RXRbeta Research Articles

Constitutively active retinoid receptors exhibit interfamily and intrafamily promoter specificity.

Retinoid receptors are ligand activated transcription factors that regulate gene transcription through a complex network of interactions with members of the nuclear hormone receptor superfamily. Although ligand is required for trans-activation, addition of ligand to mammalian cells in vitro complicates the study of individual activated retinoid receptors. In order to circumvent this problem we have constructed a series of retinoid receptors which do not require ligand for trans-activation. This was accomplished by fusing the acidic activation domain of the herpes simplex viral protein VP16 to the carboxyl terminus of individual retinoid receptors. All of the chimeric receptors were found to exhibit constitutive trans-activation activity in CV-1 and P19 cells when cotransfected with a reporter that contained a trimerized retinoic acid receptor-beta 2 (RAR beta 2) retinoic acid response element. Further analysis conducted on reporters containing either the RAR beta 2 promoter or the rat cellular retinol binding protein II (rCRBPII) promoter showed that promoter specificity was well conserved between the chimeric receptors in the absence of exogenous retinoid and their ligand-induced native counterparts. Moreover, on the RAR beta 2 promoter reporter construct, the chimeric retinoid receptors displayed both cell type and inter- and intrafamily differences in trans-activation, whereas, trans-activation of the rCRBPII in the absence of exogenous ligand in CV-1 and P19 cells was found to be stimulated only by chimeric retinoid X receptor-alpha (RXR alpha). In P19 cells trans-activation of the rCRBPII promoter by RXR alpha v in the absence of exogenous ligand was inhibited by RAR alpha and the constitutive forms of RAR alpha, RAR beta, RAR gamma, RXR beta, and to a lesser extent RXR gamma.

In vitro transcriptional studies of the roles of the thyroid hormone (T3) response elements and minimal promoters in T3-stimulated gene transcription.

Thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that are encoded by two different genes, TR alpha and TR beta, and bind to thyroid hormone response elements (TREs) in the promoters of thyroid hormone (T3)-regulated genes. Retinoid X receptors (RXRs), major members of the thyroid hormone receptor auxiliary proteins, have recently been shown to enhance the binding of TRs to TREs. We previously showed that TRs extracted from rat pituitary GH3 cells retain ligand (T3) and DNA binding specificity and stimulate rat growth hormone (rGH) promoter activity in a cell-free in vitro transcription system. In this report, we have studied further how T3 activates endogenous TRs and stimulates transcription from different TRE-containing promoters. We found that T3 (10(-8) M) selectively stimulates transcription from rGH-TRE- and TREpal-, but not ME-TRE- and F2-TRE-, containing templates in which these TREs are linked in front of the rGH minimal promoter containing only the TATA box binding protein, but not any other proximal binding protein, sequence. In contrast, only the TREpal/AdML template, in which TREpal oligonucleotide was linked in front of the adenovirus major late gene (AdML) minimal promoter, was stimulated by T3. Electrophoretic mobility shift assay (EMSA) demonstrates that endogenous TR complexes specifically bind to either natural or idealized TRE (rGH-TRE, TREpal, ME-TRE, and F2-TRE) oligonucleotides. To further understand these receptor-DNA complexes formed on various TREs, isoform-specific anti-receptor antisera (TR alpha, TR beta 1, TR beta 2, and RXR beta) were added in the EMSA. These antisera differentially supershifted TR.DNA complexes formed on the TREs. These data suggest either that endogenous TR isoforms and RXR beta may form different complexes on the various TREs or that TR.RXR complexes have distinct conformations when bound to the various TREs. Taken together, these data suggest that particular TREs in which specific TR.RXR complexes are formed and different minimal promoters may provide specificity in T3-mediated transcriptional stimulation of gene expression.

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.

Positive and negative regulation of retinoid X receptor gene expression by thyroid hormone in the rat. Transcriptional and post-transcriptional controls by thyroid hormone.

The 9-cis-retinoic acid receptors (RXRs), belonging to the members of the steroid/thyroid hormone receptor superfamily, act as auxiliary proteins, heterodimerizing with other nuclear receptors such as retinoic acid receptors (RARs), vitamin D receptor, thyroid hormone receptors, and peroxisome-proliferator activated receptor, thereby transactivating target genes in a ligand-dependent manner. We have previously reported that in the rat, thyroid hormone (TH) positively and negatively regulates the hepatic mRNA levels of RXR beta and RXR gamma, respectively. In the present study, we have tried to elucidate the level at which TH regulates the gene expression of RXR beta and RXR gamma in the rat. A RNA synthesis inhibitor (actinomycin D), but not a protein synthesis inhibitor (cycloheximide), blocked the induction of RXR beta mRNA by TH. On the other hand, none of these drugs inhibited the decrease of RXR gamma mRNA levels caused by TH. Nuclear run-on assays showed that the transcription rate of the RXR beta gene was positively regulated by TH, whereas the transcription of RXR gamma gene was not controlled by TH. Taken together, these results indicate that the gene expression of RXR beta is positively regulated by TH at transcriptional level, while the negative regulation of the RXR gamma gene expression by TH may occur at a post-transcriptional level in intact rat. Thus, the RXR-mediated signal transductions may be modulated in part through TH control of the levels of RXR beta and RXR gamma.

Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells.

Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells.

Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Characterization and tissue expression of multiple triiodothyronine receptor-auxiliary proteins and their relationship to the retinoid X-receptors.

Thyroid hormone receptor (TR) binding to thyroid hormone response elements is enhanced by heterodimerization with T3 receptor auxiliary proteins (TRAPs). Although retinoid X-receptors (RXRs) behave similarly to TRAP by heterodimerizing with TRs and enhancing TR binding to thyroid hormone response elements, it is not known whether endogenous TRAPs are RXRs. In this study, we used the electrophoretic mobility shift assay to demonstrate that at least two different TR-TRAP heterodimer complexes can be formed from nuclear extracts of various rat tissues and cell lines. Additionally, the TRAPs appear to be differentially expressed in rat tissues. In antibody supershift experiments, most of the faster migrating TR-TRAP heterodimer bands and some of the slower migrating TR-TRAP heterodimer bands were recognized in several tissues and cell lines by the anti-RXR alpha-specific antibody. Immunodepletion assays showed that the slower migrating TR-TRAP heterodimer bands in most tissues and cell lines were recognized by the anti-RXR beta-specific antibody. Therefore, we have demonstrated that RXR alpha and RXR beta are endogenous TRAPs in a variety of tissues and cell lines, and are differentially expressed. We speculate that this heterogeneity of TRAP distribution may contribute to tissue and cell-specific expression of T3-regulated genes.

Characterization and tissue expression of multiple triiodothyronine receptor-auxiliary proteins and their relationship to the retinoid X- receptors

Thyroid hormone receptor (TR) binding to thyroid hormone response elements is enhanced by heterodimerization with T3 receptor auxiliary proteins (TRAPs). Although retinoid X-receptors (RXRs) behave similarly to TRAP by heterodimerizing with TRs and enhancing TR binding to thyroid hormone response elements, it is not known whether endogenous TRAPs are RXRs. In this study, we used the electrophoretic mobility shift assay to demonstrate that at least two different TR-TRAP heterodimer complexes can be formed from nuclear extracts of various rat tissues and cell lines. Additionally, the TRAPs appear to be differentially expressed in rat tissues. In antibody supershift experiments, most of the faster migrating TR-TRAP heterodimer bands and some of the slower migrating TR-TRAP heterodimer bands were recognized in several tissues and cell lines by the anti-RXR alpha-specific antibody. Immunodepletion assays showed that the slower migrating TR-TRAP heterodimer bands in most tissues and cell lines were recognized by the anti-RXR beta-specific antibody. Therefore, we have demonstrated that RXR alpha and RXR beta are endogenous TRAPs in a variety of tissues and cell lines, and are differentially expressed. We speculate that this heterogeneity of TRAP distribution may contribute to tissue and cell-specific expression of T3-regulated genes.

Retinoic acid receptor γ 2 gene expression is up-regulated by retinoic acid in 3T3-L1 preadipocytes

Retinoids, especially all-trans retinoic acid (RA), have been shown to inhibit the differentiation of preadipose cells. In the present study, the expression of retinoic acid receptors (RAR alpha, beta and gamma) and retinoid X receptors (RXR alpha, beta and gamma) was examined by Northern blot analysis in rat adipose tissue and mouse 3T3-L1 adipose cells. The adipose tissue and/or 3T3-L1 cells expressed mRNAs for a number of nuclear retinoid receptors, including RAR alpha, beta and gamma, and RXR alpha, beta and gamma. RAR alpha, RAR gamma, RXR alpha and RXR beta mRNAs were abundant in adipose tissue and 3T3-L1 cells. RXR gamma mRNA was detected in adipose tissue but not in 3T3-L1 cells. Treatment of 3T3-L1 cells with 1 microM RA led to a 4-5-fold increase in the RAR gamma mRNA level, but only a trace amount of RAR beta mRNA was detected. RAR gamma mRNA expression was rapidly (within 2 h) induced by physiological concentrations of RA in a dose-dependent manner. The response of RAR gamma mRNA expression to RA was reversible; rapid disappearance of RAR gamma mRNA occurred on RA removal. In addition, the induction of RAR gamma expression did not require de novo protein synthesis, but was completely abolished by an inhibitor of RNA synthesis. Using RAR gamma 1 and gamma 2 isoform-specific probes, the patterns of RAR gamma 1 and gamma 2 mRNA expression in 3T3-L1 cells in the presence and absence of RA were examined. RAR gamma 1 mRNA was detected in 3T3-L1 cells but was not affected by RA treatment; however, RAR gamma 2 mRNA was strongly induced by RA.

Mapping of RXRB to human chromosome 6p21.3.

Retinoid X Receptor beta (RXRB) is a member of the retinoid X receptor (RXR) family of nuclear receptors which are involved in mediating the effects of retinoic acid (RA). We have confirmed the localization of RXRB to chromosome 6 and we have mapped the gene to chromosome 6p21.3-p21.1 by PCR amplification of 5' untranslated sequence in panels of rodent-human somatic cell hybrids and to 6p21.3 by fluorescent in situ hybridization.

Mechanisms of transactivation by retinoic acid receptors

Retinoids play an important role in development and differentiation. Their effect is mediated through nuclear receptors, RAR (alpha, beta and gamma) and RXR (alpha, beta and gamma), which are members of a distinct subclass (hereafter referred to as type II) of the nuclear receptor superfamily that includes the thyroid hormone receptor (T3R), the vitamin D3 receptor (VD3R) and the peroxisome proliferator activated receptor (PPAR). Type II receptors transactivate through binding sites composed of closely related half-sites (consensus sequence AGG/TTCA) arranged as direct repeats and, with the possible exception of RXR, do not bind to their cognate binding sites as homodimers but require RXR for high affinity binding. RXR thus provides a link between biologically distinct ligand induced pathways and is a potential target for cross-regulation. In addition, RAR can utilize alternative routes to enhance transcription initiation mediated through transcriptional co-activators which are expressed in a cell-type specific manner.

Inhibition of estrogen-responsive gene activation by the retinoid X receptor beta: evidence for multiple inhibitory pathways.

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Inhibition of Estrogen-Responsive Gene Activation by the Retinoid X Receptor β: Evidence for Multiple Inhibitory Pathways

The retinoid X receptor beta (RXR beta; H-2RIIBP) forms heterodimers with various nuclear hormone receptors and binds multiple hormone response elements, including the estrogen response element (ERE). In this report, we show that endogenous RXR beta contributes to ERE binding activity in nuclear extracts of the human breast cancer cell line MCF-7. To define a possible regulatory role of RXR beta regarding estrogen-responsive transcription in breast cancer cells, RXR beta and a reporter gene driven by the vitellogenin A2 ERE were transfected into estrogen-treated MCF-7 cells. RXR beta inhibited ERE-driven reporter activity in a dose-dependent and element-specific fashion. This inhibition occurred in the absence of the RXR ligand 9-cis retinoic acid. The RXR beta-induced inhibition was specific for estrogen receptor (ER)-mediated ERE activation because inhibition was observed in ER-negative MDA-MB-231 cells only following transfection of the estrogen-activated ER. No inhibition of the basal reporter activity was observed. The inhibition was not caused by simple competition of RXR beta with the ER for ERE binding, since deletion mutants retaining DNA binding activity but lacking the N-terminal or C-terminal domain failed to inhibit reporter activity. In addition, cross-linking studies indicated the presence of an auxiliary nuclear factor present in MCF-7 cells that contributed to RXR beta binding of the ERE. Studies using known heterodimerization partners of RXR beta confirmed that RXR beta/triiodothyronine receptor alpha heterodimers avidly bind the ERE but revealed the existence of another triiodothyronine-independent pathway of ERE inhibition. These results indicate that estrogen-responsive genes may be negatively regulated by RXR beta through two distinct pathways.

Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.

Divergent effects of 9-cis-retinoic acid receptor on positive and negative thyroid hormone receptor-dependent gene expression.

The thyroid hormone (TH)-inducible expression of some genes has recently been shown to be enhanced by 9-cis-retinoic acid (9-cis-RA) receptor (RXR). This effect appears to be at least partially elicited by the ability of RXR to heterodimerize with TH receptor (THR) and enhance its binding to the cis-acting thyroid hormone responsive elements (TREs) found within those genes. However, whether RXR beta enhances TH/THR-mediated transactivation of all Tre-containing genes, and if RXR has any effect on TH-dependent negative regulation are not known. In the present study, we show that the TH/THR-inducible expression of the myelin basic protein (MBP) gene is not enhanced by RXR beta, despite high affinity binding of the RXR beta.THR alpha heterodimer to the MBP-TRE. We also demonstrate that RXR beta reverses the TH/THR-dependent down-regulation mediated by the negative TRE found within the promoter of the mouse thyroid stimulating hormone gene (TSH). The ligand for RXR beta (9-cis-RA), either alone or in combination with TH, did not enhance the transcription mediated by either the MBP-TRE, TSH-TRE, or the malic enzyme (ME)-TRE. However, the ME-TRE is known to confer RXR beta-dependent enhancement of TH-induced gene expression. Thus, the capacity of RXR beta to modulate TH-dependent transcriptional regulation depends upon the nature of the TRE.

Cloning and chromosome mapping of human retinoid X receptor ?: selective amino acid sequence conservation of a nuclear hormone receptor in mammals

The murine retinoid X receptor beta (mRXR beta) is a nuclear hormone receptor that activates transcription of murine major histocompatibility complex (MHC) class I genes in response to retinoic acid. In this study, the human RXR beta gene was mapped onto the short arm or centromeric region of chromosome 6 (6pter-q13), which also harbors the MHC. Chromosomal localization was performed by Southern hybridization of genomic DNA from human rodent cell hybrids with the mRXR beta gene as a probe. In addition, a full-length cDNA clone encoding a human RXR beta was isolated by nucleic acid screening of a human cDNA library with a fragment of the mRXR beta gene as a probe. Comparison of the nucleotide-coding sequences of the human and the murine RXR beta revealed a predominance of third base substitutions, resulting in selective conservation of the predicted amino acid sequence of the proteins. The overall sequence homology was 97.6% on the amino acid level as opposed to 91.6% on the nucleotide level. In Northern hybridization experiments with the human cDNA as a probe, RXR beta gene transcripts were detected in a variety of human tumor cell lines, regardless of whether these cell lines expressed MHC class I genes.

Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.

The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors. Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.