A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors.

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

Interplay of Protein Disorder in Retinoic Acid Receptor Heterodimer and Its Corepressor Regulates Gene Expression.

In its unliganded form, the retinoic acid receptor (RAR) in heterodimer with the retinoid X receptor (RXR) exerts a strong repressive activity facilitated by the recruitment of transcriptional corepressors in the promoter region of target genes. By integrating complementary structural, biophysical, and computational information, we demonstrate that intrinsic disorder is a required feature for the precise regulation of RAR activity. We show that structural dynamics of RAR and RXR H12 regions is an essential mechanism for RAR regulation. Unexpectedly we found that, while mainly disordered, the corepressor N-CoR presents evolutionary conserved structured regions involved in transient intramolecular contacts. In the presence of RXR/RAR, N-CoR exploits its multivalency to form a cooperative multisite complex that displays equilibrium between different conformational states that can be tuned by cognate ligands and receptor mutations. This equilibrium is key to preserving the repressive basal state while allowing the conversion to a transcriptionally active form.

Computer-Assisted Discovery of Retinoid X Receptor Modulating Natural Products and Isofunctional Mimetics.

Natural products (NPs) are progressively recognized as invaluable source of pharmacological tools and lead structures. To enable NP-inspired retinoid X receptor (RXR) modulator design, three novel RXR-targeting NPs were computationally identified. Among them, valerenic acid was found to be selective for RXRß, rendering it a unique pharmacological tool compound. The NPs then served as templates for automated, ligand-based de novo design of innovative, easily accessible mimetics that inherited the biological activities of their natural templates.

An antagonist of the retinoid X receptor reduces the viability of Trichuris muris in vitro.

BACKGROUND: Trichuriasis is a parasitic disease caused by the human whipworm, Trichuris trichiura. It affects millions worldwide, particularly in the tropics. This nematode parasite burrows into the colonic epithelium resulting in inflammation and morbidity, especially in children. Current treatment relies mainly on general anthelmintics such as mebendazole but resistance to these drugs is increasingly problematic. Therefore, new treatments are urgently required. METHODS: The prospect of using the retinoid X receptor (RXR) antagonist HX531 as a novel anthelmintic was investigated by carrying out multiple viability assays with the mouse whipworm Trichuris muris. RESULTS: HX531 reduced both the motility and viability of T. muris at its L3, L4 and adult stages. Further, bioinformatic analyses show that the T. muris genome possesses an RXR-like receptor, a possible target for HX531. CONCLUSIONS: The study suggested that Trichuris-specific RXR antagonists may be a source of much-needed novel anthelmintic candidates for the treatment of trichuriasis. The identification of an RXR-like sequence in the T. muris genome also paves the way for further research based on this new anthelmintic lead compound.

Retinoid X receptor ligands: a patent review (2007 - 2013).

INTRODUCTION: Retinoid X receptors (RXRs) are nuclear receptors that act as ligand-dependent transcription factors. RXRs function as homodimers or as heterodimers with other nuclear receptors, such as retinoic acid receptors, PPARs, liver X receptors, farnesoid X receptor, vitamin D receptor or thyroid hormone receptors. RXR ligands (agonists or antagonists) show various physiological effects, depending on their partner receptors. RXR agonist bexarotene (Targretin®) is used for the treatment of cutaneous T-cell lymphoma in clinical practice. RXR agonists were also reported to be useful for treatment of type 2 diabetes, autoimmune disease and Alzheimer's disease. RXR antagonists were also reported to be effective in type 2 diabetes treatment. AREAS COVERED: Here patent applications (2007 - 2013) concerning RXR ligands are summarized, and the usefulness of RXR ligands as pharmaceutical agents is discussed. EXPERT OPINION: RXR agonists show a wide variety of biological effects. However, they cause serious side effects, such as blood triglyceride elevation, hypothyroidism and others. Thus, for clinical application of RXR agonists, abrogation of these side effects is required. RXR heterodimer-selective agonists and RXR partial agonists exhibiting desired effects without side effects are expected to find clinical application.

Farnesoic acid and methyl farnesoate production during lobster reproduction: possible functional correlation with retinoid X receptor expression.

Farnesoic acid (FA) and methyl farnesoate (MF) are juvenile hormone-related compounds secreted by the mandibular organ (MO) of crustaceans and play an important role in stimulation of ovarian maturation. To better understand how the MO activity influences female reproduction by secretion of FA and MF, the biosynthesis and release of these two compounds were measured in vitro by the incorporation of l-[(3)H-methyl]methionine into MF and [2-(14)C]acetate into FA by the MO of Homarus americanus. The production of FA is 7.5 times that of MF, and most FA and MF synthesized remained within the gland, and was not released into the surrounding medium. Most FA and MF were synthesized in the anterior fan-fold region of the MO. The rates of biosynthesis of FA and MF were stage-related, with maximal production occurring during secondary vitellogenesis (i.e. stages 4 and 5). A potential juvenoid receptor, retinoid X receptor (RXR), HaRXR, was characterized using PCR cloning techniques. HaRXR belongs to the nuclear hormone receptor superfamily and its deduced amino acid sequence shares a high homology to other RXRs of crustaceans, insects, and vertebrates. Transcripts of HaRXR can be detected in many tissues, and significant high expression level was detected in the MO, especially in the anterior fan-fold region. Expression of HaRXR was also related to reproductive stage, and maximal level of expression was observed at stage 4, in which secondary vitellogenesis is occurring. Changes in transcript level of HaRXR and the rates of FA/MF biosynthesis in the female reproductive cycle indicate that HaRXR and FA/MF may play important roles in crustacean reproduction.

Induction of CYP3A4 and MDR1 gene expression by baicalin, baicalein, chlorogenic acid, and ginsenoside Rf through constitutive androstane receptor- and pregnane X receptor-mediated pathways.

The herbal products baicalin, baicalein, chlorogenic acid, and ginsenoside Rf have multiple pharmacological effects and are extensively used in alternative and/or complementary therapies. The present study investigated whether baicalin, baicalein, chlorogenic acid, and ginsenoside Rf induced the expression of the cytochrome P450 3A4 (CYP3A4) and multi-drug resistance 1 (MDR1) genes through the pregnane X receptor and constitutive androstane receptor pathways. Real time PCR, western blotting, and a luminescent assay were used to assess the induction of gene expression and activity of CYP3A4 and MDR1 by the test compounds. The interactions of baicalein/chlorogenic acid/ginsenoside Rf with constitutive androstane receptor and pregnane X receptor were evaluated using luciferase reporter and gel shift assays. Baicalein induced the expression of CYP3A4 and MDR1 mRNA by activating pregnane X receptor and constitutive androstane receptor. Chlorogenic acid and ginsenoside Rf showed a relatively weak effect on CYP3A4 promoter activation only in HepG2 cells cotransfected with constitutive androstane receptor and demonstrated no effects on MDR1 via either the constitutive androstane receptor or pregnane X receptor pathway. Baicalin had no effect on either CYP3A4 or MDR1 gene expression. In conclusion, baicalein has the potential to up-regulate CYP3A4 and MDR1 through the direct activation of the constitutive androstane receptor and pregnane X receptor pathways. Chlorogenic acid and ginsenoside Rf only induced constitutive androstane receptor-mediated CYP3A4 expression.

Interactions of methyl farnesoate and related compounds with a crustacean retinoid X receptor.

While a functional role for the sesquiterpenoid hormone methyl farnesoate in arthropods has been recognized for decades, the identification of a receptor that mediates the action of this hormone remains equivocal. Luciferase reporter assays were used in the present study to evaluate the ability of methyl farnesoate and other putative ligands to activate gene transcription associated with the retinoid X receptor (RXR) and RXR:EcR heterodimeric complexes from the crustacean (Daphnia magna). The daphnid RXR constructs, transfected into HepG2 cells along with the reporter construct, significantly activated luciferase gene expression in response to tributyltin indicating that the crustacean RXR is indeed ligand activated. However, RXR was not activated by methyl farnesoate or other putative RXR ligands. Cells co-transfected with the daphnid RXR and EcR produced luciferase in response to ecdysteroids and this activation was significantly enhanced when cells were also provided either methyl farnesoate or other putative RXR ligands. This synergy among RXR and EcR ligands was not dependent upon the co-activator SRC-1 and did not correlate to a physiological response of daphnids to juvenoid hormones (male sex determination). Results indicate that methyl farnesoate, along with compounds that are functionally similar to methyl farnesoate synergize with ecdysteroids to activate the RXR:EcR receptor complex. However, this effect appears to be unrelated to the ability of these compounds to stimulate male sex determination.

A complex between 6-iodolactone and the peroxisome proliferator-activated receptor type gamma may mediate the antineoplastic effect of iodine in mammary cancer.

Recently we and other groups have shown that molecular iodine (I(2)) exhibits potent antiproliferative and apoptotic effects in mammary cancer models. In the human breast cancer cell line MCF-7, I(2) treatment generates iodine-containing lipids similar to 6-iodo-5-hydroxy-eicosatrienoic acid and the 6-iodolactone (6-IL) derivative of arachidonic acid (AA), and it significantly decreases cellular proliferation and induces caspase-dependent apoptosis. Several studies have shown that AA is a natural ligand of the peroxisome proliferator-activated receptors (PPARs), which are nuclear transcription factors thought to participate in regulating cancer cell proliferation. Our results show that in MCF-7 cells: (1) 6-IL binds specifically and with high affinity to PPAR proteins (EMSA assays), (2) 6-IL activates both transfected (by transactivation assays) and endogenous (by lipid accumulation) peroxisome proliferator response elements, and (3) 6-IL supplementation increases PPAR gamma and decreases PPAR alpha expression. These results implicate PPARs in a molecular mechanism by which I(2), through formation of 6-IL, inhibits the growth of human breast cancer cells.

Molecular cloning of the ecdysone receptor and the retinoid X receptor from the scorpion Liocheles australasiae.

cDNAs of the ecdysone receptor and the retinoid X receptor were cloned from the Japanese scorpion Liocheles australasiae, and the amino acid sequences were deduced. The full-length cDNA sequences of the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor were 2881 and 1977 bp in length, respectively, and the open reading frames encoded proteins of 560 and 414 amino acids. The amino acid sequence of the L. australasiae ecdysone receptor was similar to that of the ecdysone receptor-A of the soft tick, Ornithodoros moubata (68%) and to that of the ecdysone receptor-A1 of the lone star tick, Amblyomma americanum (66%), but showed lower similarity to the ecdysone receptors of Orthoptera and Coleoptera (53-57%). The primary sequence of the ligand-binding region of the L. australasiae ecdysone receptor was highly homologous to that of ticks (85-86%). The amino acid sequence of the L. australasiae retinoid X receptor was also homologous to the amino acid sequence of ultraspiracles of ticks (63%) and insects belonging to the orders Orthoptera and Coleoptera (60-64%). The identity of both the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor to their lepidopteran and dipteran orthologs was less than 50%. The cDNAs of both the L. australasiae ecdysone receptor (L. australasiae ecdysone receptor-A) and the L. australasiae retinoid X receptor were successfully translated in vitro using a rabbit reticulocyte lysate system. An ecdysone analog, ponasterone A, bound to L. australasiae ecdysone receptor-A (K(D) = 4.2 nM), but not to L. australasiae retinoid X receptor. The L. australasiae retinoid X receptor did not enhance the binding of ponasterone A to L. australasiae ecdysone receptor-A, although L. australasiae retinoid X receptor was necessary for the binding of L. australasiae ecdysone receptor-A to ecdysone response elements.

Screening for ligands of human retinoid X receptor-alpha using ultrafiltration mass spectrometry.

Retinoid X receptors (RXRs) function as ligand-activated transcription factors and are obligatory components of a large number of nuclear receptor heterodimers. RXRs help regulate diverse physiological responses including the cancer prevention responses of cell proliferation, inflammation, cell differentiation, and apoptosis. Since RXRs represent important targets for cancer chemoprevention, an ultrafiltration mass spectrometry-based assay was developed to facilitate the discovery of potential chemoprevention agents that bind to human RXRalpha. Natural and synthetic ligands for RXRalpha including 9-cis-retinoic acid, docosahexaenoic acid, and LG100268 could be detected and identified in DMSO (dimethyl sulfoxide) or even complex matrixes such as extracts of marine bacteria. Specific binding of ligands to RXRalpha was demonstrated through competitive binding using ultrafiltration LC-MS/MS (liquid chromatography-tandem mass spectrometry), and ligands could be ranked in order of affinity for RXRalpha. Therefore, ultrafiltration LC-MS/MS is suitable for the screening of complex mixtures such as natural product extracts for the discovery of new ligands to RXRalpha.

Molecular cloning and expression analysis of ecdysone receptor and retinoid X receptor from the kuruma prawn, Marsupenaeus japonicus.

Two cDNAs encoding EcR (MjEcR) and RXR (MjRXR) were cloned and sequenced from the kuruma prawn Marsupenaeus japonicus using PCR techniques. The amino acid sequence of MjEcR was similar to that of known EcR especially in the ligand binding domain (LBD) of insect EcR. The DNA binding domain of MjRXR showed higher homology with that of insect USP (>90% identity) than vertebrate RXR ( approximately 85% identity), while LBD of MjRXR is more homologous with that of vertebrate RXR ( approximately 65% identity) than that of insect USP (30-60% identity). The transcripts of MjEcR and MjRXR were expressed in all tissues examined and in particular, highly in Y-organ and heart and in ovary and heart, respectively. Quantitative real-time PCR analyses revealed that the expression level of MjEcR in hepatopancreas and thoracic muscle increased from intermolt to premolt stages. The analyses also showed that the expressions of MjEcR and MjRXR were regulated in a tissue-specific manner. No significant changes were observed in reproductive organs throughout the molting stages, and MjRXR was expressed much more than MjEcR at all stages. These data suggest that MjRXR mediates a certain hormonal signal related to reproduction.

Ecdysteroid-responsive genes, RXR and E75, in the tropical land crab, Gecarcinus lateralis: differential tissue expression of multiple RXR isoforms generated at three alternative splicing sites in the hinge and ligand-binding domains.

In order to study the potential role of the steroid molting hormone (20-hydroxyecdysone) in regulating molt-induced claw muscle atrophy, full-length cDNAs encoding retinoid-X receptor (Gl-RXR) and E75 early ecdysone inducible gene (Gl-E75) were obtained from land crab (Gecarcinus lateralis) skeletal muscle mRNA using RT-PCR and 3' and 5' RACE. Gl-E75A (3528bp), which encoded a protein of 828 amino acids, had highest sequence identity to Me-E75A from a shrimp (Metapenaeus ensis). It was expressed in skeletal muscle and gonads. The deduced amino acid sequence of Gl-RXR was highly similar to that of the fiddler crab RXR (Up-RXR) and insect ultraspiracle (USP). Nine variant sequences occurred in Gl-RXR mRNAs at three alternative splicing sites, one in the "T box" in the linker D domain and two in the ligand-binding domain (LBD). The three T-box variants, termed T(+8), T(+7), and T(+12), contained insertions of 8, 7, or 12 amino acids, respectively. Four variants were generated at the first site in the LBD. Two of the LBD site 1 variants differed in the presence (+33) or absence (-33) of a 33-amino acid sequence; the other two were LBD truncations with or without the 33 amino acid sequence (+33DeltaE/F and -33DeltaE/F, respectively). Two variants differing in the presence (+35) or absence (-35) of a 35-amino acid sequence were generated at the second site in the LBD. The Gl-RXRa isoform (1516 bp) with the longest open reading frame (+12/+33/+35) encoded a protein of 436 amino acids. Thoracic muscle expressed only isoforms with the T(+12) sequence. In contrast, claw muscle expressed isoforms with T(+7) or T(+12) and fewer isoforms with T(+8). Ovary and testis expressed a greater number of RXR isoforms than skeletal muscle. All tissues expressed full-length and truncated RXR isoforms. These data suggest that differences in response of claw and thoracic muscles to elevated ecdysteroid are due in part to differences in the expression of RXR isoforms.

Trialkyltin compounds bind retinoid X receptor to alter human placental endocrine functions.

Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells, tributyltin chloride and triphenyltin hydroxide, which are typical environmental contaminants and cause masculinization in female mollusks, are potent stimulators of human chorionic gonadotropin production and aromatase activity, which play key endocrine functions in maintaining pregnancy and fetal development. However, the molecular mechanism through which these compounds stimulate these endocrine functions remains unclear. Our current study shows that trialkyltin compounds, including tributyltin chloride and triphenyltin hydroxide, function as RXR agonists. Trialkyltins directly bind to the ligand-binding domain of RXR with high affinity and function as transcriptional activators. Unlike the natural RXR ligand, 9-cis-retinoic acid, the activity of trialkyltins is RXR specific and does not activate the retinoic acid receptor pathway. In addition, trialkyltins activate RXR to stimulate the expression of a luciferase reporter gene containing the human placental promoter I.1 sequence of aromatase, suggesting that trialkyltins stimulate human placental endocrine functions through RXR-dependent signaling pathways. Therefore, our results suggest that activation of RXR may be a novel mechanism by which trialkyltins alter human endocrine functions.

Expression of vitamin D3 receptor and retinoid receptors in human breast cancer: identification of potential heterodimeric receptors.

Vitamin D3 (VD) and all-trans-retinoic acid (ATRA) have been postulated as a novel treatment option for breast carcinoma. Since the combined effects of retinoids and VD derivatives are attributed to heterodimeric interactions between members of the nuclear receptor family, the expression patterns of the heterodimers formed by vitamin D3 receptor (VDR) and the retinoid receptors RARs (RAR-alpha, RAR-beta and RAR-gamma) and RXRs (RXR-alpha, RXR-beta and RXR-gamma) have been studied by immunohistochemistry in benign and malignant breast tissues. Present results revealed that immunoexpressions to all receptor types studied were higher in both in situ and infiltrative carcinomas than in benign breast diseases. In a variable number of cases of infiltrative carcinoma, immunostaining appeared in the nucleus, whereas in the other two disorders immunostaining was only cytoplasmic. The correlation established between VDR and the different isoforms of retinoid receptors revealed that VDR seems to select mainly RAR-alpha to form heterodimers and to exert their properties as transcription factor. The results of this study suggest that this heterodimer plays a critical role in cancer malignancy, and its presence indicates those patient groups presenting a better response to adjuvant therapies based on the combination of vitamin D and ATRA.