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.     

Retinoic acid-induced developmental defects are mediated by RARbeta/RXR heterodimers in the pharyngeal endoderm

Fusion and hypoplasia of the first two branchial arches, a defect typically observed in retinoic acid (RA) embryopathy, is generated in cultured mouse embryos upon treatment with BMS453, a synthetic compound that exhibits retinoic acid receptor beta (RARbeta) agonistic properties in transfected cells. By contrast, no branchial arch defects are observed following treatment with synthetic retinoids that exhibit RARalpha or RARgamma agonistic properties. The BMS453-induced branchial arch defects are mediated through RAR activation, as they are similar to those generated by a selective pan-RAR agonist, are prevented by a selective pan-RAR antagonist and cannot be mimicked by exposure to a pan-RXR agonist alone. They are enhanced in the presence of a pan-RXR agonist, and cannot be generated in Rarb-null embryos. Furthermore, they are accompanied, in the morphologically altered region, by ectopic expression of Rarb and of several other direct RA target genes. Therefore, craniofacial abnormalities characteristic of the RA embryopathy are mediated through ectopic activation of RARbeta/RXR heterodimers, in which the ligand-dependent activity of RXR is subordinated to that of RARbeta. Endodermal cells lining the first two branchial arches respond to treatment with the RARbeta agonist, in contrast to neural crest cells and ectoderm, which suggests that a faulty endodermal regionalization is directly responsible for RA-induced branchial arch dysmorphologies. Additionally, we provide the first in vivo evidence that the synthetic RARbeta agonist BMS453 exhibits an antagonistic activity on the two other RAR isotypes.     

A new class of RAR subtype selective retinoids: correlation of pharmacological effects with receptor activity

The synthesis and biological activity of a series of structurally related retinoids with different RAR subtype selectivities are described. These retinoids bind to all three RAR subtypes but in functional transactivation assays, they show RARbeta or RARbeta,gamma selectivity with weak RARalpha activity. The subtype selectivity of these retinoids was found to correlate with their efficacy (ODC inhibition) and toxicity (topical irritation and teratogenicity) profiles. The degree of RARgamma transactivation activity correlates with their topical toxicity and teratogenicity as measured by the inhibition of chondrogenesis. Of the RARbeta selective retinoids reported here, retinoid 12 is the most promising, as it is completely devoid of two common retinoid related toxicities, namely topical irritation and teratogenesis.     

Retinoid X receptors and retinoid response in neuroblastoma cells

Retinoic acid (RA) modulates differentiation and apoptosis of neural cells via RA receptors (RARs) and retinoid X receptors (RXRs). Neuroblastoma cells are potentially useful models for elucidating the molecular mechanisms of RA in neural cells, and responses to different isomers of RA have been interpreted in terms of differential homo- and heterodimerization of RXRs. The aim of this study was to identify the RXR types expressed in neuroblast and substrate-adherent neuroblastoma cells, and to study the participation of these RXRs in RAR heterodimers. RXRbeta was the predominant RXR type in N-type SH SY 5Y cells and S-type SH EP cells. Gel shift and supershift assays demonstrated that RARbeta and RARgamma predominantly heterodimerize with RXRbeta. In SH SY 5Y cells, RARgamma/RXRbeta was the predominant heterodimer binding to the DR5 RARE in the absence of 9-cis RA (9C), whereas the balance shifted in favor of RARbeta/RXRbeta in the presence of ligand. There was a marked difference between the N- and S-type neuroblastoma cells in retinoid receptor-DNA interactions, and this may underlie the differential effects of retinoids in these neuroblastoma cell types. There was no evidence to indicate that 9C functions via RXR homodimers in either SH SY 5Y or SH EP neuroblastoma cells. The results of this study suggest that interactions between retinoid receptors and other nuclear proteins may be critical determinants of retinoid responses in neural cells.     

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.     

Rational design of RAR-selective ligands revealed by RARbeta crystal stucture

The crystal structure of the ligand-binding domain of RARbeta, a suspect tumour suppressor, reveals important features that distinguish it from the two other RAR isotypes. The most striking difference is an extra cavity allowing RARbeta to bind more bulky agonists. Accordingly, we identified a ligand that shows RARbeta selectivity with a 100-fold higher affinity to RARbeta than to alpha or gamma isotypes. The structural differences between the three RAR ligand-binding pockets revealed a rationale explaining how a single retinoid can be at the same time an RARalpha, gamma antagonist and an RARbeta agonist. In addition, we demonstrate how to generate an RARbeta antagonist by gradually modifying the bulkiness of a single substitution. Together, our results provide structural guidelines for the synthesis of RARbeta-selective agonists and antagonists, allowing for the first time to address pharmacologically the tumour suppressor role of RARbeta in vitro and in animal models.