RXRbeta Research Articles

New retinoid X receptor subtypes in zebra fish (Danio rerio) differentially modulate transcription and do not bind 9-cis retinoic acid.

Retinoid X receptors (RXRs), along with retinoic acid (RA) receptors (RARs), mediate the effects of RA on gene expression. Three subtypes of RXRs (alpha, beta, and gamma) which bind to and are activated by the 9-cis stereoisomer of RA have been characterized. They activate gene transcription by binding to specific sites on DNA as homodimers or as heterodimers with RARs and other related nuclear receptors, including the vitamin D receptor, thyroid hormone receptors (TRs), and peroxisome proliferator-activated receptors. Two additional RXR subtypes (delta and epsilon) isolated from zebra fish cDNA libraries are described here; although both subtypes form DNA-binding heterodimers with RARs and TR, neither binds 9-cis RA, and both are transcriptionally inactive on RXR response elements. In cotransfection studies with TR, the delta subtype was found to function in a dominant negative manner, while the epsilon subtype had a slight stimulatory effect on thyroid hormone (T3)-dependent transcriptional activity. The discovery of these two novel receptors in zebra fish expands the functional repertoire of RXRs to include ligand-independent and dominant negative modulation of type II receptor function.

Exclusive Homodimerization of the Orphan Receptor Hepatocyte Nuclear Factor 4 Defines a New Subclass of Nuclear Receptors

Hepatocyte nuclear factor 4 (HNF-4), a highly conserved member of the steroid hormone receptor superfamily critical for development and liver-specific gene expression, is very similar to another superfamily member, retinoid X receptor alpha (RXR alpha), in overall amino acid sequence and DNA binding specificity. Since RXR alpha is known to heterodimerize with many other nuclear receptors, the formation of heterodimers between HNF-4 and RXR alpha was examined. With the electrophoretic mobility shift assay, coimmunoprecipitation, and transient transfection assays, it is shown that, unlike other nuclear receptors, HNF-4 does not form heterodimers with RXR alpha either in the presence or in the absence of DNA. We also show that in vitro-translated HNF-4 does not form heterodimeric complexes on DNA with a number of other receptors, including RXR beta, RXR gamma, retinoic acid receptor alpha, or thyroid hormone receptor alpha. To investigate the hypothesis that the lack of heterodimerization between HNF-4 and RXR alpha is due to a strong homodimerization activity of HNF-4, glycerol gradient sedimentation and kinetic analysis were used to show that HNF-4 is in fact a stable homodimer in solution. Finally, immunohistochemistry is used to show that the HNF-4 protein is found exclusively in the nuclei in both HepG2 cells, which express endogenous HNF-4, and transfected COS cells, which overexpress HNF-4. These findings lead us to propose that HNF-4 defines a new subclass of nuclear receptors which reside primarily in the nucleus and which bind DNA and regulate transcription as homodimers.

Efficient Inhibition of Activation-induced Fas Ligand Up-regulation and T Cell Apoptosis by Retinoids Requires Occupancy of both Retinoid X Receptors and Retinoic Acid Receptors

Two retinoic acid (RA) receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been identified. All-trans-RA and its 9-cis-isomer are ligands for RARs, but only 9-cis-RA binds RXRs with high affinity. Activation-induced T cell hybridoma death is mediated via the engagement of Fas by activation-up-regulated Fas ligand, and RA prevents this type of apoptosis by inhibiting the induction of Fas ligand expression. To investigate the mechanism of RA action, T hybridoma cells were transfected with cDNA encoding RXR beta or dominant-negative RXR beta. Cells that overexpressed RXR beta were more sensitive to 9-cis-RA rescue from activation-induced death than cells transfected with vector alone. In contrast, cells expressing the dominant-negative RXR beta could not be rescued from death with 9-cis-RA. In wild type cells, an RAR-selective synthetic retinoid had little effect on activation-induced apoptosis, while an RXR-selective agonist prevented apoptosis but only at concentrations about approximately 10-fold greater than that required for 9-cis-RA. Simultaneous addition of the RAR- and RXR-selective retinoids completely prevented activation-induced apoptosis at concentrations where either alone had relatively little protective effect. The same hierarchy of efficacy was found for activation-induced Fas ligand expression. These data demonstrate that binding of both RARs and RXRs is required for efficient inhibition of activation-induced Fas ligand upregulation and T cell apoptosis by retinoic acid.

Retinoic acid directly stimulates osteoclastic bone resorption and gene expression of cathepsin K/OC-2

Vitamin A metabolites such as all-trans-retinoic acid (all-trans-RA) affect several steps of metabolic processes in vertebrates. In the last few years, several studies have shown the effect of RA on bone formation and metabolism. However, mechanisms of its action still remain unclear, especially with respect to the regulation of bone cells. Therefore, this study was carried out to clarify how RA regulates the activity of osteoclasts. Using a pit assay involving unfractionated bone cells, including osteoclasts obtained from rabbits, we found that RA stimulated an increase in the bone-resorbing activity in a dose- and time-dependent manner. Furthermore, this effect occurred more rapidly than that of treatments with 1 alpha,25-dihydroxyvitamin D3. However, this effect of RA may be partly related to cross-talk between osteoclasts and other types of cells. Therefore we studied the effect of RA on isolated osteoclasts. We found that all-trans-RA regulates the gene expression of cathepsin K/OC-2, a dominant cysteine proteinase, at the transcriptional level in mature osteoclasts isolated from rabbits. Moreover, retinoic acid-receptor alpha mRNA and retinoid X-receptor beta mRNA were expressed in these mature osteoclasts. Our results indicate that osteoclasts are target cells for RA and that RA might regulate a part of bone formation and metabolism through osteoclasts.

Evidence that Retinoid X Receptors Mediate Retinoid-dependent Transcriptional Activation of the Retinoic Acid Receptor β Gene in S91 Melanoma Cells

S91 melanoma cells are growth arrested and differentiate when treated with retinoids. These processes correlate with expression of the retinoic acid receptor (RAR) beta gene, which is induced through a retinoic acid response element (beta RARE). We wished to determine which endogenous retinoid receptors (RARs and retinoid X receptors, RXRs) mediate induction of the RAR beta gene. We show that RXR alpha and RXR beta are constitutively expressed. Electrophoretic mobility shift assays with nuclear extracts show specific binding to the beta RARE (Complex I) in untreated cells, which can be supershifted by antibodies against RXRs but not by anti-RAR antibodies. After 48 h of treatment with retinoic acid, Complex I is replaced by a faster migrating Complex II, which can be supershifted by anti-RAR beta and anti-RXR alpha antibodies. This suggests that induction of the RAR beta gene is largely mediated by RXRs only. Accordingly, we also find that 9-cis RA, which activates both RAR and RXR, is a more potent inducer of the RAR beta gene than RA, which only activates RAR. After 48 h, all RXRs appear to be titrated by the newly synthesized RAR beta into an RAR beta.RXR heterodimer complex. Thus, it appears that the beta RARE is sequentially occupied by RXR dimers and RAR-RXR heterodimers.

Synthesis and structure-activity relationships of stilbene retinoid analogs substituted with heteroaromatic carboxylic acids.

Retinoids elicit biological responses by activating a series of nuclear receptors. Six retinoid receptors belonging to two families are currently known: retinoic acid receptors (RAR alpha,beta,and gamma) and retinoid X receptors (RXR alpha,beta,and gamma). Stilbene retinoid analogs of retinoic acid (RA), such as (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)prope n-1- yl]benzoic acid (TTNPB, 1) and (E)-4-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)pro pen-1- yl]benzoic acid (3-methyl-TTNPB, 2), display differential RAR and RXR activities, depending on the substituent at C3 of the naphthalene ring. We report here structural modifications of the benzoate moiety of 2 that result in analogs with greater RXR selectivity as well as those with pan-agonist (activate both RAR and RXR receptors) activities, analyze the structural features that impart receptor selectivity, and describe a stereoselective method for the synthesis of these analogs. The biological activities associated with the RAR and RXR receptors were examined by testing representative examples with different receptor activation profiles for their ability to induce tissue transglutaminase (Tgase) activity in a human promyelocytic leukemia cell line (HL-60 cdm-1) and to inhibit tumor-promoter-induced ornithine decarboxylase (ODC) activity in hairless mouse skin. These results suggest that RAR agonists and RXR agonists may have different therapeutic applications. Finally, we show that RXR agonists are significantly reduced in teratogenic potency relative to RAR agonists and may therefore have significant advantages in clinical practice.

Isoform-specific retinoid-X receptor (RXR) antibodies detect differential expression of RXR proteins in the pituitary gland.

There are three known isoforms of the retinoid-X receptor (RXR): RXR alpha, RXR beta, and RXR gamma. RXR alpha and RXR beta messenger RNAs are widely expressed, whereas RXR gamma messenger RNA is restricted to only a few tissues, including embryonic pituitary gland. Little is known about the level of expression and cell distribution of RXR proteins in the adult pituitary gland. To examine these issues further, we raised isoform-specific polyclonal antibodies against each of the known mouse RXR isoforms using synthetic peptides containing isoform-specific epitopes from the amino-terminal region. The specificity of each antibody was confirmed by immunoprecipitation, Western immunoblot analysis, and electrophoretic mobility shift assay with supershift studies of in vitro translated RXR isoforms. Immunocytochemical analysis showed that anti-RXR alpha and anti-RXR beta antisera stained the nuclei of most pituitary cells. In contrast, anti-RXR gamma antiserum stained the nuclei of only a few cells throughout the pituitary. In the hypothyroid state, however, a marked increase in both the number and density of RXR gamma-immunostained nuclei were observed compared to those in the euthyroid state. Double immunostaining studies of hypothyroid rat pituitary with antibodies against pituitary hormones indicated that RXR gamma protein was predominantly expressed in thyrotropes. Antibody supershift experiments using nuclear extracts of adult rat whole pituitary and rodent pituitary cell lines showed that anti-RXR gamma antibody could alter the mobility of protein-DNA complexes formed only from nuclear extracts of rat whole pituitary and thyrotropic TtT-97 cells. In contrast, anti-RXR alpha and anti-RXR beta antibodies could supershift protein-DNA complexes formed from nuclear extracts of all cell lines tested. RXR gamma protein expression in TtT-97 cells also was observed by Western immunoblot analyses. Therefore, there is thyrotrope-predominant expression of RXR gamma protein. We speculate that RXR gamma may play a role in the regulation of thyroid hormone target genes in thyrotropes and possibly cell type differentiation in the pituitary.

Evidence for the Involvement of Retinoic Acid Receptor RARα-dependent Signaling Pathway in the Induction of Tissue Transglutaminase and Apoptosis by Retinoids

In this study, we show that all-trans-retinoic acid (RA) is a potent inducer of tissue transglutaminase (TGase II) and apoptosis in the rat tracheobronchial epithelial cell line SPOC-1. We demonstrate that these cells express the retinoid receptors RAR alpha, RAR gamma, and RXR beta. To identify which of these receptors are involved in regulating these processes, we analyzed the effects of several receptor-selective agonists, an antagonist, and a dominant-negative RAR alpha. We show that the RAR-selective retinoid SRI-6751-84 strongly increased TGase II expression at both the protein and mRNA levels, whereas the RXR-selective retinoid SR11217 had little effect. The RAR alpha-selective retinoid Ro40-6055 was also able to induce TGase II, whereas the RAR gamma-selective retinoid CD437 was inactive. The induction of TGase II by the RAR-selective retinoid was completely inhibited by the RAR alpha-antagonist Ro41-5253. Overexpression of a truncated RAR alpha gene with dominant-negative activity also inhibited the induction of TGase II expression. The increase in TGase II is associated with an induction of apoptosis as revealed by DNA fragmentation and the generation of apoptotic cells. We demonstrate that apoptosis is affected by retinoids in a manner similar to TGase II. Our results suggest that the induction of TGase II expression and apoptosis in SPOC-1 cells are mediated through an RAR alpha-dependent signaling pathway.

The Ligand-Binding Domains of the Thyroid Hormone/Retinoid Receptor Gene Subfamily Function in Vivo To Mediate Heterodimerization, Gene Silencing, and Transactivation

The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LBD of RXRbeta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad or T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity. This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements. These studies indicate that the LBD has diverse functional roles in gene regulation.

Ligand occupancy is not required for vitamin D receptor and retinoid receptor-mediated transcriptional activation.

Although steroid hormone receptor activation has been known to be dependent on ligand binding, we report here ligand-independent transcriptional activation of the vitamin D receptor and retinoid receptors. In these studies, CV1 cells were transiently transfected with a human vitamin D receptor (VDR) expression vector and a reporter plasmid that contains multiple copies of the rat osteocalcin vitamin D response element up-stream of the bacterial chloramphenicol acetyltransferase (CAT) gene [osteocalcin (OC)VDREtkCAT]. Treatment of cells with 10(-8) M 1,25-dihydroxyvitamin D3 resulted in a 25-fold induction of CAT activity. When cells were treated with 5-50 nM okadaic acid (OA), an inhibitor of protein phosphatase-1 and -2A, significant inductions of CAT activity (18- to 57-fold) were observed. As VDR and dopamine receptors are colocalized in certain brain regions, we also examined whether VDR-mediated transcription can be activated by dopamine. VDR was found to activate CAT gene expression in cells treated with 200-500 microM dopamine (3- to 11-fold induction) or the selective D1 agonist SKF38393 (20-fold induction). Cells were also transfected with retinoic acid receptor (RAR) or retinoid-X receptor (RXR) expression vectors and reporter plasmids that contain either a retinoic acid response element or an RXR-specific response element. OA alone induced chloramphenicol acetyltransferase (CAT) activity in cells transfected with RAR alpha, RAR beta, RXR alpha, RXR beta, or RXR gamma (3- to 18-fold induction). However, OA did not affect transcription by RAR gamma, suggesting specificity of activation by OA among the retinoid receptors. Although the retinoid receptors have been detected in brain, maximum stimulation of transcription was not greater than 1.6-fold in the presence of 100-500 microM dopamine or 100 microM SKF38393 treatment. These data suggest specificity for dopamine activation among steroid hormone receptors and that phosphorylation alone, in the absence of ligand, can activate VDR- and retinoid receptor-mediated transcription.

A Dynamic Balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and Retinoic Acid Receptor:Retinoid X Receptor Heterodimers Regulates Oct-3/4 Expression in Embryonal Carcinoma Cells

The Oct-3/4 transcription factor is a member of the POU family of transcription factors and, as such, probably plays a crucial role in mammalian embryogenesis and differentiation. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Similarly, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repressed in retinoic acid (RA)-differentiated EC cells. Previously we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for negative regulators. Our present results demonstrate that in P19 and RA-treated P19 cells, the orphan receptors ARP-1/COUP-TFII and EAR-3/COUP-TFI repress Oct-3/4 promoter activity through the RAREoct site in a dose-dependent manner. While the N-terminal region of the ARP-1/COUP-TFII receptor is dispensable for this repression, the C-terminal domain harbors the silencing region. Interestingly, three different RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha:RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bind and activate Oct-3/4 promoter through the RAREoct site in a ligand-dependent manner. We have shown that antagonism between ARP-1/COUP-TFII or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellular balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repression by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RAR:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by ARP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affinity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to compete with and even displace RAR:RXR from the RAREoct site and subsequently to actively silence the Oct-3/4 promoter. We have shown that RA treatment of EC cells results in up-regulation of ARP-1/COUP-TFII and EAR-3/COUP-TFI expression. Most interestingly, in RA-treated EC cells, the kinetics of Oct-3/4 repression inversely correlates with the kinetics of ARP-1/COUP-TFII and EAR-3/COUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors participate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene expression during development.

1,25-Dihydroxyvitamin D3 and retinoid X receptor expression in human colorectal neoplasms.

Epidemiological studies suggest that 1,25-dihydroxyvitamin D3 (D3) protects against colorectal carcinogenesis. Animal and in vitro studies show an antiproliferative effect of D3 in a variety of tumours including those of large bowel origin. D3 actions are mediated by D3 receptors (VDR) alone or by VDR in conjunction with retinoid X receptors (RXRs) in all D3 responsive tissues. The expression of mRNAs encoding VDR and RXRs in normal and malignant human colorectum was determined. Full length VDR (4.6 kB), RXR alpha (5.5 kB), and RXR gamma (3.5 and 7 kB) mRNAs were expressed in all tissues, but RXR beta mRNA was not expressed in any. VDR expression was reduced in 12 carcinomas relative to paired normal mucosa, and RXR alpha expression was reduced in nine. There was no correlation between VDR or RXR alpha expression and the site, grade of differentiation, or Dukes's staging of the tumour. The finding of persistent VDR and RXR coexpression in all colorectal tumours provides a rational basis for exploring a role for D3 in the treatment of colorectal malignancy.

Retinoid-X receptor (RXR) differentially augments thyroid hormone response in cell lines as a function of the response element and endogenous RXR content.

Retinoid-X receptor (RXR) forms heterodimers with thyroid hormone receptor (TR) and significantly enhances binding to thyroid hormone response elements (TREs). Expression of RXR in a transient transfection assay augments the T3 response, but the influences of the specific cell line and TRE used have not been systematically studied. We determined RXR alpha and -beta augmentation of the TR alpha-mediated T3 response in transient transfection assays of COS, JEG, and mouse embryonic stem (ES) cell lines for a series of eight wild-type thyroid hormone (T3) and retinoic acid response elements (previously shown to bind TR). RXR augmented T3-induced expression in COS and ES cells (1.5- to 4-fold greater expression with added RXR compared to TR alone), but had minimal effect on augmentation of response in JEG cells. For most elements studied there was a proportional augmentation of basal and T3-stimulated expression. TREs from rat GH and laminin-B1, however, had relatively higher levels of T3-induced expression as a result of RXR cotransfection (T3 induction ratios increased 2-fold or greater). Previous characterization of these elements demonstrates that they contain more than two hexameric binding domains, all of which can simultaneously bind TR. The influence of endogenous RXR expression in a cell line on RXR augmentation of the T3 response was determined. RXR alpha and -beta messenger RNA (mRNA) expression was quantitated by Northern blot in each cell line. COS and JEG cells expressed almost exclusively RXR alpha mRNA, although expression was almost 2-fold higher in JEG compared to COS cells (12 +/- 2.5 vs. 6.8 +/- 0.5 density units relative to actin; mean +/- SE; P < 0.05). ES cells expressed only RXR beta mRNA, but at a very low level (0.4 +/- 0.1). Nuclear extracts prepared from JEG and COS cells augmented TR binding proportional to the endogenous RXR mRNA expression, and the heterodimer band was supershifted by the addition of antibody to RXR alpha. Nuclear extracts from ES cells had no detectable TR heterodimer binding to a range of response elements. RXR augmentation of the T3 response differs among cell lines and is greater in those with reduced endogenous RXR. Furthermore, the functional augmentation of the T3 response ratio by RXR is likely to require additional sequences contained in only a subset of elements in which RXR augments TR binding.

Retinoid-X receptor (RXR) differentially augments thyroid hormone response in cell lines as a function of the response element and endogenous RXR content

Retinoid-X receptor (RXR) forms heterodimers with thyroid hormone receptor (TR) and significantly enhances binding to thyroid hormone response elements (TREs). Expression of RXR in a transient transfection assay augments the T3 response, but the influences of the specific cell line and TRE used have not been systematically studied. We determined RXR alpha and -beta augmentation of the TR alpha-mediated T3 response in transient transfection assays of COS, JEG, and mouse embryonic stem (ES) cell lines for a series of eight wild-type thyroid hormone (T3) and retinoic acid response elements (previously shown to bind TR). RXR augmented T3-induced expression in COS and ES cells (1.5- to 4-fold greater expression with added RXR compared to TR alone), but had minimal effect on augmentation of response in JEG cells. For most elements studied there was a proportional augmentation of basal and T3-stimulated expression. TREs from rat GH and laminin-B1, however, had relatively higher levels of T3-induced expression as a result of RXR cotransfection (T3 induction ratios increased 2-fold or greater). Previous characterization of these elements demonstrates that they contain more than two hexameric binding domains, all of which can simultaneously bind TR. The influence of endogenous RXR expression in a cell line on RXR augmentation of the T3 response was determined. RXR alpha and -beta messenger RNA (mRNA) expression was quantitated by Northern blot in each cell line. COS and JEG cells expressed almost exclusively RXR alpha mRNA, although expression was almost 2-fold higher in JEG compared to COS cells (12 +/- 2.5 vs. 6.8 +/- 0.5 density units relative to actin; mean +/- SE; P < 0.05). ES cells expressed only RXR beta mRNA, but at a very low level (0.4 +/- 0.1). Nuclear extracts prepared from JEG and COS cells augmented TR binding proportional to the endogenous RXR mRNA expression, and the heterodimer band was supershifted by the addition of antibody to RXR alpha. Nuclear extracts from ES cells had no detectable TR heterodimer binding to a range of response elements. RXR augmentation of the T3 response differs among cell lines and is greater in those with reduced endogenous RXR. Furthermore, the functional augmentation of the T3 response ratio by RXR is likely to require additional sequences contained in only a subset of elements in which RXR augments TR binding.

Transcriptional Activity of a Fluorinated Vitamin D Analog on VDR-RXR-Mediated Gene Expression

The transcriptional activity of the hexafluorinated derivative of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 [F6-1,25-(OH)2D3], was examined in cultured cells by a transient expression assay (CAT assay) using expression vectors for the rat nuclear vitamin D3 receptor (VDR) and the rat 9-cis-retinoic acid receptor (RXR beta), and a reporter plasmid containing a consensus vitamin D3 response element (VDRE) consisting of two directly repeated AGGTCA motifs spaced by 3 bp (DR3). At physiological concentrations, the transcriptional activity of F6-1,25-(OH)2D3 was 2-4 times more potent than that of 1,25-(OH)2D3 in both nontarget (HeLa) and target (UMR106) cells for 1,25-(OH)2D3. The transcriptional activity of F6-1,25-(OH)2D3 was also higher when the endogenous target gene (osteopontin), which has a VDRE related to the DR3 in its promoter, was induced. A gel-shift assay using DR3 as a probe and in vitro synthesized receptors showed that the ligand-induced DNA binding of VDR required RXR to form a heterodimer. Moreover, in this assay we found that F6-1,25-(OH)2D3 induced the receptor-DNA complex at a 10-fold lower concentration than 1,25-(OH)2D3 without influencing the dissociation kinetics. However, the binding affinity of F6-1,25-(OH)2D3 for VDR was slightly lower than that of 1,25-(OH)2D3. The increased DNA binding of ligand-bound VDR by introducing hexafluorines into 1,25-(OH)2D3 may potentiate the transcriptional activity. Thus, the higher biological activity of F6-1,25-(OH)2D3 may be exerted at least in part by enhanced transcriptional activity.

Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo.

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo.

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

Phosphorylation enhances the target gene sequence-dependent dimerization of thyroid hormone receptor with retinoid X receptor.

To understand the molecular basis of the phosphorylation-enhanced transcriptional activity of human thyroid hormone nuclear receptor subtype beta 1 (hTR beta 1), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta), we studied the effect of phosphorylation on the interaction of hTR beta 1 with the retinoid X receptor beta (RXR beta). In vitro, the extent of hTR beta 1.RXR beta heterodimer bound to various thyroid hormone response elements (TREs) was compared before and after phosphorylation of hTR beta 1. Without phosphorylation, hTR beta 1.RXR beta heterodimer was barely detectable under the experimental conditions. After phosphorylation of hTR beta 1, heterodimer bound to (i) the chicken lysozyme gene TRE, (ii) a TRE consisting of direct repeats of half-site binding motifs separated by four gaps, and (iii) a palindromic TRE was enhanced by approximately 10-, 7-, and 6-fold, respectively. The effect of phosphorylation on hTR beta 1.RXR beta heterodimerization was reversible. Dephosphorylation of the phosphorylated hTR beta 1 by alkaline phosphatase led to loss of the ability of hTR beta 1 to form a heterodimer with RXR beta in either the absence or the presence of DNA. These results indicate that the heterodimerization is enhanced by phosphorylation. To evaluate the effect of phosphorylation on the interaction of hTR beta 1 with RXR beta in vivo, we cotransfected hTR beta 1, RXR beta and TRE-chloramphenicol acetyltransferase (CAT) expression plasmids into CV-1 cells. CAT activity was assessed in the presence or absence of okadaic acid. Okadaic acid is a potent inhibitor of phosphatases 1 and 2A and increases the in vivo phosphorylation of hTR beta 1 by approximately 10-fold. Using the CAT reporter gene under control of the TRE from the malic enzyme gene, we found that RXR beta increased the okadaic acid-enhanced hTR beta 1-mediated CAT activity by 2- to 3-fold in the presence of 3,3',5-triiodo-L-thyronine. However, 9-cis-retinoic acid did not enhance the effect of okadaic acid. Our results indicate that phosphorylation is essential for the interaction of hTR beta 1 with RXR beta. Thus, phosphorylation plays a pivotal role in the gene-regulating activity of hTR beta 1.

The mouse Rxrb gene encoding RXRβ: genomic organization and two mRNA isoforms generated by alternative splicing of transcripts initiated from CpG island promoters

Two major isoforms of retinoid X receptor β (RXRβ; H-2RIIBP), encoded by the Rxrb gene, have been identified in the mouse. Northern analysis of Rxrb mRNA showed two close bands of 2.8 and 2.6 kb in many tissues and cell lines. They are designated as m Rxrβ1 and m Rxrβ2, respectively. Some rapidly growing cell lines and spleen tissue had about twofold more Rxrβ1 mRNA than Rxrβ2 whereas most adult tissues had similar amounts of both β 1 and β 2. Amino acid (aa) sequences deduced from cDNAs show an extra N-terminal domain of 72 aa for RXRβ1 that is well conserved between mouse and human, but not found in RXRβ2. These isoforms are generated from separate exons transcribed from different CpG island promoters and spliced into the common acceptor site in the transactivation domain by an alternative splicing. The Rxrb gene contains an intron in the midst of the first zinc-finger coding region. This is different from the retinoic acid receptor (RAR) and other nuclear receptor superfamily genes that contain an intron between the first and the second zinc-finger coding regions. These results, together with their unique ability to form heterodimers with other members of the superfamily, suggest a distinct phylogenic position for the Rxr genes.

Specificity of retinoid receptor gene expression in mouse cervical epithelia.

Retinoids are powerful regulators of epithelial differentiation and are essential for its maintenance. Because retinoids are necessary for cervical epithelial differentiation, they have been used as chemopreventive agents of cervical dysplasia and neoplasia. We were interested in determining whether different cervical epithelial phenotypes express specific retinoid receptors. The cervical epithelium contains the two phenotypes, stratified squamous and simple columnar, which join at the squamocolumnar junction. In addition, the simple columnar epithelium undergoes squamous metaplasia in response to vitamin A deficiency. Therefore, the cervical epithelium is suitable to study the expression pattern of the retinoid receptors in the three phenotypes, simple columnar, stratified squamous, and squamous metaplastic, simultaneously. The distribution pattern of the major retinoic acid receptor (RAR) isoforms (alpha 1, alpha 2, beta 2, beta 3, gamma 1, and gamma 2) and retinoid-X receptors (RXR alpha, -beta, and -gamma) was studied by in situ hybridization. At the tissue level, RAR alpha (1 and 2) and RXR (alpha and beta) transcripts and, to a lesser extent, RAR gamma (1 and 2) transcripts were associated with the cervical stratified squamous subjunctional epithelium. The simple columnar epithelium, which is highly responsive to vitamin A status, expressed high levels of RAR alpha (1 and 2), RAR beta (2 and 3), and RXR (alpha and beta) transcripts. Only RAR beta (2 and 3) and RXR (alpha and beta) transcripts were down-modulated by the condition of vitamin A deficiency and expressed less in squamous metaplastic foci than the simple columnar epithelium. RXR gamma was undetectable in all three cervical epithelia. At the cellular level, basal and suprabasal expression was found for RARs, and preferential localization of RXRs was seen in basal cells. RXRs are auxiliary proteins for a variety of other nuclear receptors with which they form heterodimers, including RARs. The fact that RXRs are mainly localized in basal and columnar cells of the cervix suggests the need for the regulation and diversity generated by potential heterodimeric interactions in these rapidly proliferating cells in vivo. The unique pattern of expression and localization of the RARs and RXRs in different cervical epithelial tissues and cell types supports the hypothesis that they perform specific functions in cervical epithelial differentiation. This is in contrast to the major isoforms of each RAR, which have similar patterns of expression in the different cervical epithelial phenotypes and cell types, suggesting a redundancy in function.