Rexinoid-induced expression of IGFBP-6 requires RARbeta-dependent permissive cooperation of retinoid receptors and AP-1

The synthetic rexinoid bexarotene (Targretin, LGD1069) inhibits the formation of both estrogen receptor-negative and estrogen receptor-positive breast cancer in preclinical models and controls the expression of growth-regulatory biomarkers, such as IGFBP-6 (insulin-like growth factor-binding protein 6), RARbeta, or cyclin D1. In this study, we identified a classical retinoic acid-responsive element in the first intron in the IGFBP-6 gene adjacent to a consensus AP-1 binding site, both elements essential for rexinoid-induced expression of IGFBP-6. In chromatin binding experiments, bexarotene increased the occupancy of the identified enhancer element by RXRalpha, RARbeta, cJun, cFos, and p300. In normal mammary epithelial cells and T47D breast cancer cells, small interfering RNA-mediated knockdown of all RXR isoforms or RARbeta, but not RARalpha or RARgamma alone, blocked the induction of IGFBP-6 by bexarotene. Simultaneous knockdown of RARalpha and RARgamma abrogated both the induction of RARbeta and the up-regulation and secretion of IGFBP-6. The suppression of either RARbeta or cJun by small interfering RNA blocked the recruitment of RXRalpha and cJun to the enhancer. These results demonstrate a novel cooperative interaction between retinoid receptors and AP-1 orchestrated by RARbeta and highlight a novel mechanism by which RARbeta can mediate the cancer-preventive effects of rexinoids.     

Dimerization with retinoid X receptors and phosphorylation modulate the retinoic acid-induced degradation of retinoic acid receptors alpha and gamma through the ubiquitin-proteasome pathway

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for targeted degradation of proteins. We show that, in F9 cells and in transfected COS-1 cells, the nuclear retinoid receptors, retinoic acid receptor gamma2 (RARgamma2), RARalpha1, and retinoid X receptor alpha1 (RXRalpha1) are degraded in a retinoic acid-dependent manner through the ubiquitin-proteasome pathway. The degradation of RARgamma2 is entirely dependent on its phosphorylation and on its heterodimerization with liganded RXRalpha1. In contrast, RARalpha1 degradation can occur in the absence of heterodimerization, whereas it is inhibited by phosphorylation, and heterodimerization reverses that inhibition. RXRalpha1 degradation is also modulated by heterodimerization. Thus, each partner of RARgamma/RXRalpha and RARalpha/RXRalpha heterodimers modulates the degradation of the other. We conclude that the ligand-dependent degradation of RARs and RXRs by the ubiquitin-proteasome pathway, which is regulated by heterodimerization and by phosphorylation, could be important for the regulation of the magnitude and duration of the effects of retinoid signals.     

Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes

Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.     

Expression of nuclear hormone receptors, their coregulators and type I iodothyronine 5'-deiodinase gene in mammary tissue of nonlactating and postlactating rats

The aim of the study was to test the hypothesis that expression of retinoid receptors (RARalpha, RARbeta, RARgamma), rexinoid receptors (RXRalpha, RXRbeta), thyroid hormone receptors (TRalpha, TRbeta), estrogen receptors (ERalpha, ERbeta), nuclear receptor coregulators (N-CoR, SRC-1, SMRT), and in addition type I iodothyronine 5'-deiodinase (5'-DI), EGFR and erb-B2/neu would be different in mammary postlactating tissue in comparison with that of nonlactating mammary gland. Using RT-PCR, we have shown that expression of RARalpha, RXRalpha,TRalpha, ERalpha,ERbeta,N-CoR, SRC-1, SMRT and EGFR in rat was significantly increased in postlactating mammary gland when compared to that of nonlactating mammary tissue. Postlactating mammary glands were found to express all RAR and RXR subtypes studied when compared to nonlactating mammary tissues that express exclusively RARalpha and RXRalpha subtypes. Enhanced expression of a number of nuclear hormone receptors, their coregulators in mammary tissue of postlactating rats in comparison with nonlactating animals identify a potential role for retinoid, thyroid and estrogen signalling pathways also after lactation period.     

Requirement of retinoic acid receptor isotypes alpha, beta, and gamma during the initial steps of neural differentiation of PCC7 cells

Retinoic acid (RA) is indispensable for morphogenesis and differentiation of several tissues, including the nervous system. The requirement of the RA receptor (RAR) isotypes alpha, beta, and gamma and the putative role of retinoid X receptor-(RXR) signaling in RA-induced neural differentiation, was analyzed. For this compound-selective retinoids and the murine embryonal carcinoma cell line PCC7, a model system for RA-dependent neural differentiation was used. The present paper shows that proliferating PCC7 cells primarily express RXRalpha and RARalpha, lower levels of RXRbeta, and barely detectable amounts of RARbeta, RARgamma, and RXRgamma. At receptor-selective concentrations, only a RARalpha or RARgamma agonist induced the typical tissue-like differentiation pattern consisting of neuronal and nonneuronal cells. Differentiation-associated processes, such as the down-regulation of Oct4, up-regulation of certain nuclear receptors and proneuronal genes, and the induction of neuronal markers could be triggered by receptor-selective concentrations of a RARalpha-, beta-, or gamma-selective agonist, although with distinct efficacy. The differences are only partially explained by the distinct RARalpha, beta, and gamma expression levels and the dissociation constants for the bound retinoids, suggesting differential requirement of RAR isotypes during the initial stages of neural differentiation of PCC7 cells.     

Expression of retinoid receptors during the retinoic acid-induced neuronal differentiation of human embryonal carcinoma cells

Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRalpha is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRalpha and RARs during the RA-induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRalpha, RARalpha, RARbeta, and RARgamma were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up-regulation of RXRalpha expression in NT2 cells. The induced RXRalpha was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRalpha expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase- and vasoactive intestinal peptide-positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRalpha expression in NT2 cells and suggest that RXRalpha might play a crucial role in the cellular functions of human CNS neurons.     

F9 embryocarcinoma cells: a cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling

Mouse F9 embryocarcinoma (EC) cells constitute a well established cell-autonomous model system for investigating retinoid signaling in vitro as, depending on culture conditions, retinoic acid (RA) can induce their differentiation into either primitive, parietal or visceral extraembryonic endoderm-like cells. These RA-induced differentiations are accompanied by decreases in proliferation rates, modifications of expression of subsets of RA-target genes, and induction of apoptosis. To elucidate the roles played by the multiple retinoid receptors (RARs and RXRs) in response to RA treatments, F9 EC cells lacking one or several RARs or RXRs were engineered through homologous recombination. Mutated RARs and/or RXRs were then reexpressed in given RAR or RXR null backgrounds. WT and mutant cells were also treated with different combinations of ligands selective for RXRs and/or for each of the three RAR isotypes. These studies lead to the conclusion that most RA-induced events (e.g. primitive and visceral differentiation, growth arrest, apoptosis and activation of expression of a number of genes) are transduced by RARgamma/RXRalpha heterodimers, whereas some other events (e.g. parietal differentiation) are mediated by RARalpha/RXRalpha. heterodimers. They also demonstrate that both AF-1 and AF-2 activation functions of RARs and RXRs, as well as their phosphorylation, are differentially required in these RA-induced events. In RARgamma/RXRalpha heterodimers, the phosphorylation of RARgamma is necessary for triggering primitive differentiation, while that of RXRalpha is required for growth arrest. On the other hand, phosphorylation of RARalpha is necessary for parietal differentiation. Thus, retinoid receptors are sophisticated signal integrators that transduce not only the effects of their cognate ligands, but also those of ligands that bind to membrane receptors.     

Retinoic acid biosynthetic activity and retinoid receptors in the olfactory mucosa of adult mice

Retinoids play important roles in the ontogenic development of the olfactory system in mammals, but their function in adult olfactory mucosa has not been explored. In the present study, the olfactory mucosal expression of nuclear retinoid receptors was examined in adult mice. Several retinoic acid receptor isotypes were identified in olfactory mucosa from adult C57BL/6 mice by RNA-PCR and DNA sequence analysis, including RARbeta, RXRalpha, RXRbeta, and RXRgamma. In addition, a previously unidentified mouse RXRbeta isoform containing a 12-nucleotide insertion in exon 7 was detected. Furthermore, in vitro metabolic studies demonstrated that olfactory mucosal cytosolic and microsomal preparations are active in the biosynthesis of retinoic acids from all-trans- and 9-cis-retinal. These results indicate that components of the retinoid signal transduction system are expressed in adult olfactory mucosa and may play important roles in gene regulation in this unique tissue where olfactory neuronal cells are continuously replaced.