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All trans-retinoic acid (ATRA) is used in the treatment of acute promyelocytic leukemia (APL) and it is a promising agent also in solid tumors. The pharmacological activity of ATRA is mediated by the ligand-activated RAR and RXR transcription factors. In the present study, we define the basal and ATRA dependent RARα interactome in a RARα-overexpressing breast cancer cellular model, identifying 28 nuclear proteins. We focus our attention on the S100A3 calcium-binding protein, which interacts with RARα constitutively. In ATRA-sensitive breast cancer cells, S100A3 binds to RARα in basal conditions and binding is reduced by the retinoid. The interaction of S100A3 with RARα is direct and in lung cancer, APL and acute-myeloid-leukemia (AML) cells. In APL, S100A3 interacts not only with RARα, but also with PML-RARα. The interaction surface maps to the RARα ligand-binding domain, where the I396 residue plays a crucial role. Binding of S100A3 to RARα/PML-RARα controls the constitutive and ATRA-dependent degradation of these receptors. S100A3 knockdown decreases the amounts of RARα in breast- and lung cancer cells, inducing resistance to ATRA-dependent anti-proliferative/differentiating effects. Conversely, S100A3 knockdown in PML-RARα+ APL and PML-RARα− AML cells reduces the amounts of RARα/PML-RARα and increases basal and ATRA-induced differentiation. In this cellular context, opposite effects on RARα/PML-RARα levels and ATRA-induced differentiation are observed upon S100A3 overexpression. Our results provide new insights into the molecular mechanisms controlling RARα activity and have practical implications, as S100A3 represents a novel target for rational drug combinations aimed at potentiating the activity of ATRA.

The nuclear retinoic acid (RA) receptors (RARalpha, beta and gamma) are ligand-dependent regulators of transcription. Upon activation by RA, they are recruited at the promoters of target genes together with several coregulators. Then, they are degraded by the ubiquitin proteasome system. Here, we report that the degradation of the RARalpha subtype involves ubiquitination and the tripartite motif protein TRIM24, which was originally identified as a ligand-dependent corepressor of RARalpha. We show that in response to RA, TRIM24 serves as an adapter linking RARalpha to the proteasome for its degradation. In addition, TRIM24 and the proteasome are recruited with RARalpha to the promoters of target genes and thus are inherently linked to RARalpha transcriptional activity.

Retinoic acid receptors (RARs) are classically considered as nuclear ligand-dependent regulators of transcription. Here we highlighted a novel face of the RARα subtype: RARα is present in low amounts in the cytoplasm of mouse embryonic fibroblasts (MEFs) where it interacts with profilin2a (PFN2A), a small actin-binding protein involved in filaments polymerization. The interaction involves the N-terminal proline-rich motif (PRM) of RARα and the SH3-like domain of PFN2a. When increased in the cytoplasm, RARα competes with other PFN2a-binding proteins bearing PRMs and involved in actin filaments elongation. Consequently, the actin filament network is altered and MEFs adhesion is decreased. This novel role opens novel avenues for the understanding of pathologies characterized by increased levels of cytoplasmic RARα.

Retinoic acid (RA), a major natural active metabolite of vitamin A (VA) is well known to play critical roles in embryonic development. The effects of RA are mediated by nuclear receptors (RARs), which regulate the expression of gene batteries involved in cell growth and differentiation. Since the early 1990s several laboratories have focused on understanding how RA-regulated genes and RAR binding sites operate by studying the differentiation of embryonal carcinoma cells and embryonic stem cells. The development of hybridization-based microarray technology and high performance software analysis programs has allowed the characterization of thousands of RA-regulated genes. During the two last decades, publication of the genome sequence of various organisms has allowed advances in massive parallel sequencing and bioinformatics analysis of genome-wide data sets. These new generation sequencing (NGS) technologies have revolutionized the field by providing a global integrated picture of RA-regulated gene networks and the regulatory programs involved in cell fate decisions during embryonal carcinoma and embryonic stem cells differentiation. Now the challenge is to reconstruct the RA-regulated gene networks at the single cell level during the development of specialized embryonic tissues.

Treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) is the first example of targeted therapy. In fact, the oncogenic fusion-protein (PML-RAR) typical of this leukemia contains the retinoid-nuclear-receptor RARalpha. PML-RAR is responsible for the differentiation block of the leukemic blast. Besides PML-RAR, two endogenous RARalpha proteins are present in APL blasts, i.e. RARalpha1 and RARalpha2. We developed different cell populations characterized by PML-RAR, RARalpha2 and RARalpha1 knock-down in the APL-derived NB4 cell-line. Unexpectedly, silencing of PML-RAR and RARalpha2 results in similar increases in the constitutive expression of several granulocytic differentiation markers. This is accompanied by enhanced expression of the same granulocytic markers upon exposure of the NB4 blasts to ATRA. Silencing of PML-RAR and RARalpha2 causes also similar perturbations in the whole genome gene-expression profiles of vehicle and ATRA treated NB4 cells. Unlike PML-RAR and RARalpha2, RARalpha1 knock-down blocks ATRA-dependent induction of several granulocytic differentiation markers. Many of the effects on myeloid differentiation are confirmed by over-expression of RARalpha2 in NB4 cells. RARalpha2 action on myeloid differentiation does not require the presence of PML-RAR, as it is recapitulated also upon knock-down in PML-RAR-negative HL-60 cells. Thus, relative to RARalpha1, PML-RAR and RARalpha2 exert opposite effects on APL-cell differentiation. These contrasting actions may be related to the fact that both PML-RAR and RARalpha2 interact with and inhibit the transcriptional activity of RARalpha1. The interaction surface is located in the carboxy-terminal domain containing the D/E/F regions and it is influenced by phosphorylation of Ser-369 of RARalpha1.

To determine whether hepatic depletion of vitamin A (VA) stores has an effect on the postnatal heart, studies were carried out with mice lacking liver retinyl ester stores fed either a VA-sufficient (LRVAS) or VA-deficient (LRVAD) diet (to deplete circulating retinol and extrahepatic stores of retinyl esters). There were no observable differences in the weights or gross morphology of hearts from LRVAS or LRVAD mice relative to sex-matched, age-matched, and genetically matched wild-type (WT) controls fed the VAS diet (WTVAS), but changes in the transcription of functionally relevant genes were consistent with a state of VAD in LRVAS and LRVAD ventricles. In silico analysis revealed that 58/67 differentially expressed transcripts identified in a microarray screen are products of genes that have DNA retinoic acid response elements. Flow cytometric analysis revealed a significant and cell-specific increase in the number of proliferating Sca-1 cardiac progenitor cells in LRVAS animals relative to WTVAS controls. Before myocardial infarction, LRVAS and WTVAS mice had similar cardiac systolic function and structure, as measured by echocardiography, but, unexpectedly, repeat echocardiography demonstrated that LRVAS mice had less adverse remodeling by 1 wk after myocardial infarction. Overall, the results demonstrate that the adult heart is responsive to retinoids, and, most notably, reducing hepatic VA stores (while maintaining circulating levels of VA) impacts ventricular gene expression profiles, progenitor cell numbers, and response to injury.

Retinoid receptors (RRs) play a key role in cell proliferation and differentiation. We characterized the expression of RA receptors and retinoid X receptors (RARs and RXRs) in a series of 111 thyroid tumors and investigated the mechanisms responsible for their deregulation: hypermethylation of the RARB2 promoter, loss of heterozygosity (LOH) in the regions of RARB and RXRA, and altered expression of CRBP1 and enzymes involved in RA biosynthesis (RDH10 and RALDH2). Expression of RALDH2 and RDH10 was conserved in 100 % of adenomas and in 90 and 98 %, respectively, of carcinomas, whereas staining for CRBP1 was decreased in 9 % of FAs and 28 % of carcinomas, mainly anaplastic carcinomas (55 %). We found an abnormal expression of RARA, RARB, RXRA, and RXRB in 67, 69, 66, and 73 %, respectively, of thyroid carcinomas (n = 78) and in 9, 9, 9, and 33 % of follicular adenomas (n = 33) (p < 0.001). An abnormal staining pattern of at least two of these markers had 90 % sensitivity and 91 % specificity for a diagnosis of malignancy. Promoter hypermethylation of RARB2 was observed in some anaplastic carcinomas (14 %). LOH was found to be common at the RARB locus (3p24–3p25) and the RXRA locus (9q34), respectively, in 44 and 55 % of carcinomas and in 27 and 43 % of adenomas. In conclusion, immunohistochemical staining for RARs and RXRs may help in the differential diagnosis between well-differentiated carcinoma and follicular adenoma. Further investigation should be carried out to determine whether the characterization of RR expression might identify patients who could benefit from therapy with RA derivatives.

Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the "kinome". Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression.

The vitamin A derivative, retinoic acid (RA), is essential for embryonic development through the activation of cognate nuclear receptors, RARs, which work as ligand dependent regulators of transcription. In vitro studies revealed how RARs control gene expression at the molecular level and now it appears that it is fine-tuned by a phosphorylation code. In addition, several genetic approaches provided valuable insights on the functions of RARs during development and on the influence of other actors such as the enzymes involved in RA synthesis and degradation and other signaling pathways. It appears that RARs are the conductors of the RA signaling symphony through controlling the dynamics and the coordination of the different players and development steps.

Retinoic acid (RA) regulates several gene programs by nuclear RA receptors (RARs) that are ligand-dependent transcriptional transregulators. The basic mechanism for switching on transcription of cognate-target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes. In addition to these classical genomic effects, we recently demonstrated that RA also induces the rapid activation of the p38MAPK/MSK1 pathway, with characteristic downstream consequences on the phosphorylation of RARs and the expression of their target genes. Here, we aimed at deciphering the underlying mechanism of the rapid non-genomic effects of RA. We highlighted a novel paradigm in which a fraction of the cellular RARα pool is present in membrane lipid rafts, where it forms complexes with G protein alpha Q (Gαq) in response to RA. This rapid RA-induced formation of RARα/Gαq complexes in lipid rafts is required for the activation of p38MAPK that occurs in response to RA. Accordingly, in RA-resistant cancer cells, characterized by the absence of p38MAPK activation, RARα present in membrane lipid rafts does not associate with Gαq, pointing out the essential contribution of RARα/Gαq complexes in RA signaling.

Pluripotent mouse embryonal carcinoma (mEC) and mouse embryonic stem (mES) cells differentiate into several cell lineages upon retinoic acid (RA) addition. Differentiation is facilitated, in part, by RA activation of nuclear RA receptors (RARs) that bind to DNA response elements located in the promoters of target genes. The purpose of the studies reported here was to immunolocalize RARα and RARγ protein in mEC and mES cells and in their RA-induced differentiated progeny. Fixed cells were reacted with three different RARα antibodies and one RARγ antibody. Pluripotent and differentiated mEC and mES cells showed positive nuclear immunoreactivity with all antibodies tested. Two RARα antibodies also showed positive reactivity in the cytoplasm. Surprisingly, our results revealed variability in immunofluorescence intensity and in RARα and RARγ distribution from one cell to the other, suggesting that RARα and RARγ protein levels were not synchronous throughout the cell population. The results indicate that RARα and RARγ are present in pluripotent and differentiating mEC and mES cells and suggest that the expression of these proteins is dynamic.

The transcriptional activity of nuclear retinoic acid receptors (RARs) relies on the association/dissociation of coregulators at the ligand-binding domain. However, we determined that the N-terminal domain (NTD) also plays a role through its phosphorylation, and we isolated vinexinβ, a cytoskeleton protein with three SH3 domains, as a new partner of the RARγ NTD. Here we deciphered the mechanism of the interaction and its role in RARγ-mediated transcription. By combining molecular and biophysical (surface plasmon resonance, NMR, and fluorescence resonance energy transfer) approaches, we demonstrated that the third SH3 domain of vinexinβ interacts with a proline-rich domain (PRD) located in RARγ NTD and that phosphorylation at a serine located in the PRD abrogates the interaction. The affinity of the interaction was also evaluated. In vivo, vinexinβ represses RARγ-mediated transcription and we dissected the underlying mechanism in chromatin immunoprecipitation experiments performed with F9 cells expressing RARγ wild type or mutated at the phosphorylation site. In the absence of retinoic acid (RA), vinexinβ does not occupy RARγ target gene promoters and sequesters nonphosphorylated RARγ out of promoters. In response to RA, RARγ becomes phosphorylated and dissociates from vinexinβ. This separation allows RARγ to occupy promoters. This is the first report of an RAR corepressor association/dissociation out of promoters and regulated by phosphorylation.

International audience; Nuclear retinoic acid receptor alpha (RARalpha) activates gene expression through dynamic interactions with coregulatory protein complexes, the assembly of which is directed by the ligand and the AF-2 domain of RARalpha. Then RARalpha and its coactivator SRC-3 are degraded by the proteasome. Recently it has emerged that the proteasome also plays a key role in RARalpha-mediated transcription. Here we show that SUG-1, one of the six ATPases of the 19 S regulatory complex of the 26 S proteasome, interacts with SRC-3, is recruited at the promoters of retinoic acid (RA) target genes, and thereby participates to their transcription. In addition, SUG-1 also mediates the proteasomal degradation of SRC-3. However, when present in excess amounts, SUG-1 blocks the activation of RARalpha target genes and the degradation of RARalpha that occurs in response to RA, via its ability to interfere with the recruitment of SRC-3 and other coregulators at the AF-2 domain of RARalpha. We propose a model in which the ratio between SUG-1 and SRC-3 is crucial for the control of RARalpha functioning. This study provides new insights into how SUG-1 has a unique role in linking the transcription and degradation processes via its ability to interact with SRC-3.

Vitamin A or retinol is a multifunctional vitamin that is essential at all stages of life from embryogenesis to adulthood. Up to now, it has been accepted that the effects of vitamin A are exerted by active metabolites, the major ones being 11-cis retinal for vision, and all trans-retinoic acid (RA) for cell growth and differentiation. Basically RA binds nuclear receptors, RARs, which regulate the expression of a battery of target genes in a ligand dependent manner. During the last decade, new scenarios have been discovered, providing a rationale for the understanding of other long-noted but not explained functions of retinol. These novel scenarios involve: (i) other nuclear receptors such as PPAR β/δ, which regulate the expression of other target genes with other functions; (ii) extranuclear and nontranscriptional effects, such as the activation of kinases, which phosphorylate RARs and other transcription factors, thus expanding the list of the RA-activated genes; (iii) finally, vitamin A is active per se and can work as a cytokine that regulates gene transcription by activating STRA6. New effects of vitamin A and RA are continuously being discovered in new fields, revealing new targets and new mechanisms thus improving the understanding the pleiotropicity of their effects.

Retinoic acid (RA), the major bioactive metabolite of retinol or vitamin A, induces a spectrum of pleiotropic effects in cell growth and differentiation that are relevant for embryonic development and adult physiology. The RA activity is mediated primarily by members of the retinoic acid receptor (RAR) subfamily, namely RARα, RARβ and RARγ, which belong to the nuclear receptor (NR) superfamily of transcription factors. RARs form heterodimers with members of the retinoid X receptor (RXR) subfamily and act as ligand-regulated transcription factors through binding specific RA response elements (RAREs) located in target genes promoters. RARs also have non-genomic effects and activate kinase signaling pathways, which fine-tune the transcription of the RA target genes. The disruption of RA signaling pathways is thought to underlie the etiology of a number of hematological and non-hematological malignancies, including leukemias, skin cancer, head/neck cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, pancreatic cancer, liver cancer, glioblastoma and neuroblastoma. Of note, RA and its derivatives (retinoids) are employed as potential chemotherapeutic or chemopreventive agents because of their differentiation, anti-proliferative, pro-apoptotic, and anti-oxidant effects. In humans, retinoids reverse premalignant epithelial lesions, induce the differentiation of myeloid normal and leukemic cells, and prevent lung, liver, and breast cancer. Here, we provide an overview of the biochemical and molecular mechanisms that regulate the RA and retinoid signaling pathways. Moreover, mechanisms through which deregulation of RA signaling pathways ultimately impact on cancer are examined. Finally, the therapeutic effects of retinoids are reported.

International audience; Nuclear retinoic acid (RA) receptors (RARs) activate gene expression through dynamic interactions with coregulators in coordination with the ligand and phosphorylation processes. Here we show that during RA-dependent activation of the RARalpha isotype, the p160 coactivator pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3 is phosphorylated by p38MAPK. SRC-3 phosphorylation has been correlated to an initial facilitation of RARalpha-target genes activation, via the control of the dynamics of the interactions of the coactivator with RARalpha. Then, phosphorylation inhibits transcription via promoting the degradation of SRC-3. In line with this, inhibition of p38MAPK markedly enhances RARalpha-mediated transcription and RA-dependent induction of cell differentiation. SRC-3 phosphorylation and degradation occur only within the context of RARalpha complexes, suggesting that the RAR isotype defines a phosphorylation code through dictating the accessibility of the coactivator to p38MAPK. We propose a model in which RARalpha transcriptional activity is regulated by SRC-3 through coordinated events that are fine-tuned by RA and p38MAPK.

Nuclear retinoic acid receptors (RARs) are ligand-dependent transcription factors that regulate the expression of retinoic acid target genes. Although the importance of RAR phosphorylation in their N-terminal domain is clearly established, the underlying mechanism for the phosphorylation-dependent transcriptional activity of the receptors had not been elucidated yet. Here, using a yeast two-hybrid system, we report the isolation of vinexin β as a new cofactor that interacts with the N-terminal A/B domain of the RARγ isotype. Vinexin β is a multiple SH3 motif-containing protein associated with the cytoskeleton and also present in the nucleus. We demonstrate that vinexin β colocalizes with RARγ in the nucleus and interacts with the non-phosphorylated form of the AF-1 domain of RARγ. We also show that this interaction is prevented upon phosphorylation of the AF-1 domain. Using F9 cells stably overexpressing vinexin β or vinexin knockdown by RNA interference, we demonstrate that vinexin β is an inhibitor of RARγ-mediated transcription. We propose a model in which phosphorylation of the AF-1 domain controls RARγ-mediated transcription through triggering the dissociation of vinexin β.

Inhibition of phosphodiesterase IV by N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (piclamilast) enhances the myeloid differentiation induced by all-trans-retinoic acid (ATRA), retinoic acid receptor α (RARα), or retinoic acid receptor X agonists in NB4 and other retinoid-sensitive myeloid leukemia cell types. ATRA-resistant NB4.R2 cells are also partially responsive to the action of piclamilast and retinoic acid receptor X agonists. Treatment of NB4 cells with piclamilast or ATRA results in activation of the cAMP signaling pathway and nuclear translocation of cAMP-dependent protein kinase. This causes a transitory increase in cAMP-responsive element-binding protein phosphorylation, which is followed by down-modulation of the system. ATRA + piclamilast have no additive effects on the modulation of the cAMP pathway, and the combination has complex effects on cAMP-regulated genes. Piclamilast potentiates the ligand-dependent transactivation and degradation of RARα through a cAMP-dependent protein kinase-dependent phosphorylation. Enhanced transactivation is also observed in the case of PML-RARα. In NB4 cells, increased transactivation is likely to be at the basis of enhanced myeloid maturation and enhanced expression of many retinoid-dependent genes. Piclamilast and/or ATRA exert major effects on the expression of cEBP and STAT1, two types of transcription factors involved in myeloid maturation. Induction and activation of STAT1 correlates directly with enhanced cytodifferentiation. Finally, ERK and the cAMP target protein, Epac, do not participate in the maturation program activated by ATRA + piclamilast. Initial in vivo studies conducted in severe combined immunodeficiency mice transplanted with NB4 leukemia cells indicate that the enhancing effect of piclamilast on ATRA-induced myeloid maturation translates into a therapeutic benefit.

In eukaryotic cells, liganded RARγ2/RXRα 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 RARγ2 and RXRα partners as well as of their respective AF-1 and AF-2 domains in the processes of transactivation and degradation. RARγ2 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 RXRα. In contrast, RXRα plays only a modulatory role, cooperating with RARγ2 through its AF-2 domain and its phosphorylated AF-1 domain in both the transcription activity and the degradation of the RARγ2/RXRα 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.

PPAR-gamma/RXR heterodimers play a key regulatory role in murine placental development. We report here that PPAR-gamma and RXR-alpha are highly expressed in cultured primary extravillous cytotrophoblasts (EVCT) isolated from human first-trimester placenta. Using this cell culture model, we showed that PPAR-gamma agonists inhibit EVCT invasion, whereas PPAR-gamma or pan-RXR antagonists promoted EVCT invasion by themselves and abolished PPAR-gamma agonist-mediated effect. Together these data underscore an important function of PPAR/RXR heterodimers in the modulation of trophoblast invasion.

Nuclear receptors recruit coregulator complexes through both their AF-1 and AF-2 activation domains. Here we demonstrate that TIF2, a p160 coactivator, is able to bridge the two activation domains of the retinoic acid (RA) receptor isotype RARalpha1, resulting in synergistic activation of transcription. Bridging requires the presence of motifs in region A of RARalpha1 and in the activation domain AD1 of TIF2. Notably, only RARalpha1 exerted this interaction, which requires additional unknown factors. This is the first observation of a RAR isotype-selective coactivator interaction. Because another p160 coactivator, SRC-1, has no effect, this is also the first demonstration of a difference between the members of this coactivator family.

We have previously shown that all-trans retinoic acid (atRA), the active metabolite of vitamin A, enhances the activation of the inducible nitric oxide synthase (NOS II) pathway, a component of innate immunity, in rats in vivo. We investigated the relative contribution of retinoic acid receptor-alpha (RARalpha) and retinoid X receptors (RXRs) to NOS II activation triggered by LPS. Five-day supplementation with 10 mg/kg of either atRA or the RARalpha selective agonist Ro-40-6055, but not with 10 mg/kg of the pan-RXR agonist Ro-25-7386, enhanced the LPS-induced NOS II mRNA, protein expression in liver, and plasma nitrite/nitrate concentration. Both atRA and the RARalpha agonist (but not the RXR agonist) increased the number of peripheral T helper lymphocytes and plasma interferon-gamma concentration. Synergism between retinoids and LPS on NOS II activation within an organ coincided with synergism on interferon regulatory factor-1 mRNA expression but not with the level of expression of the RARalpha protein. These results suggest that, in vivo, atRA activates NOS II through RARalpha and contributes to characterizing the complex effect of retinoids on the host inflammatory/immune response.

Nuclear retinoic acid (RA) receptors (RARs) are phosphorylated at conserved serine residues located in their N-terminal domain. Phosphorylation of RARgamma2 at these residues is increased in response to RA subsequently to the activation of p38MAPK. We show here that this RA-induced phosphorylation of RARgamma2 resulted from the down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. By overexpressing Akt and by using agents that activated or inhibited the PI3K/Akt pathway, we also demonstrated that the RA-induced down-regulation of the PI3K/Akt pathway targeted not only the phosphorylation of RARgamma2 but also the turnover and transcriptional activity of the receptor. Altogether these data indicate that the PI3K/Akt pathway plays an important role in retinoic acid signaling.

The nuclear retinoic acid receptor RAR gamma 2 undergoes proteasome-dependent degradation upon ligand binding. Here we provide evidence that the domains that signal proteasome-mediated degradation overlap with those that activate transcription, i.e. the activation domains AF-1 and AF-2. The AF-1 domain signals RAR gamma 2 degradation through its phosphorylation by p38MAPK in response to RA. The AF-2 domain acts via the recruitment of SUG-1, which belongs to the 19S regulatory subunit of the 26S proteasome. Blocking RAR gamma 2 degradation through inhibition of either the p38MAPK pathway or the 26S proteasome function impairs its RA-induced transactivation activity. Thus, the turnover of RAR gamma 2 is linked to transactivation.

The discovery of retinoic acid receptors arose from research into how vitamins are essential for life. Early studies indicated that Vitamin A was metabolized into an active factor, retinoic acid (RA), which regulates RNA and protein expression in cells. Each step forward in our understanding of retinoic acid in human health was accomplished by the development and application of new technologies. Development cDNA cloning techniques and discovery of nuclear receptors for steroid hormones provided the basis for identification of two classes of retinoic acid receptors, RARs and RXRs, each of which has three isoforms, α, β and ɣ. DNA manipulation and crystallographic studies revealed that the receptors contain discrete functional domains responsible for binding to DNA, ligands and cofactors. Ligand binding was shown to induce conformational changes in the receptors that cause release of corepressors and recruitment of coactivators to create functional complexes that are bound to consensus promoter DNA sequences called retinoic acid response elements (RAREs) and that cause opening of chromatin and transcription of adjacent genes. Homologous recombination technology allowed the development of mice lacking expression of retinoic acid receptors, individually or in various combinations, which demonstrated that the receptors exhibit vital, but redundant, functions in fetal development and in vision, reproduction, and other functions required for maintenance of adult life. More recent advancements in sequencing and proteomic technologies reveal the complexity of retinoic acid receptor involvement in cellular function through regulation of gene expression and kinase activity. Future directions will require systems biology approaches to decipher how these integrated networks affect human stem cells, health, and disease.

The nuclear retinoic acid receptors (RAR α, β and γ) and their isoforms are ligand-dependent regulators of transcription Transcription , which mediate the effects of all-trans retinoic acid (RA), the active endogenous metabolite of Vitamin A. They heterodimerize with Retinoid X Receptors (RXRs α, β and γ), and regulate the expression of a battery of target genes Target genes involved in cell growth and differentiation Differentiation . During the two last decades, the description of the crystallographic structures of RARs, the characterization of the polymorphic response elements of their target genes Target genes , and the identification of the multiprotein complexes involved in their transcriptional activity have provided a wealth of information on their pleiotropic effects. However, the regulatory scenario became even more complicated once it was discovered that RARs are phosphoproteins and that RA can activate kinase signaling cascades via a pool of RARs present in membrane lipid rafts. Now it is known that these RA-activated kinases Kinases translocate to the nucleus where they phosphorylate RARs and other retinoid signaling factors. The phosphorylation Phosphorylation state of the RARs dictates whether the transcriptional programs which are known to be induced by RA are facilitated and/or switched on. Thus, kinase signaling pathways appear to be crucial for fine-tuning the appropriate physiological activity of RARs.

Retinoic acid (RA), the active derivative of vitamin A, a fat-soluble vitamin, plays key roles in cell growth and differentiation by activating nuclear receptors, RARs (α, β and γ), which are ligand dependent regulators of transcription. The past years highlighted several novelties in the field that increased the complexity of RA effects. Indeed, in addition to its classical genomic effects, RA also has extranuclear and non-transcriptional effects. RA induces the rapid and transient activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of RARs at a conserved serine residue located in the N-terminal domain and their coregulators. In order to investigate the relevance of RARs' phosphorylation in cell differentiation, mouse embryonic stem (mES) cells were used as a model. When treated with RA, these pluripotent cells give rise to neuronal cells. Cells invalidated for each RAR were generated as well as stable rescue lines expressing RARs mutated in phosphor acceptor sites. Such a strategy revealed that RA-induced neuronal differentiation involves the RARγ2 subtype and requires RARγ2 phosphorylation. Moreover, in gene expression profiling experiments, the phosphorylated form of RARγ2 was found to regulate a small subset of genes through binding a novel RA response element consisting of two direct repeats with a 7 base pair spacer. These new findings suggest an important role for RAR phosphorylation during cell differentiation, and pave the way for further investigations with other cell types and during embryonic development. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.

Gene activation by retinoic acid nuclear receptors (RAR) is regulated by a number of molecular events such as ligand binding, interaction with cognate DNA sequences and co-regulatory proteins, and phosphorylation. Among the several phosphorylation sites that are involved in the non-genomic regulatory pathways of the RAR, two are located in a proline rich domain (PRD) within the N-terminal domain (NTD) of the receptor. This region is predicted to be intrinsically disordered, complicating its production and purification. We present here an approach enabling the high yield production of RAR fragments encompassing the PRD and the DNA binding domain (DBD). We found that expression levels were dependent on where the position of the N-terminal boundary of the fragment was placed within the RAR sequence. The purification protocol involves the use of maltose binding protein as a solubilising tag and extensive centrifugation steps at critical points of the purification process. This protocol is suitable to express 15 N, 13 C labeled proteins enabling nuclear magnetic resonance studies. The resulting proteins were characterized by biophysical methods including Small Angle X-ray Scattering and NMR. These studies showed that PRD extension of RARγ is disordered in solution, a state that is compatible with modifications such as phosphorylation.

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest by activating nuclear receptors, RARs (α, β and γ), which are ligand dependent transcriptional factors. RARs are also phosphorylated in response to RA. Here we investigated the in vivo relevance of RARs phosphorylation during RA-induced neuronal differentiation of mouse embryonic stem (mES) cells. Using ES cells where the genes encoding each RAR subtype have been inactivated and stable rescue lines expressing RARs mutated in phosphor-acceptor sites, we show that RA-induced neuronal differentiation involves RARγ2 and requires RARγ2 phosphorylation. By gene expression profiling, we found that the phosphorylated form of RARγ2 regulates a small subset of genes through binding an unusual RA response element consisting of two direct repeats with a 7 base pair spacer. These new findings suggest an important role for RARγ phosphorylation during cell differentiation, and pave the way for further investigations during embryonic development.

We identify the RARalpha, RXRalpha and CRABPII domains required for the physical interaction of these proteins. On RARalpha and RXRalpha, the sequences correspond to the DEF and DE domains, respectively, but the interaction with CRABPII does not require the AF-2AD 'core'. On CRABPII, two interacting domains are identified (NRID1 and NRID2), one of which contains the only enhancement transactivation domain of CRABPII. The interaction is ligand-independent and does not require the ligand-binding domain of CRABPII. These results further stress that interaction of CRABPII with the nuclear receptors defines a novel level of transcriptional control.

The ligand-dependent nuclear receptors PPARgamma and RXRalpha/beta were recently determined to be essential for murine placental development and trophoblast differentiation. In the current study we examined the expression and role of the PPARgamma/RXRalpha heterodimers in human invasive trophoblasts. We first report that in human first trimester placenta, PPARgamma and RXRalpha are highly expressed in cytotrophoblasts at the feto-maternal interface, especially in the extravillous cytotrophoblasts involved in uterus invasion. The coexpression of PPARgamma and RXRalpha genes in extravillous cytotrophoblast nuclei were then confirmed by immunocytochemistry, immunoblot, and real-time quantitative PCR using cultured purified primary extravillous cytotrophoblasts. We next examined, using the extravillous cytotrophoblast culture model, the biological role of PPARgamma/RXRalpha heterodimers in vitro, and we showed that both synthetic (rosiglitazone) and natural [15-deoxy-delta-(12,14)PGJ(2)] PPARgamma agonists inhibit extravillous cytotrophoblast invasion in a concentration-dependent manner and synergize with pan-RXR agonists. Conversely, PPARgamma or pan-RXR antagonists promoted extravillous cytotrophoblast invasion. Furthermore, the pan-RXR antagonist abolished the inhibitory effect of the PPARgamma agonists. Together these data underscore an important function of PPARgamma/RXRalpha heterodimers in the modulation of trophoblast invasion.

The ligand-dependent nuclear receptors PPARγ and RXRα/β were recently determined to be essential for murine placental development and trophoblast differentiation. In the current study we examined the expression and role of the PPARγ/RXRα heterodimers in human invasive trophoblasts. We first report that in human first trimester placenta, PPARγ and RXRα are highly expressed in cytotrophoblasts at the feto-maternal interface, especially in the extravillous cytotrophoblasts involved in uterus invasion. The coexpression of PPARγ and RXRα genes in extravillous cytotrophoblast nuclei were then confirmed by immunocytochemistry, immunoblot, and real-time quantitative PCR using cultured purified primary extravillous cytotrophoblasts. We next examined, using the extravillous cytotrophoblast culture model, the biological role of PPARγ/RXRα heterodimers in vitro, and we showed that both synthetic (rosiglitazone) and natural [15-deoxy-δ-(12,14)PGJ2] PPARγ agonists inhibit extravillous cytotrophoblast invasion in a conc...

Recent studies performed with null mice suggested a role of either RXRα or PPARγ in murine placental development. We report here that both PPARγ and RXRα are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPARγ (but not for PPARα or PPARδ) increase both human CGβ transcript levels and the secretion of human CG and its free β-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPARγ ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPARγ/RXRα heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CGβ gene (β5) were found to bind RXRα and PPARγ from human cytotrophoblast nuclear extracts, suggesting that PPARγ/RXRα heterodimers directly regulate human CGβ transcription. Altogether, these data show that PPARγ/RXRα heterodimers play an important role in human placental development.

In monolayers cultures, retinoic acid (RA) induces the differentiation of F9 embryonal carcinomal (EC) cells into primitive endoderm-like cells, while a combination of RA and dibutyryl cAMP leads to parietal endoderm-like differentiation. Knock out of all RARgamma isoforms (RARgamma(-/-) line) drastically impairs primitive and subsequent parietal endodermal differentiation and affects the induction of many endogenous RA-responsive genes. Using lines that reexpress RARgamma2 or overexpress RARalpha1 lacking their AF-2AD core (RARgammadeltaAF2 and RARalphadeltaAF2, respectively), we show that this conserved amphipatic alpha-helical motif (helix 12) of the ligand binding domain, and therefore the activation function AF-2 of both receptors, is required for the induction of differentiation and target gene expression upon RA treatment of F9 EC cells. We also show that these deletion mutants behave as dominant negatives.

The syncytiotrophoblast (ST), which forms the outer layer of the chorionic villi, is the endocrine unit of the human placenta. Bathing in the maternal blood of the intervillous space, the ST secretes its hormonal products directly into the maternal circulation. Leptin is expressed in the ST and is secreted into the maternal circulation. However, its regulation and physiological role during pregnancy remain poorly known. In the present work we used the in vitro model of human cytotrophoblast differentiation into ST to study the effect of physiological and synthetic retinoids on leptin synthesis and secretion. Using specific antibodies we first illustrated by immunocytochemistry the expression of retinoic acid (RA) receptor α and retinoid X receptor α (RXRα) in ST. We then observed that leptin messenger ribonucleic acid and protein expression increased with in vitro ST formation. The 9-cis isomer of RA and the synthetic retinoid specific for RXRs (BMS 649) stimulated leptin messenger ribonucleic acid expres...

Retinoids reverse potentially malignant lesions and inhibit the development of second primary cancers in patients with head-and-neck cancer. Many of the effects of retinoids result from modulation of gene expression by 2 distinct classes of nuclear receptor, RARs and RXRs; alterations in their expression can lead to tumorigenesis. To determine whether aberrations in expression of the receptors are related to the development of betel- and tobacco-related oral cancer, we used specific monoclonal antibodies against RARα and RARβ to detect expression of these proteins in 30 histopathologically normal tissues, 45 potentially malignant lesions (leukoplakia) with histological evidence of either hyperplasia (31 cases) or dysplasia (14 cases) and 64 oral squamous-cell carcinomas (SCCs) by immunohistochemistry. Of the 30 normal oral tissues analysed, 8 cases showed detectable levels of RARα protein, while 10 cases did not show detectable RARβ immunoreactivity. Immunostaining for RARα protein was observed in 12/31 (39%) hyperplastic lesions, 6/14 (43%) dysplastic lesions and 43/64 (67%) oral SCCs. Expression of RARα in oral SCC was significantly associated with the histological differentiation status of tumours (p = 0.016). In contrast, lack of detectable immunoreactivity was observed in 19/31 (61%) hyperplastic lesions, 8/14 (57%) dysplastic lesions and 21/64 (33%) oral SCCs. The hallmark of the study was the significant increase in RARα immunopositivity in oral SCCs compared to normal tissue (p = 0.0005) and hyperplastic lesions (p = 0.016). One intriguing feature was the significant decrease in RARβ immunopositivity in hyperplastic lesions compared with normal oral mucosa (p = 0.05) as well as in oral SCCs compared with normal tissues (p = 0.0008). Int. J. Cancer 91:27–31, 2001. © 2001 Wiley-Liss, Inc.

Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor α (PML/RAR α ) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RAR α cleavage, RA triggers degradation of both PML/RAR α and RAR α . Similarly, in non-APL cells, RA directly targeted RAR α and RAR α fusions to the proteasome degradation pathway. Activation of either RAR α or RXR α by specific agonists induced degradation of both proteins. Conversely, a mutation in RAR α that abolishes heterodimer formation and DNA binding, blocked both RAR α and RXR α degradation. Mutations in the RAR α DNA-binding domain or AF-2 transcriptional activation region also impaired RAR α catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation.

The nuclear receptor mouse retinoid X receptor alpha (mRXRalpha) was shown to be constitutively phosphorylated in its NH2-terminal A/B region, which contains potential phosphorylation sites for proline-directed Ser/Thr kinases. Mutants for each putative site were generated and overexpressed in transfected COS-1 cells. Constitutively phosphorylated residues identified by tryptic phosphopeptide mapping included serine 22 located in the A1 region that is specific to the RXRalpha1 isoform. Overexpression and UV activation of the stress-activated kinases, c-Jun NH2-terminal kinases 1 and 2 (JNK1 and JNK2), hyperphosphorylated RXRalpha, resulting in a marked decrease in its electrophoretic mobility. This inducible hyperphosphorylation involved three residues (serines 61 and 75 and threonine 87) in the B region of RXRalpha and one residue (serine 265) in the ligand binding domain (E region). Binding assays performed in vitro with purified recombinant proteins demonstrated that JNKs did not interact with RXRalpha but bound to its heterodimeric partners, retinoic acid receptors alpha and gamma (RARalpha and RARgamma). Hyperphosphorylation by JNKs did not affect the transactivation properties of either RXRalpha homodimers or RXRalpha/RARalpha heterodimers in transfected cultured cells.

BACKGROUND Retinoids can suppress carcinogenesis in high-risk non-neoplastic bronchial lesions and can reduce the risk of second primary non-small-cell lung cancer (NSCLC). The effects of retinoids are mediated by nuclear receptors, i.e., the retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and the retinoid X receptors (RXRalpha, RXRbeta, and RXRgamma). We investigated whether abnormalities in the in vivo expression of retinoid receptors are observed in NSCLC. METHODS Expression of retinoid receptors in paired specimens of normal and cancerous tissues from the lungs of 76 patients with NSCLC was studied by use of antiretinoid receptor antibodies (except those against RXRgamma) and immunohistochemistry. RAR messenger RNAs were analyzed by use of in situ hybridization and by reverse transcription-polymerase chain reaction (RT-PCR). Samples were also studied for loss of heterozygosity (LOH) at chromosome 3p24. All P values are two-sided. RESULTS All studied receptors were expressed in normal lung cells and in high- risk non-neoplastic lesions. In tumor cells, overexpression of RXRalpha and RARalpha was frequently observed. In contrast, RXRbeta expression decreased in 18% of the tumor specimens. Furthermore, there was a marked decrease in the expression of RARbeta in 63% of the tumors (P

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG-3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG-3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG-3 cells expressed RAR alpha and RXR alpha, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all-trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RAR alpha and RXR alpha. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 +/- 2%) being observed after 4 days of treatment with Ro 25, a RXR alpha specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 +/- 10%) occurred at 48 h with the RXR alpha-specific ligand. The RAR alpha-specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXR alpha in mediating the biological effects of retinoids on JEG-3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA.

To investigate the role of retinoids on human placental development and functions, we characterized the spatial distribution of retinoid receptors in human term chorionic villi. In situ hybridization with 35S labeled sense and antisense probes for the RARs, alpha, beta, gamma and RXRs, alpha, beta, gamma, specifically detected only RAR alpha and RXR alpha. Both RAR alpha and RXR alpha mRNA were preferentially expressed in the trophoblast cell layer. This syncytiotrophoblast expression was confirmed by immunohistochemical analyses using anti-RAR alpha and RXR alpha antibodies. Using trophoblast cells in culture, we then studied the effect on hCG secretion of 0.1 microM RA physiological forms and of selective RAR alpha and RXR alpha synthetic agonists. Only RXR alpha specific ligands such as physiological 9-cis RA and synthetic Ro 25-7386 stimulated hCG secretion (doubled). These results suggest an important role for RXR alpha in human placental development and function.

The nuclear retinoic acid receptors (RAR α, β and γ) and their isoforms are ligand-dependent regulators of transcription, which mediate the effects of all-trans retinoic acid (RA), the active endogenous metabolite of Vitamin A. They heterodimerize with Retinoid X Receptors (RXRs α, β and γ), and regulate the expression of a battery of target genes involved in cell growth and differentiation. During the two last decades, the description of the crystallographic structures of RARs, the characterization of the polymorphic response elements of their target genes, and the identification of the multiprotein complexes involved in their transcriptional activity have provided a wealth of information on their pleiotropic effects. However, the regulatory scenario became even more complicated once it was discovered that RARs are phosphoproteins and that RA can activate kinase signaling cascades via a pool of RARs present in membrane lipid rafts. Now it is known that these RA-activated kinases translocate to the nucleus where they phosphorylate RARs and other retinoid signaling factors. The phosphorylation state of the RARs dictates whether the transcriptional programs which are known to be induced by RA are facilitated and/or switched on. Thus, kinase signaling pathways appear to be crucial for fine-tuning the appropriate physiological activity of RARs.