RESUMEN
OBJECTIVES: ANTXR2 variants have been associated with ankylosing spondylitis (AS) in two previous genome-wide association studies (GWAS) (pâ¼9×10(-8)). However, a genome-wide significant association (p<5×10(-8)) was not observed. We conducted a more comprehensive analysis of ANTXR2 in an independent UK sample to confirm and refine this association. METHODS: A replication study was carried out with 2978 cases and 8365 controls. Then, these were combined with non-overlapping samples from the two previous GWAS in a meta-analysis. Human leukocyte antigen (HLA)-B27 stratification was also performed to test for ANTXR2-HLA-B27 interaction. RESULTS: Out of nine single nucleotide polymorphisms (SNP) in the study, five SNPs were nominally associated (p<0.05) with AS in the replication dataset. In the meta-analysis, eight SNPs showed evidence of association, the strongest being with rs12504282 (OR=0.88, p=6.7×10(-9)). Seven of these SNPs showed evidence for association in the HLA-B27-positive subgroup, but none was associated with HLA-B27-negative AS. However, no statistically significant interaction was detected between HLA-B27 and ANTXR2 variants. CONCLUSIONS: ANTXR2 variants are clearly associated with AS. The top SNPs from two previous GWAS (rs4333130 and rs4389526) and this study (rs12504282) are in strong linkage disequilibrium (r(2)≥0.76). All are located near a putative regulatory region. Further studies are required to clarify the role played by these ANTXR2 variants in AS.
Asunto(s)
Receptores de Péptidos/genética , Espondilitis Anquilosante/genética , Estudios de Casos y Controles , Niño , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Antígeno HLA-B27/análisis , Humanos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido SimpleRESUMEN
Retinoids exert their pleiotropic effects on cell differentiation and proliferation through specific nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Two biologically highly active natural retinoids have been identified, all-trans-retinoic acid (t-RA) and 9-cis-retinoic acid (9-cis-RA). The RXRs exclusively bind 9-cis-RA, whereas the RARs bind both isomers of RA with comparable affinity. Recently published results suggest that RARs have the same binding site for t-RA and 9-cis-RA but with different determinants (1-3). Antagonist binding on RARalpha has been suggested to induce distinct conformational changes in comparison with agonist binding. To elucidate the region minimally required for efficient binding of agonist (t-RA and 9-cis-RA) and antagonist Ro 41-5253 to the RARalpha, we generated N- and C-terminally truncated mutants of the receptor. Characterization of these deletion mutant proteins using protease mapping and ligand binding experiments revealed that different parts of the ligand-binding domain are necessary for t-RA, 9-cis-RA, and antagonist binding. Three distinct regions of the ligand-binding domain of the human retinoic acid receptor-alpha are required for binding of t-RA (RARalpha187-402), 9-cis-RA (RARalpha188-409), and the antagonist Ro 41-5253 (RARalpha226-414).
Asunto(s)
Benzoatos/metabolismo , Cromanos/metabolismo , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Tretinoina/metabolismo , Alitretinoína , Sitios de Unión , Análisis Mutacional de ADN , Cartilla de ADN , Humanos , Cinética , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Receptor alfa de Ácido Retinoico , Retinoides/metabolismo , Eliminación de SecuenciaRESUMEN
The broad spectrum of physiological activities of retinoids is mediate d by two types of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Though they have 9-cis retinoic acid as a common ligand, the amino acid sequence of their ligand binding domains is only distantly related (27%). This fact makes it probable that the ligand binding pockets of RARs and RXRs differ significantly with respect to their three dimensional structure. Therefore, one can expect that selective ligands for these receptor subclasses do exit. A clear example of a naturally existing RAR-selective retinoid is all-trans retinoic acid. Here we report on two synthetic retinoids which are very closely related to retinoic acid in structure yet show good receptor subclass selectivity. These compounds have a saturated double bond in the polyene side chain between either the 7, 8 or 9, 10 carbon atoms and are highly RAR or RXR selective, respectively (as shown by receptor binding, transactivation activity and the ability to induce RXR homodimer formation). In addition, we present compounds of the synthetic arotinoid class which are highly RAR selective. Interestingly the corresponding '9-cis analogs' are not able to bind or activate RXR alpha and show greatly reduced activity on the RARs.
Asunto(s)
Receptores de Ácido Retinoico/efectos de los fármacos , Retinoides/farmacología , Factores de Transcripción/efectos de los fármacos , Animales , Núcleo Celular/metabolismo , Ligandos , Ratones , Receptores X Retinoide , Relación Estructura-ActividadRESUMEN
Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator-activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression. Known activators of peroxisome proliferator-activated receptor include polyunsaturated fatty acids and, for RXR, the 9-cis isomer of retinoic acid. Here we report that phytanic acid is also a natural ligand for RXR alpha, being able to activate a RXR-responsive promoter. We present evidence that phytanic acid binds to RXR alpha, promotes formation of an RXR alpha/RXR response element complex (as detected by gel retardation), and induces a RXR alpha conformational change similar to that induced by 9-cis-retinoic acid (as detected by protease sensitivity). These results suggest an involvement of RXR alpha in the control of fatty acid metabolism and could imply that RXRs have a role in the disease effects resulting from defective phytanic acid catabolism.
Asunto(s)
Ácido Fitánico/metabolismo , Receptores de Ácido Retinoico/agonistas , Factores de Transcripción/agonistas , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Humanos , Ligandos , Datos de Secuencia Molecular , Receptores de Ácido Retinoico/genética , Proteínas Recombinantes/metabolismo , Enfermedad de Refsum/metabolismo , Receptores X Retinoide , Factores de Transcripción/genética , Activación Transcripcional/efectos de los fármacos , Tretinoina/metabolismoRESUMEN
Cellular responsiveness to retinoic acid and its metabolites is conferred through two distinct families of receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report on the identification and characterization of several conformationally restricted retinoids, which selectively bind and activate RX receptors. Under the influence of retinoids, HL-60 myelocytic leukemia cells differentiate into granulocytes. This effect is mediated by RAR alpha, as has been demonstrated through the use of a selective RAR alpha antagonist (Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7129-7133). Here, we show that conformationally restricted RXR-specific retinoids, at doses that are per se inactive, are able to potentiate by up to one order of magnitude the pro-differentiating effects of all-trans retinoic acid and an RAR alpha-selective synthetic retinoid. We also present evidence that these RXR-selective ligands are able to bind to a DNA RXR.RAR heterodimer complex. This finding demonstrates that agonists for RARs and RXRs can synergistically promote HL-60 differentiation, which could be mediated through a heterodimer of these receptors.
Asunto(s)
Diferenciación Celular/fisiología , Receptores de Ácido Retinoico/fisiología , Retinoides/farmacología , Factores de Transcripción/fisiología , Activación Transcripcional/efectos de los fármacos , Unión Competitiva , Diferenciación Celular/efectos de los fármacos , ADN Complementario , Células HL-60 , Humanos , Cinética , Estructura Molecular , Receptores de Ácido Retinoico/biosíntesis , Receptores de Ácido Retinoico/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Retinoides/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/efectos de los fármacos , TransfecciónRESUMEN
The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/química , Receptores de Ácido Retinoico/química , Factores de Transcripción , Animales , Benzoatos/farmacología , Cromanos/farmacología , Clonación Molecular , Endopeptidasas , Humanos , Ratones , Modelos Biológicos , Naftalenos/farmacología , Mapeo Peptídico , Conformación Proteica/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/genética , Receptores de Ácido Retinoico/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Receptores X Retinoide , Retinoides/farmacología , Eliminación de Secuencia , Tretinoina/farmacologíaRESUMEN
Several known and some new retinoids were synthesized and their in vivo activity was investigated by an assay, based on induction of alkaline phosphatase in P19 teratocarcinoma cells, human prostate carcinoma cells and primary cultures of neonatal rat heart cells. The assay used in this study was found to be reproducible and useful for rapid screening of retinoids for biological activity. Two newly synthesized compounds exhibit high biological activity. The biological potency of the compounds was compared to their ability to bind to recombinant retinoic-acid receptor alpha and to cellular retinoic-acid-binding protein I determined by Charsorb-binding assay. mRNA of both retinoic-acid-binding proteins could be detected in the three cell lines investigated. As expected from the number of different retinoic-acid receptors, the results suggest that retinoids do not need to bind retinoic-acid receptor alpha nor cellular retinoic-acid-binding protein I in order to exhibit biological activity, but most retinoids investigated show a clear correlation between binding to these proteins and their biological activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Retinoides/farmacología , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/genética , Animales , Bioensayo , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/genética , Inducción Enzimática/efectos de los fármacos , Humanos , Masculino , Ratones , ARN Mensajero/análisis , Ratas , Receptores de Ácido Retinoico , Proteínas Recombinantes/metabolismo , Retinoides/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The human retinoic acid receptor alpha was expressed in Escherichia coli. The recombinant protein was found to be very unstable in several E. coli strains, probably due to proteolysis. Conditions were established to obtain reasonable amounts of active protein for ligand and DNA binding studies. The recombinant receptor showed the expected DNA binding activities in gel-retardation assays. Ligand binding properties were measured in a charcoal absorption assay. The dissociation constant for highly specific bound retinoic acid was found to be 0.67 nM. The affinity of several synthetic retinoids to the recombinant protein was determined and compared to their biological activity. Some of the values presented here differ significantly from those published earlier for the receptor or its isolated hormone-binding domain.