. PXR is also known to regulate the expression of drug transporters including the efflux pump P-glycoprotein (MDR1). Together, we hypothesize that an individual's response to tamoxifen therapy may depend on the interplay between PXR and MDR1. Although, MDR1 does not transport tamoxifen, its role in mediating transport of endoxifen is unknown. Therefore, we assessed the ability of MDR1 to transport endoxifen in vitro by comparing endoxifen flux using a polarized model system of LLCPK cells and LLCPK cells over-expressing human MDR1. In contrast to LLCPK cells, we observed a markedly higher transport of endoxifen in the basal to apical direction of LMDR1 cells, which was abrogated in the presence of the potent and specific MDR1 inhibitor LY335979. To validate the in vivo importance of MDR1 to endoxifen disposition, plasma and tissue concentrations were determined in Mdr1a deficient mice after oral administration of endoxifen. Plasma endoxifen levels did not significantly differ between wildtype and Mdr1a deficient mice. However, brain concentrations of endoxifen were nearly 20-fold higher in Mdr1a deficient mice compared to wildtype mice. Together, these data indicate that endoxifen is a substrate of MDR1. As MDR1 can be overexpressed in some breast tumours, variation in expression and function of this transporter may alter the attained intra-tumour concentration of endoxifen, which may manifest as aberrant clinical response to tamoxifen. We are further investigating the contribution of PXR to the transcriptional regulation of MDR1 and its impact on cellular retention of endoxifen in breast cancer cells. To do this we will utilize the ability of tamoxifen to induce MDR1 expression through PXR activation in breast cancer cell lines and correlate the effect of MDR1 on the intracellular retention of tamoxifen and endoxifen. Finally, we plan to analyze breast tumour tissue from tamoxifen treated and non-treated patients to better understand the role of PXR and MDR1 to clinical response. Accordingly, activation of PXR by tamoxifen and subsequent induction of MDR1 may be an important prognostic factor for patients at risk for tamoxifen resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1703. doi:10.1158/1538-7445.AM2011-1703

Polymorphisms of the nuclear receptor pregnane X receptor and organic anion transporter polypeptides 1A2, 1B1, 1B3, and 2B1 are not associated with breast cancer risk

Organic anion transporter polypeptides (OATPs, SLCOs) are involved in the uptake of conjugates steroid hormones such as estrone-3-sulfate. It has been suggested that the expression of OATPs in breast tissues could impact breast carcinogenesis and tumor pathology. The nuclear receptor pregnane X receptor (PXR) is involved in the regulation of SLCO1A2 expression. We investigated 31 variants located in PXR, SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1 for an association with breast cancer risk and/or histo-pathological tumor characteristics. Polymorphisms were selected on the basis of a known or potential functional consequence and an allele frequency >2%. Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the GENICA population-based breast cancer case-control collection comprising 1,021 cases and 1,015 age-matched controls. Statistical analysis was performed by SAS, and all tests were two-sided. None of the 31 analyzed transporter and PXR polymorphisms showed an association with breast cancer risk or tumor characteristics. Our data suggest that among the many known transporters common variations of PXR, SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1 do not contribute to breast carcinogenesis.

Restoring Blood-Brain Barrier P-Glycoprotein Reduces Brain Amyloid-β in a Mouse Model of Alzheimer's Disease

Reduced clearance of amyloid-beta (Abeta) from brain partly underlies increased Abeta brain accumulation in Alzheimer's disease (AD). The mechanistic basis for this pathology is unknown, but recent evidence suggests a neurovascular component in AD etiology. We show here that the ATP-driven pump, P-glycoprotein, specifically mediates efflux transport of Abeta from mouse brain capillaries into the vascular space, thus identifying a critical component of the Abeta brain efflux mechanism. We demonstrate in a transgenic mouse model of AD [human amyloid precursor protein (hAPP)-overexpressing mice; Tg2576 strain] that brain capillary P-glycoprotein expression and transport activity are substantially reduced compared with wild-type control mice, suggesting a mechanism by which Abeta accumulates in the brain in AD. It is noteworthy that dosing 12-week-old, asymptomatic hAPP mice over 7 days with pregnenolone-16alpha-carbonitrile to activate the nuclear receptor pregnane X receptor restores P-glycoprotein expression and transport activity in brain capillaries and significantly reduces brain Abeta levels compared with untreated control mice. Thus, targeting intracellular signals that up-regulate blood-brain barrier P-glycoprotein in the early stages of AD has the potential to increase Abeta clearance from the brain and reduce Abeta brain accumulation. This mechanism suggests a new therapeutic strategy in AD.

The alternatively spliced murine pregnane X receptor isoform, mPXR Δ171–211 exhibits a repressive action

The orphan nuclear receptor pregnane X receptor regulates enzymes and transport proteins involved in the detoxification and clearance of numerous endobiotic and xenobiotic compounds, including pharmaceutical agents. Multiple alternatively spliced pregnane X receptor isoforms have been identified which are significantly expressed in humans and mice (up to 30% of the total pregnane X receptor transcript), however, little is known about their biological action. We explored functional differences between the major mouse pregnane X receptor isoforms mPXR 431 and mPXR Δ171–211 that lacks 41 amino acids adjacent to the ligand-binding pocket. Transient transfection assays showed that mPXR Δ171–211 reduced the basal transcription of cytochrome P450 3A4 and the drug transporter P-glycoprotein/Multi Drug Resistance Protein 1 and directly repressed the regulatory effects of mPXR 431 on these genes. Replacement of the mPXR Δ171–211 DNA-binding domain with that of GAL4 showed mPXR Δ171–211 retained its repressive role independent of binding to PXR responsive elements located within the cytochrome P450 3A4 and Multi Drug Resistance Protein 1 regulatory regions. Use of the histone deacetylase inhibitor, trichostatin A, demonstrated that the repressive function of mPXR Δ171–211 acts independently of histone acetylation state. Protein interaction assays revealed mPXR Δ171–211 and mPXR 431 differentially bind the obligatory heterodimer partner retinoid X receptor. Furthermore, mPXR 431 and mPXR Δ171–211 proteins could heterodimerize. These studies demonstrate that the variant mouse PXR isoform, mPXR Δ171–211, has a distinct repressive function from mPXR 431 in regulating genes encoding important drug metabolizing enzymes and transport proteins.

In vivo effects of Diosgenin, isolated from Asparagus officinalis, compared to Phenobarbital in rats

Event Abstract Back to Event In vivo effects of Diosgenin, isolated from Asparagus officinalis, compared to Phenobarbital in rats Magdalena Kondeva-Burdina1*, Mitka Mitcheva1, Ilina Krasteva2 and Stefan Nikolov2 1 Medical University-Sofia, Department of Pharmacology, Pharmacotherapy and Toxicology, Bulgaria 2 Medical University-Sofia, Department of Pharmacognosy and Botany, Bulgaria Diosgenin is a well-known antihypercholesterolemic agent. Biliary cholesterol secretion is mediated by different transporters and is stimulated by cholesterol and non-cholesterol steroids – Cholate and Diosgenin. It is known that nuclear receptors CAR and PXR take part in the regulation of hepatic drug metabolism. There are data that Diosgenin increased the expression of lipid-sensing nuclear receptor Pregnane X Receptor (PXR). Phenobarbital (PB) and Phenytoin are the most well known activators of PXR and CAR. PXR has been shown to take part in the regulation of hepatic metabolism – especially biochemically and genetically activation of CYP3A genes. According to these data, it is interesting to investigate in vivo the possible effect of Diosgenin, isolated from Asparagus officinalis, on the activity of some drug-metabolizing enzyme systems, compared to Phenobarbital in rats. Rats were treated with Diosgenin (50 mg/kg p.o./6 days) and Phenobarbital (75 mg/kg i.p./4 days). The total quantity of Cytochrome P450, enzyme activity of Ethylmorphine N-demethylase (EMND) (CYP3A) and Aniline Hydroxilase (AH) (CYP2E1) were measured spectrophotometrically in isolated rat microsomes. As marker of lipid peroxidation and cell protection, we measured the quantity of malondialdehyde (MDA) and reduced glutathione (GSH). Diosgenin and Phenobarbital, administered alone, increased statistically significant, compared to control, the total amount of Cytochrome P450 – with 36% and with 85%, respectively; the activity of EMND with 58% and with 184%, respectively. Diosgenin didn’t influenced the AH activity compared to Phenobarbital. Both Diosgenin and Phenobarbital have no statistically significant effect on GSH level. On the quantity of MDA, Diosgenin has no effect, while Phenobarbital increased it with 27%. Our results showed that Diosgenin, isolated from Astragalus officinalis, exhibited inducible effect similar to the effect of Phenobarbital on the activity of some drug-metabolizing enzyme systems. Keywords: Diosgenin, Phenobarbital, induction, drug-metabolizing enzymes Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotic metabolism Citation: Kondeva-Burdina M, Mitcheva M, Krasteva I and Nikolov S (2010). In vivo effects of Diosgenin, isolated from Asparagus officinalis, compared to Phenobarbital in rats. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00145 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Magdalena Kondeva-Burdina, Medical University-Sofia, Department of Pharmacology, Pharmacotherapy and Toxicology, Sofia, Bulgaria, magdalenakondeva@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Magdalena Kondeva-Burdina Mitka Mitcheva Ilina Krasteva Stefan Nikolov Google Magdalena Kondeva-Burdina Mitka Mitcheva Ilina Krasteva Stefan Nikolov Google Scholar Magdalena Kondeva-Burdina Mitka Mitcheva Ilina Krasteva Stefan Nikolov PubMed Magdalena Kondeva-Burdina Mitka Mitcheva Ilina Krasteva Stefan Nikolov Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Activation of PXR induces hypercholesterolemia in wild-type and accelerates atherosclerosis in apoE deficient mice

The nuclear hormone receptor pregnane X receptor (PXR; also called SXR) functions as a xenobiotic sensor to coordinately regulate xenobiotic metabolism via transcriptional regulation of xenobiotic-detoxifying enzymes and transporters. Although many clinically relevant PXR ligands have been shown to affect cholesterol levels, the role of PXR in cholesterol homeostasis and atherosclerosis has not been thoroughly investigated. Here, we report that activation of PXR by feeding the PXR agonist pregnenolone 16alpha-carbonitrile (0.02%) for 2 weeks to wild-type (WT) mice significantly increased total cholesterol levels and atherogenic lipoproteins VLDL and LDL levels, but had no effect in PXR knockout (PXR(-/-)) mice. Chronic PXR activation in atherosclerosis prone apolipoprotein E deficient (ApoE(-/-)) mice was found to decrease HDL levels and increase atherosclerotic cross-sectional lesion area at both the aortic root and in the brachiocephalic artery by 54% (P < 0.001) and 116% (P < 0.01), respectively. PXR activation significantly regulated genes in the liver involved in lipoprotein transportation and cholesterol metabolism, including CD36, ApoA-IV, and CYP39A1, in both WT and ApoE(-/-) mice. Furthermore, PXR activation can increase CD36 expression and lipid accumulation in peritoneal macrophages of ApoE(-/-) mice. In summary, PXR activation in WT mice increases levels of the atherogenic lipoproteins VLDL and LDL, whereas in ApoE(-/-) mice, PXR increases atherosclerosis, perhaps by diminishing levels of the antiatherogenic ApoA-IV and increasing lipid accumulation in macrophages.

Intestinal Detoxification Limits the Activation of Hepatic Pregnane X Receptor by Lithocholic Acid

The intestinal-derived secondary bile acid (BA) lithocholic acid (LCA) is hepatotoxic and is implicated in the pathogenesis of cholestatic diseases. LCA is an endogenous ligand of the xenobiotic nuclear receptor pregnane X receptor (PXR), but there is currently no consensus on the respective roles of hepatic and intestinal PXR in mediating protection against LCA in vivo. Under the conditions reported here, we show that mice lacking Pxr are resistant to LCA-mediated hepatotoxicity. This unexpected phenotype is found in association with enhanced urinary BA excretion and elevated basal expression of drug metabolism enzymes and the hepatic sulfate donor synthesis enzyme Papss2 in Pxr(-/-) mice. By subsequently comparing molecular responses to dietary and intraperitoneal administration of LCA, we made two other significant observations: 1) LCA feeding induces intestinal, but not hepatic, drug-metabolizing enzymes in a largely Pxr-independent manner; and 2) in contrast to LCA feeding, bypassing first-pass gut transit by intraperitoneal administration of LCA did induce hepatic detoxification machinery and in a Pxr-dependent manner. These data reconcile important discrepancies in the reported molecular responses to this BA and suggest that Pxr plays only a limited role in mediating responses to gut-derived LCA. Furthermore, the route of administration must be considered in the future planning and interpretation of experiments designed to assess hepatic responses to BAs, orally administered pharmaceuticals, and dietary toxins.

The Active Form of Human Aryl Hydrocarbon Receptor (AHR) Repressor Lacks Exon 8, and Its Pro185 and Ala185 Variants Repress both AHR and Hypoxia-Inducible Factor

The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has been associated with reproductive dysfunction and tumorigenesis in humans. Previous studies have characterized the repressor function of AHRRs from mice and fish, but the human AHRR ortholog (AHRR(715)) appeared to be nonfunctional in vitro. Here, we report a novel human AHRR cDNA (AHRRDelta8) that lacks exon 8 of AHRR(715). AHRRDelta8 was the predominant AHRR form expressed in human tissues and cell lines. AHRRDelta8 effectively repressed AHR-dependent transactivation, whereas AHRR(715) was much less active. Similarly, AHRRDelta8, but not AHRR(715), formed a complex with AHR nuclear translocator (ARNT). Repression of AHR by AHRRDelta8 was not relieved by overexpression of ARNT or AHR coactivators, suggesting that competition for these cofactors is not the mechanism of repression. AHRRDelta8 interacted weakly with AHR but did not inhibit its nuclear translocation. In a survey of transcription factor specificity, AHRRDelta8 did not repress the nuclear receptor pregnane X receptor or estrogen receptor alpha but did repress hypoxia-inducible factor (HIF)-dependent signaling. AHRRDelta8-Pro(185) and -Ala(185) variants, which have been linked to human reproductive disorders, both were capable of repressing AHR or HIF. Together, these results identify AHRRDelta8 as the active form of human AHRR and reveal novel aspects of its function and specificity as a repressor.

Interplay between the Nuclear Receptor Pregnane X Receptor and the Uptake Transporter Organic Anion Transporter Polypeptide 1A2 Selectively Enhances Estrogen Effects in Breast Cancer

The ligand-activated nuclear receptor pregnane X receptor (PXR) is known to play a role in the regulated expression of drug metabolizing enzymes and transporters. Recent studies suggest a potential clinically relevant role of PXR in breast cancer. However, the relevant pathway or target genes of PXR in breast cancer biology and progression have not yet been fully clarified. In this study, we show that mRNA expression of organic anion transporter polypeptide 1A2 (OATP1A2), a transporter capable of mediating the cellular uptake of estrogen metabolites, is nearly 10-fold greater in breast cancer compared with adjacent healthy breast tissues. Immunohistochemistry revealed exclusive expression of OATP1A2 in breast cancer tissue. Interestingly, treatment of breast cancer cells in vitro with the PXR agonist rifampin induced OATP1A2 expression in a time-dependent and concentration-dependent manner. Consistent with its role as a hormone uptake transporter, induction of OATP1A2 was associated with increased uptake of estrone 3-sulfate. The rifampin response was abrogated after small interfering RNA targeting of PXR. We then identified a PXR response element in the human OATP1A2 promoter, located approximately 5.7 kb upstream of the transcription initiation site. The specificity of PXR-OATP1A2 promoter interaction was confirmed using chromatin immunoprecipitation. Importantly, we used a novel potent and specific antagonist of PXR (A-792611) to show the reversal of the rifampin effect on the cellular uptake of E(1)S. These data provide important new insights into the interplay between a xenobiotic nuclear receptor PXR and OATP1A2 that could contribute to the pathogenesis of breast cancer and may also prove to be heretofore unrecognized targets for breast cancer treatment.

Structural basis of human pregnane X receptor activation by the hops constituent colupulone.

Hops extracts are used to alleviate menopausal symptoms and as an alternative to hormone replacement therapy, but they can produce potentially harmful drug-drug interactions. The nuclear xenobiotic receptor pregnane X receptor (PXR) is promiscuously activated by a range of structurally distinct chemicals. It has a key role in the transcriptional regulation of genes that encode xenobiotic metabolism enzymes. In this study, hops extracts are shown to induce the expression of numerous drug metabolism and excretion proteins. The beta-bitter acid colupulone is demonstrated to be a bioactive component and direct activator of human PXR. The 2.8-A resolution crystal structure of the ligand binding domain of human PXR in complex with colupulone was elucidated, and colupulone was observed to bind in a single orientation stabilized by both van der Waals and hydrogen bonding contacts. The crystal structure also indicates that related alpha- and beta-bitter acids have the capacity to serve as PXR agonists as well. Taken together, these results reveal the structural basis for drug-drug interactions mediated by colupulone and related constituents of hops extracts.

Active nuclear receptors exhibit highly correlated AF-2 domain motions.

Nuclear receptor ligand binding domains (LBDs) convert ligand binding events into changes in gene expression by recruiting transcriptional coregulators to a conserved activation function-2 (AF-2) surface. While most nuclear receptor LBDs form homo- or heterodimers, the human nuclear receptor pregnane X receptor (PXR) forms a unique and essential homodimer and is proposed to assemble into a functional heterotetramer with the retinoid X receptor (RXR). How the homodimer interface, which is located 30 Å from the AF-2, would affect function at this critical surface has remained unclear. By using 20- to 30-ns molecular dynamics simulations on PXR in various oligomerization states, we observed a remarkably high degree of correlated motion in the PXR–RXR heterotetramer, most notably in the four helices that create the AF-2 domain. The function of such correlation may be to create “active-capable” receptor complexes that are ready to bind to transcriptional coactivators. Indeed, we found in additional simulations that active-capable receptor complexes involving other orphan or steroid nuclear receptors also exhibit highly correlated AF-2 domain motions. We further propose a mechanism for the transmission of long-range motions through the nuclear receptor LBD to the AF-2 surface. Taken together, our findings indicate that long-range motions within the LBD scaffold are critical to nuclear receptor function by promoting a mobile AF-2 state ready to bind coactivators.

The Phytoestrogen Coumestrol Is a Naturally Occurring Antagonist of the Human Pregnane X Receptor

Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.

The Antiapoptotic Role of Pregnane X Receptor in Human Colon Cancer Cells

The orphan nuclear receptor pregnane X receptor (PXR) plays an important role in the detoxification of foreign and endogenous chemicals, including bile acids. PXR promotes bile acid elimination by activating bile acid-detoxifying enzymes and transporters. Certain bile acids are known to promote colonic carcinogenesis by inducing colon cancer cell apoptosis. However, whether and how PXR plays a role in colon cancer apoptosis has not been reported. In this study, we showed that activation of PXR by genetic (using a constitutively activated PXR) or pharmacological (using PXR agonist rifampicin) means protected the PXR-overexpressing colon cancer HCT116 cells from deoxycholic acid-induced apoptosis. Interestingly, activation of PXR also protected HCT116 cells from adriamycin-induced cell death, suggesting that the antiapoptotic effect of PXR was not bile acid specific. Moreover, the antiapoptotic effect of PXR in HCT116 cells appeared to be independent of xenobiotic enzyme regulation, because these cells had little basal and inducible expression of bile acid-detoxifying enzymes. Instead, SuperArray analysis showed that PXR-mediated deoxycholic acid resistance was associated with up-regulation of multiple antiapoptotic genes, including BAG3, BIRC2, and MCL-1, and down-regulation of proapoptotic genes, such as BAK1 and TP53/p53. Treatment with rifampicin in colon cancer LS180 cells, a cell line known to express endogenous PXR, also inhibited apoptosis. Activation of PXR in transgenic mice inhibited bile acid-induced colonic epithelial apoptosis and sensitized mice to dimethylhydrazine-induced colonic carcinogenesis, suggesting that the antiapoptotic effect of PXR is conserved in normal colon epithelium. In summary, our results have established the antiapoptotic role of PXR in both human colon cancer cells and normal mouse colon epithelium.

Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis.

Human PXR Forms a Tryptophan Zipper-Mediated Homodimer

The human nuclear receptor pregnane X receptor (PXR) responds to a wide variety of potentially harmful chemicals and coordinates the expression of genes central to xenobiotic and endobiotic metabolism. Structural studies reveal that the PXR ligand binding domain (LBD) uses a novel sequence insert to form a homodimer unique to the nuclear receptor superfamily. Terminal beta-strands from each monomeric LBD interact in an ideal antiparallel fashion to bury potentially exposed surface beta-strands, generating a 10-stranded intermolecular beta-sheet. Conserved tryptophan and tyrosine residues lock across the dimer interface and provide the first tryptophan-zipper (Trp-Zip) interaction observed in a native protein. We show using analytical ultracentrifugation that the PXR LBD forms a homodimer in solution. We further find that removal of the interlocking aromatic residues eliminates dimer formation but does not affect PXR's ability to interact with DNA, RXRalpha, or ligands. Disruption of the homodimer significantly reduces receptor activity in transient transfection experiments, however, and effectively eliminates the receptor's recruitment of the transcriptional coactivator SRC-1 both in vitro and in vivo. Taken together, these results suggest that the unique Trp-Zip-mediated PXR homodimer plays a role in the function of this nuclear xenobiotic receptor.