RXRalpha Research Articles

Cholecalciferol (Vitamin D3) Inhibits Growth and Invasion by Up-regulating Nuclear Receptors and 25-Hydroxylase (CYP27A1) in Human Prostate Cancer Cells

Epidemiological evidence suggests an inverse relationship between prostate cancer and serum vitamin D levels. We examined the ability of cholecalciferol (vitamin D(3)), a calcitriol precursor, to inhibit or reverse cellular changes associated with malignant transformation and invasion and explored its mechanisms of action. The RWPE2-W99 human prostate epithelial cell line, which forms slow-growing tumors in nude mice, was used because it mimics the behavior of the majority of primary human prostate cancers. Cholecalciferol, at physiological levels: (i) inhibited anchorage-dependent and -independent growth; (ii) induced differentiation by decreasing vimentin expression with a concomitant decrease in motility/chemotaxis; (iii) decreased MMP-9 and MMP-2 activity with concomitant decrease in invasion; and (iv) exerted its effects by up-regulating vitamin D receptor (VDR), retinoid-X receptor-alpha (RXR-alpha), and androgen receptor (AR) in a dose-dependent manner. Furthermore, we found that RWPE2-W99 prostate cancer cells, similar to RWPE-1 cells (Tokar and Webber. Clin Exp Metast 2005; 22: 265-73), constitutively express the enzyme 25-hydroxylase CYP27A1 which is markedly up-regulated by cholecalciferol. Cholecalciferol has effects similar to those of calcitriol on growth, MMP activity, and VDR. The ability of CYP27A1 to catalyze the conversion of cholecalciferol to 25(OH)D(3) and of 25(OH)D(3) to calcitriol has been reported. RWPE2-W99 cells, similar to RWPE-1 cells, appear to have the rare ability to locally convert cholecalciferol to the active hormone calcitriol. Because it can inhibit cellular changes associated with malignant transformation and invasion, we propose that cholecalciferol may be an effective agent for the treatment of prostate cancer.

Changes in expression levels of genes involved in fatty acid metabolism: upregulation of all three members of the PPAR family (?, ?, ?) and the newly described adiponectin receptor 2, but not adiponectin receptor 1 during neonatal cardiac development of the rat

During neonatal cardiac development, the heart changes its substrate preference from glucose to fatty acids. The aim of this study was to investigate the changes in mRNA expression levels of genes involved in the control of cardiac fatty acid metabolism in the transition from neonatal to adult life. mRNA expression levels for peroxisome proliferator activated receptor (PPAR) alpha, gamma and delta, PPARgamma co-factor 1 alpha and beta (PGC-1 alpha and beta), 9-cis retinoc-acid-activated receptor alpha, beta and gamma (RXR alpha, beta, gamma), 5'-AMP activated protein kinase (AMPK) alpha1 and alpha2, adiponectin receptor 1 and 2 (AR 1 and AR 2) were measured in heart tissue of neonatal 0-day, 7-day and 21- day old rats. mRNA expression of all three members of the PPAR family were upregulated significantly from day 0 to day 21 (alpha +117%, gamma +133%, delta +203%). In addition, m-RNA expression of all RXR isoforms increased from day 0 to day 7 (alpha +125%, beta +69%; gamma +41%). AR 2 exhibited a small but significant increase in mRNA expression (+ 46%). We were able to demonstrate for the first time that in addition to PPARalpha, also PPARgamma and delta, as well as all RXR isoforms and AR 2 are upregulated in the heart during neonatal development.

Retinoic acid and arsenic trioxide cooperate for apoptosis through phosphorylated RXR alpha

Arsenite trioxide (As2O3) induces apoptosis in several cell lines by disturbing key signal transduction pathways through its oxidative properties. Here, we report that As2O3 also induces the phosphorylation of the retinoid receptor RXRalpha, subsequent to oxidative damages and the activation of the stress-activated protein kinases cascade (JNKs). We also report that RA amplifies both As2O3-induced phosphorylation of RXRalpha and apoptosis. Taking advantage of 'rescue' F9 cell lines expressing RXRalpha mutated at its phosphorylation sites, in an RXRalpha null background, we provide evidence that RXRalpha is a key element involved in that potentiating effect. Finally, we demonstrate that As2O3 also abrogates the transactivation of RA-target genes.

AhR and PPARa: antagonistic effects on CYP2B and CYP3A, and additive inhibitory effects on CYP2C11

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. The peroxisome proliferator-activated receptor alpha (PPARα) is a member of the nuclear receptor super-family of ligand-activated transcription factors and it functions as an obligate heterodimer with retinoid X-receptor alpha RXRα. The aim was to investigate whether the negative cross-talk recently proposed by the present authors between AhR and PPARα on CYP4A and CYP1A has any impact on other cytochrome P450 enzymes. Treatment of male Wistar rats with a PPARα ligand clofibric acid (CA) induced CYP2B1/2 and CYP3A proteins, activities, and the mRNA expression of CYP2B1, CYP2B2, CYP3A1 and CYP3A2, and suppressed CYP2C11 protein, activities and mRNA expression. AhR ligand Sudan III (S.III) treatment decreased basal and CA-induced CYP2B, CYP3A and CYP2C11 protein, activities and mRNA expression. To the best of the authors’ knowledge, this is the first study showing the presence of mutual effects of AhR and PPARα on CYP2B and CYP3A and an additive inhibitory effect on CYP2C11 in the livers of male rats.

Breast cancer progression in MCF10A series of cell lines is associated with alterations in retinoic acid and retinoid X receptors and with differential response to retinoids.

In most breast carcinomas and in breast cancer cell lines, retinoic acid receptor beta (RARbeta) is lost or down-regulated, whereas retinoic acid receptor alpha and gamma (RARalpha, gamma) and retinoid X receptors (RXRalpha, beta, gamma) are variably expressed. Little is known about alterations of the above receptors in hyperplastic and premalignant stages of breast cancer development. In this study, we employed the MCF10A series of breast epithelial cell lines (the parental and benign MCF10A, premalignant MCF10AT, and malignant MCF10CA1a) to assess whether in the course of their malignant transformation specific alterations in RARalpha, beta, gamma and RXRalpha, beta, gamma expression occur and whether they may affect the sensitivity of cells to retinoids. Malignant properties of the above cell lines were estimated by the nude mice xenograft transplantation assay. Among the above receptors most significant alterations occurred in RARbeta2, which was detected in the normal breast epithelial cells both, at mRNA and protein levels, but expressed in the MCF10A cell lines at mRNA level only. The transformation of benign MCF10A cells into premalignant MCF10AT and malignant MCF10CA1a was also associated with increase in RARalpha, RARgamma, RXRalpha, and RXRbeta proteins expression. All-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA), and 4-(hydroxyphenyl) retinamide (4-HPR) induced RARbeta2 protein expression exclusively in the benign MCF10A cells and the former two retinoids, mRNA expression in MCF10A and MCF10AT cells, but not in malignant, MCF10CA1a cells, suggesting that the loss of inducible RARbeta expression is associated with the progression and malignant transformation of MCF10A cells. Retinoids also variable decreased the RARalpha, RARgamma and RXRalpha protein expression preferentially in the premalignant and malignant, but not in benign MCF10A cells. Among the above retinoids, 4-HPR was most efficacious in inhibiting the growth of the three cell lines and this apparently was not dependent on the levels of the RARbeta2 transcriptional activation. Thus, our data support the hypothesis that breast epithelial cells in the course of their progression and malignant transformation may differentially respond to retinoids and that not only RARbeta, but RARalpha, gamma and/or RXRalpha, beta may also serve as potential targets for retinoids in breast cancer prevention and therapy trials.

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.

INTRONE RXR ALPHA POLYMORPHISM AND A BLOOD GROUP IN CHRONIC HEART FAILURE

We have found association of introne RXR Alpha polymorphism and A blood group in chronic heart failure

Reduced intranuclear mobility of APL fusion proteins accompanies their mislocalization and results in sequestration and decreased mobility of retinoid X receptor alpha.

Acute promyelocytic leukemia (APL) cells contain one of five chimeric retinoic acid alpha-receptor (RAR alpha) genes (X-RAR alpha) created by chromosomal translocations or deletion; each generates a fusion protein thought to transcriptionally repress RAR alpha target genes and block myeloid differentiation by an incompletely understood mechanism. To gain spatiotemporal insight into these oncogenic processes, we employed fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Fluorescence microscopy demonstrated that the intracellular localization of each of the X-RAR alpha proteins was distinct from that of RAR alpha and established which portion(s) of each X-RAR alpha protein-X, RAR, or both-contributed to its altered localization. Using FRAP, we demonstrated that the intranuclear mobility of each X-RAR alpha was reduced compared to that of RAR alpha. In addition, the mobility of each X-RAR alpha was reduced further by ligand addition, in contrast to RAR alpha, which showed no change in mobility when ligand was added. Both the reduced baseline mobility of X-RAR alpha and the ligand-induced slowing of X-RAR alpha could be attributed to the protein interaction domain contained within X. RXR alpha aberrantly colocalized within each X-RAR alpha; colocalization of RXR alpha with promyelocytic leukemia (PML)-RAR alpha resulted in reduced mobility of RXR alpha. Thus, X-RAR alpha may interfere with RAR alpha through its aberrant nuclear dynamics, resulting in spatial and temporal sequestration of RXR alpha and perhaps other nuclear receptor coregulators critical for myeloid differentiation.

Retinoids in cancer chemoprevention.

We review the therapeutic and preventive applications of a retinoid analog (vitamin A and its derivatives) for human cancers. Chemoprevention of cancer is an intervention in the carcinogenic process by chemical agents that block or reverse the malignant transformation of cells. Retinoids are prime candidates for cancer chemoprevention since cancer is characterized by abnormal growth with a lack of differentiation, which could be modified by retinoids. Retinoids exert their biological functions through nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR). A number of experimental and clinical studies have been performed in the past two decades with retinoids showing that they inhibit or reverse the carcinogenic process in some organs, including hematological malignancy as well as premalignant and malignant lesions in the oral cavity, head and neck, breast, skin and liver. We particularly focus upon the therapeutic application of all-trans RA (atRA) to acute promyelocytic leukemia (APL) and on the preventive approach to hepatocellular carcinoma (HCC) by a synthetic retinoid analog, acyclic retinoid. In both malignancies, malfunction of retinoid nuclear receptors is closely related to their carcinogenic process. In APL, a chromosomal translocation produces a chimeric protein between RAR alpha and a protein called promyelocyte leukemia protein (PML). PML-RAR alpha works as a dominant negative receptor in the leukemic cells, interfering with the normal function of RAR alpha and/or PML, which in turn results in the arrest of cell maturation at the stage of promyelocytes. Oral administration of atRA induces differentiation of promyelocytic leukemic cells to mature neutrophils, and leads to a high rates (over 90%) of complete remission. AtRA therapy has become standard in the treatment of APL. In the case of HCC, post-translational modification of RXR by phosphorylation impairs its function, which leads to uncontrolled cell growth. Acyclic retinoid suppresses the phosphorylation of RXR alpha, restores its function in the presence of its endogenous ligand, 9-cis RA, and thereby induces apoptosis of the cancer cells. Acyclic retinoid given orally successfully suppresses the development of second primary tumors in cirrhotic patients who undergo curative removal of preceding HCC. Eradication of (pre)malignant clones ('clonal deletion') from the liver is suggested as a mechanism of the chemopreventive effect. Further development of more effective retinoids as well as their use in combination with other classes of anticancer agents including immunopreventive drugs like interferons may provide strategies for cancer prevention.

Tissue-specific regulation of leptin expression and secretion by all-trans retinoic acid.

In addition to hormones and the sympathetic nervous system, specific nutrients may regulate leptin mRNA expression and adipose tissue metabolism. However, little is known regarding the effect of nutrients on leptin mRNA expression. Retinoic acid (RA) is a ligand of some nuclear receptors and previous reports have demonstrated contradictory effects on plasma leptin levels. Thus, we examined the effect of RA on expression of leptin in adipocytes of murine and human origin. After 48 h incubation of murine 3T3-L1 adipocytes with 1 and 10 microM all-trans RA, the expression of leptin mRNA was reduced by 56% and 65%, respectively, whereas the secretion of leptin was reduced by 38% and 77%, respectively. In human adipose tissue explants, 1 microM all-trans RA reduced leptin mRNA expression levels by 55% and leptin secretion by 25% after 24 h incubation. We observed an increased mRNA expression level of the transcription factors peroxisomal proliferator activated receptor gamma (PPAR gamma), retinoid X receptor alpha (RXR alpha), and RA receptor alpha (RAR alpha) in 3T3-L1 cells, whereas the mRNA level of these transcription factors was unchanged in human adipose tissue explants after incubation with RA. In two other leptin-expressing cell systems, the human placental trophoblast cell line BeWo and normal human primary osteoblasts, there was no effect of RA on leptin mRNA expression, but leptin secretion was reduced by 64% after 24 h incubation with 10 microM all-trans RA in BeWo cells. In conclusion, all-trans RA reduced both expression and secretion of leptin in human and rodent adipose tissue. In human BeWo cells or primary osteoblasts, leptin mRNA expression levels was not changed by all-trans RA, indicating a tissue-specific regulation of leptin mRNA expression by all-trans RA.

The RING Finger Protein, RNF8, Interacts with Retinoid X Receptor α and Enhances Its Transcription-stimulating Activity

Retinoid X receptor alpha (RXR alpha) is a member of the steroid hormone receptor superfamily. Using yeast two-hybrid screening, beta-galactosidase assays, and pull-down assays, we show that RNF8, a RING finger protein recently isolated as a protein binding to a ubiquitin-conjugating enzyme, binds to RXR alpha through the N-terminal regions of both proteins. In COS7 cells, overexpressed RNF8 colocalized and interacted with RXR alpha in the nucleus, as shown by fluorescence resonance energy transfer. A point mutation of RNF8, Cys-403 to Ser (C403S), which disrupts the RING finger structure, or deletion of the N-terminal region (Delta N) of RNF8 prevented localization of RNF8 to the nucleus without affecting nuclear localization of RXR alpha. Although transient overexpression of RNF8 had little effect on RXR alpha ubiquitination, RNF8 dose-dependently enhanced RXR alpha-mediated transactivation of the RXR-responsive element (RXRE)-bearing gene promoter without the addition of its ligand, 9-cis-retinoic acid (RA), and up-regulated the expression of the genes downstream of RXRE as well as an RA-response element. This transactivation-enhancing activity was not seen with either the C403S point mutant or the Delta N deletion mutant of RNF8. These results suggest a novel function of RNF8 as a regulator of RXR alpha-mediated transcriptional activity through interaction between their respective N-terminal regions.

Molecular determinants of human adipose tissue: differences between visceral and subcutaneous compartments in obese women.

The adipose tissue is playing an important role in the development of human obesity and its related comorbidities, but little is known about the mechanisms governing its differentiation and proliferation. In this work, we studied the expression of transcription factors involved in fat storage and metabolic regulations in adipose tissue of 50 well-characterized obese women. In multivariate analyses, 80% of c enhancer binding protein alpha (cEBP alpha), c and a sterol regulatory element binding protein 1 (c and a SREBP1), and retinoid X receptor (RXR alpha) levels in sc adipose tissue (SAT) could be explained by other transcription factors. In addition, RXR alpha was the major determinant of peroxisome proliferator and activated receptor-gamma 1 variability in SAT, with the two factors being involved in the determination of the variability of insulin resistance. In contrast, the levels of all these transcription factors, together with various phenotypic and biological characteristics of the patients, seemed to participate only marginally in the regulation of visceral adipose tissue activity. In similar multivariate analyses, they could explain only a minor part of the variability of cEBP alpha, c and a SREBP1, or RXR alpha, suggesting the involvement of other regulators. Overall, our results demonstrate a different regulation of visceral adipose tissue and SAT and a different role of both tissues in insulin resistance and lipid storage.

Retinal Dystrophy Resulting from Ablation of RXRα in the Mouse Retinal Pigment Epithelium

Vitamin A (retinol) actions in eye development are mediated by retinoic acid receptors (RARs and RXRs). Using the Cre/loxP system, we have selectively ablated RXR alpha in the retinal pigment epithelium (RPE), a cell monolayer critically involved in visual retinoid renewal and phagocytosis of photoreceptor outer segments. In the mutant (RXR alpha (rpe-/-)) mice, RPE cells are morphologically and functionally abnormal and display decreased expression of proteins involved in the visual retinoid cycle, namely RPE65, CRALBP, and RGR. RXR alpha (rpe-/-) mice also show alterations of photoreceptor cells including: 1) decrease in their number; 2) outer segment shortening and disorganization, and 3) reduced light responses in electroretinograms. These results indicate that RXR alpha is required for normal maturation of the RPE, which is known to play essential roles in photoreceptor cell function and survival, and point to a possible involvement of RXR alpha signaling pathways in the RPE in human retinal diseases.

Novel Roles of Retinoid X Receptor (RXR) and RXR Ligand in Dynamically Modulating the Activity of the Thyroid Hormone Receptor/RXR Heterodimer

Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand.

The AF-1 and AF-2 Domains of RARγ2 and RXRα Cooperate for Triggering the Transactivation and the Degradation of RARγ2/RXRα Heterodimers

In eukaryotic cells, liganded RAR gamma 2/RXR alpha heterodimers activate the transcription of retinoic acid (RA) target genes and then are degraded through the ubiquitin-proteasome pathway. In this study, we dissected the role of the RAR gamma 2 and RXR alpha partners as well as of their respective AF-1 and AF-2 domains in the processes of transactivation and degradation. RAR gamma 2 is the "engine" initiating transcription and its own degradation subsequent to ligand binding. Integrity of its AF-2 domain and phosphorylation of its AF-1 domain are required for both the degradation and the transactivation of the receptor. Deletion of the whole AF-1 domain does not impair these processes but shifts the receptor toward other proteolytic pathways through RXR alpha. In contrast, RXR alpha plays only a modulatory role, cooperating with RAR gamma 2 through its AF-2 domain and its phosphorylated AF-1 domain in both the transcription activity and the degradation of the RAR gamma 2/RXR alpha heterodimers. Our results underline that the AF-1 and AF-2 domains of each heterodimer partner cooperate with one other and that this cooperation is relevant for both the transcription and degradation processes.

Adenomatous Polyposis Coli (APC)-independent Regulation of β-Catenin Degradation via a Retinoid X Receptor-mediated Pathway

Beta-catenin is a component of stable cell adherent complexes whereas its free form functions as a transcription factor that regulate genes involved in oncogenesis and metastasis. Free beta-catenin is eliminated by two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways regulated by glycogen synthase kinase 3beta (GSK3 beta) or p53-inducible Siah-1. Dysregulation of beta-catenin turnover consequent to mutations in critical genes of the APC-dependent pathways is implicated in cancers such as colorectal cancer. We have identified a novel retinoid X receptor (RXR)-mediated APC-independent pathway in the regulation of beta-catenin. In this proteasomal pathway, RXR agonists induce degradation of beta-catenin and RXR alpha and repress beta-catenin-mediated transcription. In vivo, beta-catenin interacts with RXR alpha in the absence of ligand, but RXR agonists enhanced the interaction. RXR agonist action was not impaired by GSK3 beta inhibitors or deletion of the GSK3 beta-targeted sequence from beta-catenin. In APC- and p53-mutated colorectal cancer cells, RXR agonists still inactivated endogenous beta-catenin via RXR alpha. Interestingly, deletion of the RXR alpha A/B region abolished ligand-induced beta-catenin degradation but not RXR alpha-mediated transactivation. RXR alpha-mediated inactivation of oncogenic beta-catenin paralleled a reduction in cell proliferation. These results suggest a potential role for RXR and its agonists in the regulation of beta-catenin turnover and related biological events.

Retinoid X Receptor α Represses GATA-4-mediated Transcription via a Retinoid-dependent Interaction with the Cardiac-enriched Repressor FOG-2

Dietary vitamin A and its derivatives, retinoids, regulate cardiac growth and development. To delineate mechanisms involved in retinoid-mediated control of cardiac gene expression, the regulatory effects of the retinoid X receptor alpha (RXR alpha) on atrial naturietic factor (ANF) gene transcription was investigated. The transcriptional activity of an ANF promoter-reporter in rat neonatal ventricular myocytes was repressed by RXR alpha in the presence of 9-cis-RA and by the constitutively active mutant RXR alpha F318A indicating that liganded RXR confers the regulatory effect. The RXR alpha-mediated repression mapped to the proximal 147 bp of the rat ANF promoter, a region lacking a consensus retinoid response element but containing several known cardiogenic cis elements including a well characterized GATA response element. Glutathione S-transferase "pull-down" assays revealed that RXR alpha interacts directly with GATA-4, in a ligand-independent manner, via the DNA binding domain of RXR alpha and the second zinc finger of GATA-4. Liganded RXR alpha repressed the activity of a heterologous promoter-reporter construct containing GATA-response element recognition sites in cardiac myocytes but not in several other cell types, suggesting that additional cardiac-enriched factors participate in the repression complex. Co-transfection of liganded RXR alpha and the known cardiac-enriched GATA-4 repressor, FOG-2, resulted in additive repression of GATA-4 activity in ventricular myocytes. In addition, RXR alpha was found to bind FOG-2, in a 9-cis-RA-dependent manner. These data reveal a novel mechanism by which retinoids regulate cardiogenic gene expression through direct interaction with GATA-4 and its co-repressor, FOG-2.

Cyclin D3 Is a Cofactor of Retinoic Acid Receptors, Modulating Their Activity in the Presence of Cellular Retinoic Acid-binding Protein II

Ligand-induced transcription activation of retinoic acid (RA) target genes by nuclear receptors (retinoic acid (RAR) and retinoid X (RXR) receptors) depends on the recruitment of coactivators. We have previously demonstrated that the small 15-kDa cellular RA-binding protein II (CRABPII) is a coactivator present in the RA-dependent nuclear complex. As identifying cell-specific partners of CRABPII might help to understand the novel control of RA signaling, we performed a yeast two-hybrid screen of a hematopoietic HL-60 cDNA library using human CRABPII as bait and have subsequently identified human cyclin D3 as a partner of CRABPII. Cyclin D3 interacted with CRABPII in a ligand-independent manner and equally bound RAR alpha, but not RXR alpha, and only in the presence of RA. We further show that cyclin D3 positively modulated RA-mediated transcription through CRABPII. Therefore, cyclin D3 may be part of a ternary complex with CRABPII and RAR. Finally, we show that cyclin D3 expression paralleled HL-60 differentiation and arrest of cell growth. These findings led us to speculate that control of cell proliferation during induction of differentiation may directly involve, at the transcriptional level, nuclear receptors, coactivators, and proteins of the cell cycle in a cell- and nuclear receptor-specific manner.

Immunohistochemical detection of the retinoid X receptors alpha, beta, and gamma in human prostate.

The aim of this study was to evaluate the presence and distribution of retinoid X receptors (RXRs) alpha, beta, and gamma in normal, hyperplastic (nodular, basal cell, and atrophic hyperplasia), and carcinomatous human prostates in order to elucidate the relationship among these receptors and the onset and development of prostatic adenocarcinoma. RXRalpha and RXRgamma were immunodetected in all samples of normal, nodular, and basal cell hyperplasia, as well as carcinomatous prostates. In atrophic glands, the expression of both receptors was found in 22.5% of samples. Positive immunostaining for RXRbeta was observed in 53.3% of normal prostates, 100% of samples showed basal cell hyperplasia, and were negative in nodular and atrophic hyperplasia. In prostatic adenocarcinoma, only 3 of 25 samples (the 3 diagnosed as well-differentiated) were positive for RXRbeta. Results suggest that diminished RXRbeta expression might be related to prostate cancer progression and because the responsiveness to retinoic acid treatments depends on the expression of different receptors, it is important to study their expression before therapy.

1098 The VIP receptor VPAC-1 in highly expressed and regulated by FXR and RXR alpha nuclear receptors in the human gallbladder epithelium

1098 The VIP receptor VPAC-1 in highly expressed and regulated by FXR and RXR alpha nuclear receptors in the human gallbladder epithelium