Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.     

Altered expression of retinoic acid (RA) receptor mRNAs in the fetal mouse secondary palate by all-trans and 13-cis RAs: implications for RA-induced teratogenesis

Retinoic acid (RA) is mandatory for various biological processes and normal embryonic development but is teratogenic at high concentrations. In rodents, one of the major malformations induced by RA is cleft palate (CP). RA mediates its effects by RA receptors (RARs), but the expression patterns of RARs in the developing palate are still unclear. We investigated the normal expression of RAR alpha, beta, and gamma messenger RNAs (mRNAs) in the fetal mouse secondary palate and the effects of all-trans and 13-cis RAs on the expression of RAR mRNAs by Northern blot analysis. RAR alpha (2.8, 3.8 kb), RAR beta (3.3 kb), and RAR gamma (3.7 kb) mRNAs were detected in the fetal palate on gestational days (GD) 12.5-14.5. The expression of RAR alpha and gamma mRNAs did not show apparent sequential changes, but that of RAR beta mRNA increased at GD 13.5. Treatment of pregnant mice with 100 mg/kg all-trans RA induced CP in 94% of the fetuses and elevated the levels of RAR beta and gamma mRNAs in the fetal palate. The up-regulation of RAR beta mRNA by all-trans RA was more marked than that of RAR gamma mRNA. Treatment with 100 mg/kg 13-cis RA induced CP in only 19% of the fetuses. Although 13-cis RA elevated the RAR beta and gamma mRNA levels in fetal palates, its up-regulation was slower and less marked than that induced by all-trans RA. These findings indicate that the induction of RAR beta mRNA in the fetal palate correlates well with the tissue concentration of all-trans RA after RA treatment, and RAR beta may be one of the most influential candidate molecules for RA-induced teratogenesis.     

Regulation of alpha 2(I) collagen expression in stellate cells by retinoic acid and retinoid X receptors through interactions with their cofactors

Retinoic acid (RA) suppresses alpha 2(I) collagen expression in hepatic stellate cells through the binding of retinoic acid receptor beta (RAR beta) and retinoid X receptor alpha (RXR alpha) to RA response elements (RAREs) in the alpha 2(I) collagen promoter. This study determined the influence of coactivators and corepressors to RAR beta and RXR alpha on the regulation of the alpha 2(I) collagen promoter. The coactivators, steroid receptor coactivator-1 (SRC-1) and growth hormone receptor interacting protein-1 (GRIP-1), enhanced, while the nuclear receptor corepressor (N-CoR) abolished the inhibitory effect of RAR beta and RXR alpha on the promoter activity. In the presence of RA, the coactivators SRC-1 and GRIP-1 formed complexes with RAR beta and RXR alpha which are bound to an oligonucleotide specifying a RARE site in the promoter. In conclusion, this study shows that in the presence of retinoic acid, the coactivators SRC-1 and GRIP-1 augment, while the corepressor N-CoR abolishes, the suppressive effects of RAR beta and RXR alpha on alpha 2(I) collagen promoter activity.     

Roles of retinoic acid receptors in early embryonic morphogenesis and hindbrain patterning

Mutants mice carrying targeted inactivations of both retinoic acid receptor (RAR) alpha and RAR gamma (A alpha/A gamma mutants) were analyzed at different embryonic stages, in order to establish the timing of appearance of defects that we previously observed during the fetal period. We show that embryonic day (E)9.5 A alpha/A gamma embryos display severe malformations, similar to those already described in retinaldehyde dehydrogenase 2 null mutants. These malformations reflect early roles of retinoic acid signaling in axial rotation, segmentation and closure of the hindbrain; formation of otocysts, pharyngeal arches and forelimb buds; and in the closure of the primitive gut. The hindbrain of E8.5 A alpha/A gamma embryos shows a posterior expansion of rhombomere 3 and 4 (R3 and R4) markers, but fails to express kreisler, a normal marker of R5 and R6. This abnormal hindbrain phenotype is strikingly different from that of embryos lacking RAR alpha and RAR beta (A alpha/A beta mutants), in which we have previously shown that the territory corresponding to R5 and R6 is markedly enlarged. Administration of a pan-RAR antagonist at E8.0 to wild-type embryos cultured in vitro results in an A alpha/A beta-like hindbrain phenotype, whereas an earlier treatment at E7.0 yields an A alpha/A gamma-like phenotype. Altogether, our data suggest that RAR alpha and/or RAR gamma transduce the RA signal that is required first to specify the prospective R5/R6 territory, whereas RAR beta is subsequently involved in setting up the caudal boundary of this territory.     

Murine RARbeta4 displays reduced transactivation activity, lower affinity for retinoic acid, and no anti-AP1 activity

The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta, and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Each of the RARs is expressed as four to seven different isoforms. Four isoforms of RAR beta (beta1, beta2, beta3, and beta4), which differ only in their N-terminal sequence (A domain) have been described. These RARbeta isoforms display a specific pattern of expression in developing and adult animals and are highly evolutionarily conserved suggesting that they mediate distinct cellular effects of vitamin A. Experiments were performed to examine directly the RA-binding activity, transactivation activity, and anti-AP1 activity of each of these four RARbeta isoforms. The results demonstrate that RARbeta1, beta2, and beta3 bind RA with a similar K(d) value, have a similar EC(50) value in RA-dependent transactivation assays and inhibit AP1 activity to a similar level. By contrast, RARbeta4 has an elevated K(d) for RA, an increased EC(50) value in RA-dependent transactivation assays and does not display the ability to inhibit AP1 activity. This provides additional evidence that at least one RAR isoform, RARbeta4, may mediate distinct activities within a cell. Furthermore, these data suggest that the presence of an A domain in RARbeta is important for modulating these activities of RARs.     

A role for retinoic acid in regulating the regeneration of deer antlers

Deer antlers are the only mammalian organs that can be repeatedly regenerated; each year, these complex structures are shed and then regrow to be used for display and fighting. To date, the molecular mechanisms controlling antler regeneration are not well understood. Vitamin A and its derivatives, retinoic acids, play important roles in embryonic skeletal development. Here, we provide several lines of evidence consistent with retinoids playing a functional role in controlling cellular differentiation during bone formation in the regenerating antler. Three receptors (alpha, beta, gamma) for both the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families show distinct patterns of expression in the growing antler tip, the site of endochondral ossification. RAR alpha and RXR beta are expressed in skin ("velvet") and the underlying perichondrium. In cartilage, which is vascularised, RXR beta is specifically expressed in chondrocytes, which express type II collagen, and RAR alpha in perivascular cells, which also express type I collagen, a marker of the osteoblast phenotype. High-performance liquid chromatography analysis shows significant amounts of Vitamin A (retinol) in antler tissues at all stages of differentiation. The metabolites all-trans-RA and 4-oxo-RA are found in skin, perichondrium, cartilage, bone, and periosteum. The RXR ligand, 9-cis-RA, is found in perichondrium, mineralised cartilage, and bone. To further define sites of RA synthesis in antler, we immunolocalised retinaldehyde dehydrogenase type 2 (RALDH-2), a major retinoic acid-generating enzyme. RALDH-2 is expressed in the skin and perichondrium and in perivascular cells in cartilage, although chondroprogenitors and chondrocytes express very low levels. At sites of bone formation, differentiated osteoblasts which express the bone-specific protein osteocalcin express high levels of RALDH2. The effect of RA on antler cell differentiation was studied in vitro; all-trans-RA inhibits expression of the chondrocyte phenotype, an effect that is blocked by addition of the RAR antagonist Ro41-5253. In monolayer cultures of mesenchymal progenitor cells, all-trans-RA increases the expression of alkaline phosphatase, a marker of the osteoblastic phenotype. In summary, this study has shown that antler tissues contain endogenous retinoids, including 9-cis RA, and the enzyme RALDH2 that generates RA. Sites of RA synthesis in antler correspond closely with the localisation of cells which express receptors for these ligands and which respond to the effects of RA.     

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells.

Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.     

Control of retinoic acid receptor heterodimerization by ligand-induced structural transitions. A novel mechanism of action for retinoid antagonists

Heterodimerization of retinoic acid receptors (RARs) with 9-cis-retinoic receptors (RXRs) is a prerequisite for binding of RXR.RAR dimers to DNA and for retinoic acid-induced gene regulation. Whether retinoids control RXR/RAR solution interaction remains a debated question, and we have used in vitro and in vivo protein interaction assays to investigate the role of ligand in modulating RXR/RAR interaction in the absence of DNA. Two-hybrid assay in mammalian cells demonstrated that only RAR agonists were able to increase significantly RAR interaction with RXR, whereas RAR antagonists inhibited RXR binding to RAR. Quantitative glutathione S-transferase pull-down assays established that there was a strict correlation between agonist binding affinity for the RAR monomer and the affinity of RXR for liganded RAR, but RAR antagonists were inactive in inducing RXR recruitment to RAR in vitro. Alteration of coactivator- or corepressor-binding interfaces of RXR or RAR did not alter ligand-enhanced dimerization. In contrast, preventing the formation of a stable holoreceptor structure upon agonist binding strongly altered RXR.RAR dimerization. Finally, we observed that RAR interaction with RXR silenced RXR ligand-dependent activation function. We propose that ligand-controlled dimerization of RAR with RXR is an important step in the RXR.RAR activation process. This interaction is dependent upon adequate remodeling of the AF-2 structure and amenable to pharmacological inhibition by structurally modified retinoids.     

Differential contributions of AF-1 and AF-2 activities to the developmental functions of RXR alpha

We have engineered a mouse mutation that specifically deletes most of the RXR alpha N-terminal A/B region, which includes the activation function AF-1 and several phosphorylation sites. The homozygous mutants (RXR alpha af1(o)), as well as compound mutants that further lack RXR beta and RXR gamma, are viable and display a subset of the abnormalities previously described in RXR alpha-null mutants. In contrast, RXR alpha af1(o)/RAR(-/-)(alpha, beta or gamma) compound mutants die in utero and exhibit a large array of malformations that nearly recapitulate the full spectrum of the defects that characterize the fetal vitamin A-deficiency (VAD) syndrome. Altogether, these observations indicate that the RXR alpha AF-1 region A/B is functionally important, although less so than the ligand-dependent activation function AF-2, for efficiently transducing the retinoid signal through RAR/RXR alpha heterodimers during embryonic development. Moreover, it has a unique role in retinoic acid-dependent involution of the interdigital mesenchyme. During early placentogenesis, both the AF-1 and AF-2 activities of RXR alpha, beta and gamma appear to be dispensable, suggesting that RXRs act as silent heterodimeric partners in this process. However, AF-2 of RXR alpha, but not AF-1, is required for differentiation of labyrinthine trophoblast cells, a late step in the formation of the placental barrier.