Retinoic Acid Receptor Isotypes Research Articles

Synergistic activation of RARβ and RARγ nuclear receptors restores cell specialization during stem cell differentiation by hijacking RARα-controlled programs.

How cells respond to different external cues to develop along defined cell lineages to form complex tissues is a major question in systems biology. Here, we investigated the potential of retinoic acid receptor (RAR)-selective synthetic agonists to activate the gene regulatory programs driving cell specialization during nervous tissue formation from embryonic carcinoma (P19) and mouse embryonic (E14) stem cells. Specifically, we found that the synergistic activation of the RARβ and RARγ by selective ligands (BMS641 or BMS961) induces cell maturation to specialized neuronal subtypes, and to astrocytes and oligodendrocyte precursors. Using RAR isotype knockout lines exposed to RAR-specific agonists, interrogated by global transcriptome landscaping and in silico modeling of transcription regulatory signal propagation, revealed major RARα-driven gene programs essential for optimal neuronal cell specialization and hijacked by the synergistic activation of the RARβ and RARγ receptors. Overall, this study provides a systems biology view of the gene programs accounting for the previously observed redundancy between RARs, paving the way toward their potential use for directing cell specialization during nervous tissue formation.

Assessing the Safety and Efficacy of Trifarotene in the Treatment of Acne Vulgaris.

Acne vulgaris is a common skin condition of the face and trunk that negatively impacts quality of life. Trifarotene is a new first-in-class fourth-generation topical retinoid that has been uniquely studied in the treatment of both facial and truncal acne. Through selective agonism of retinoic acid receptor (RAR)-gamma, the most predominant RAR isotype in the epidermis, trifarotene exerts more targeted, skin-specific effects than earlier generation retinoids. This narrative review summarizes all currently available literature regarding the use of trifarotene in acne vulgaris. We focus on efficacy, safety, and tolerability data and highlight quality of life outcomes and patient-reported satisfaction. Future clinical trials and the clinical applicability of this novel medication in the treatment of acne are also discussed.

Abstract 5204: Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor

Abstract Migration and adhesion are two critical processes highly related to metastasic dissemination and all trans-retinoic acid (ATRA) is known to diminished migration and metalloprotease (MMP) secretion in breast cancer cell lines. Our objective was to evaluate the effect of activating each retinoic acid receptor (RAR) isotype in adhesion, migration and MMPs secretion using two hormone-independent murine cell lines: LM38-LP y 4T1. First we determined that both lines express the different RAR isotypes with the exception of RARβ in 4T1 cell line. Cells were treated for 96h with AM580 (RARα agonist, 200nM), AC55649 (RARβ agonist, 2μM), BMS961 (RARγ agonist, 50nM) or vehicle (DMSO). The migratory potential was evaluated by a “wound healing” assay. LM38-LP cell line reduced its migratory capacity in response to AM580 and AC55649 (AM580: 108.0±24.3μm, AC55649: 114.1±12.8μm vs. vehicle: 186.9±31.0μm, p<0.05). An inverse effect was observed in 4T1 cells which increased its migratory potential after the treatment with AM580 (378.9±32.0μm vs. vehicle 302.2±31.9μm, p<0.05). The same agonist treatments were used to obtain conditioned media in order to evaluate soluble MMPs activity by zymography. AM580, AC55649 and BMS961 pre-treatments decreased soluble MMP2 activity in LM38-LP cells (0.53±0.03au (arbitrary units), 0.47±0.01 au, and 0.75±0.04 au respectively, fold change vs. control). Conversely, AM580 pre-treatment increased MMP2 and MMP9 in 4T1 cells (1.57±0.10 au and 2.04±0.26 au respectively, fold change vs. control). Regarding adhesion assay, after the above mentioned treatments cells were detached, incubated 2h at 37°C in order to recover cell surface proteins and finally seeded. After 2h incubation, non-adherent cells were washed out with PBS and adherent cells were fixed, stained and quantified by densitometry. We observed that AM580 and AC55649 diminished LM38-LP adhesive capacity (0.51±0.02 au, 0.83±0.00 au respectively, fold change vs. control) while AC 55649 increased this parameter in 4T1 cells (1.28±0.06 au fold change vs. control). Finally we performed an experimental lung metastasis assay using LM38-LP cells. Cells where treated with the different agonist during 144h and then inoculated into the tail vein of syngeneic mice. Lungs were removed 21 days later and number of superficial lung metastasis was determined. AC55649 treatment induced an increase in the metastatic potential (Md [Rg]: 143, [86-314] vs. control Md [Rg]: [21.5, 2-73], p<0.05). In conclusion, the activation of RARα and RARβ isotypes leads to opposite responses in different cell lines. We hypothesized that the differences in RARβ expression between this two cell lines should be responsible of this effect. Surprisingly, the activation of RARβ increased the metastatic potential of LM38-LP cells in spite of the negative regulation of parameters associated with the metastatic process. Citation Format: Carolina Flumian, Damián E. Berardi, Stefano M. Cirigliano, María I. Díaz Bessone, Elisa D. Bal de Kier Joffé, Alejandro J. Urtreger, Laura B. Todaro. Parameters associated with metastatic dissemination are differentially modulated by specific isotypes of the retinoic acid receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2015-5204

Responsiveness of stem-like human glioma cells to all-trans retinoic acid and requirement of retinoic acid receptor isotypes α, β and γ

Retinoic acid (RA) is required for development and homeostasis of the normal mammalian brain and may play a role in the initiation and progression of malignant brain tumors, such as the glioblastoma multiforme (GBM) and the gliosarcoma (Gsarc). The subpopulation of stem-like glioma cells (SLGCs) was shown to resist standard glioma radio-/chemotherapy and to propagate tumor regrowth. We used phenotypically distinct, self-renewing SLGC lines from six human GBMs, two Gsarcs, and two subcloned SLGC derivatives in order to investigate their responsiveness to all-trans retinoic acid (atRA) and to identify the RA-receptor (RAR) isotypes involved. In general, atRA exerted a pro-proliferative and pro-survival effect on SLGCs, though the efficacy was distinct. By means of RAR isotype-selective retinoids we disclosed that these effects were mediated by RARα and RARγ, except for one SLGC line, in which the pro-proliferative signal was induced by the RARβ-selective retinoid. Only one GBM-derived cell line (T1338) and a subpopulation of another (T1389) displayed neural differentiation in response to atRA. Differentiation of T1338 was induced by RARα and RARγ isotype-selective retinoids, associated with down-regulation of Sox2, and the failure to induce orthotopic tumors in the brains of SCID mice. The differential responsiveness of the SLGC lines appeared unrelated to the expression of RARβ, as (i) atRA augmented RAR isotype mRNA expression and particularly rarβ mRNA in all SLGC lines, (ii) rarβ promoter hypomethylation in the SLGC lines was not related to differentiation and (iii) the induction of T1338 differentiation was by RARα- and RARγ-selective ligands.

Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis

IntroductionRetinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, β and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARβ are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/β in favor of RARγ.MethodsThe effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis.ResultsModulation of the RARα/β to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death.ConclusionsThe functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/β balance suggests that prevalence of RARγ over-RARα/β expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials.See related editorial by Garattini et al http://breast-cancer-research.com/content/14/5/111

Histone H3 Methylation Mediates All-Trans-Retinoic Acid Responsiveness in Acute Myeloid Leukemia

Abstract 224During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARα and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL), ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have previously hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARα isoforms, particularly ATRA-inducible RARα2, are down-regulated in AML (Blood. 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARα2 in AML is associated with a diminution in levels of histone histone H3 lysine 4 dimethylation (H3K4me2) on the ATRA-responsive RARA2 promoter (a modification associated with transcriptional activation). Interestingly, the H3K4me1/me2 demethylase LSD1/KDM1 (AOF2) is highly expressed in AML patients (www.proteinatlas.org). A number of small molecules that target this enzyme (LSD1i) are in development and, collectively, these data predict that the use of LSD1i will facilitate induction of expression of genes that are required for differentiation of AML cells. In this study we used tranylcypromine (TCP, a monoamine oxidase used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders, respectively), which functions a time-dependent, mechanism-based inhibitor of LSD1.Here we show that TCP unlocked the ATRA-driven therapeutic differentiation response in non-APL AML cell lines including the TEX cell line, which is derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. TEX cells are >90% CD34+, respond poorly to ATRA and mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). Consistent with this, ATRA/TCP treatment increased differentiation in primary patient samples. ATRA alone had in general only small effects in primary AML samples and TCP showed minimal activity in most cases. Furthermore, shRNA-mediated knockdown of LSD1 confirmed a critical role for this enzyme in blocking the ATRA response in AML cells.The effects of ATRA/TCP on AML cell maturation were paralleled by enhanced induction of genes associated with myelomonocytic differentiation, including direct ATRA targets. LSD1i treatment did not lead to an increase in genome-wide H3K4me2, but did increase H3K4 dimethylation of myelomonocytic differentiation-associated genes. Importantly, treatment with ATRA/TCP dramatically diminished the clonogenic capacity of AML cells in vitro and engraftment of cells derived from AML patients in vivo, suggesting that ATRA/TCP may also target leukemic stem cells. These data strongly suggest that LSD1 may, at least in part, contribute to AML pathogenesis by inhibiting the normal function of ATRA in myelomonocytic development and pave the way for effective differentiation therapy of AML. Disclosures:No relevant conflicts of interest to declare.

Modulation of Histone H3K4 and H3K27 Methylation Levels Via Pharmacological Inhibition of LSD1 and Degradation of the EZH2-Containing Polycomb Repressive Complex 2 Stimulates ATRA-Mediated Differentiation of AML Cells.

Abstract 1046Poster Board I-68During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARαa and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL) ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARαa isoforms, particularly ATRA-inducible RARαa2, are down-regulated in AML (Blood 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARαa2 in AML is associated with a diminution in histone H3K4me2 and an increase H3K27me3 on the RARA2 promoter (modifications associated with transcriptional activation and silencing, respectively). Interestingly, H3K4 demethylase LSD1 (AOF2) and the polycomb represive complex 2 (PCR2)-associated H3K27 methyltransferase EZH2 are highly expressed in AML (www.proteinatlas.org). Small molecules that target these enzymes are in development and, given the above results, we predict that the use of such agents in combination with ATRA will enhance the effects of ATRA-mediated induction of gene expression and differentiation of AML cells. To test this hypothesis, we used ATRA-responsive HL-60 AML cells and the TEX cell line. TEX cells are derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. These cells, the ATRA-responsiveness of which is not known, mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). LSD1 activity was inhibited using monoaminoxidase inhibitor (MAOI) trans-2-phenylcyclopropylamine (Parnate, 1μM) in combination with pharmacological (1μM) and sub-optimal (0.1μM) concentrations of ATRA. Co-treatment with Parnate potentiated the HL-60 response to sub-optimal ATRA concentration. While ATRA appeared to be a less potent inducer of TEX cell differentiation, Parnate nevertheless enhanced their maturation at pharmacological ATRA concentrations and sensitized these cells to differentiation induction under sub-optimal ATRA levels. Additionally, we investigated the biguanide polyamine analogue 1,15-bis[N5-[3,3-(diphenyl) propyl]-N1-biguanido]-4,12-diazapentadecane (2d), which is structurally unrelated to Parnate, obtaining similar results. Biguanide polyamine analogue inhibitors of LSD1 may have several benefits over MAOIs, including DNA targeting due their cationic nature. We also tested 3-deazaneplanocin A (DZNep), which diminishes levels of H3K27 trimethylation via depletion of the EZH2 catalytic subunit of the PCR2. Consistent with our hypothesis and the above data, co-treatment of HL-60 and TEX cells with DZNep (0.05μM) and ATRA (0.1μM and/or 1μM) led to more robust differentiation response than when ATRA was used as a single agent. The use of ATRA in combination with DZNep and LSD1 inhibitors at the same time led to a better differentiation response, as measured by CD11b/CD11c expression, morphology and superoxide production (NBT assay), than when either drug alone was used with ATRA. The effects of these drug combinations on AML cell maturation were paralleled by synergistic induction of endogenous ATRA target genes and expected changes in the levels of H3K4/K27 methylation. At the concentrations used with ATRA neither Parnate, 2d nor DZNep induced differentiation when used as single agents, however, when used at higher concentrations both singly and in combination with ATRA, these drugs exerted cytotoxic effects. Importantly, the above described combination treatments were specific for AML blasts as they had no cytotoxic effects on normal CD33+/CD34+ cell populations. These data demonstrate existence of therapeutically relevant crosstalks between the ATRA-induced differentiation pathway and histone H3K4 and K27 methylation and that targeting LSD1 and/or EZH2 in combination with ATRA may represent a promising treatment for AML. Disclosures:Marton:Progen Pharmaceuticals: Employment. Woster:Progen Pharmaceuticals: Consultancy, Research Funding. Casero:Progen Pharmaceuticals: Consultancy, Research Funding.

Commitment of Mouse Embryonic Stem Cells to the Adipocyte Lineage Requires Retinoic Acid Receptor β and Active GSK3

Key events leading to terminal differentiation of preadipocytes into adipocytes have been identified in recent years. However, signaling pathways involved in the decision of stem cells to follow the adipogenic lineage have not yet been characterized. We have previously shown that differentiating mouse embryonic stem (mES) cells give rise to functional adipocytes upon an early treatment with retinoic acid (RA). The goal of this work was to identify regulators of RA-induced commitment of mES cells to the adipocyte lineage. First, we investigated the role of RA receptor (RAR) isotypes in the induction of mES cell adipogenesis. Using synthetic retinoids selective of RAR isotypes, we show that RARbeta activation is both sufficient and necessary to trigger commitment of mES cells to adipocytes. Then, we performed a small-scale drug screening to find signaling pathways involved in RARbeta-induced mES cell adipogenesis. We show that pharmacological inhibitors of glycogen synthase kinase (GSK) 3, completely inhibit RARbeta-induced adipogenesis in mES cells. This finding uncovers the requirement of active GSK3 in RARbeta-induced commitment of mES cells toward the adipocyte lineage. Finally, we investigated the role of the Wnt pathway, in which GSK3 is a critical negative regulator, in adipocyte commitment by analyzing Wnt pathway activity in RA- and RARbeta-induced mES cell adipogenesis. Our results suggest that although RARbeta and active GSK3 are required for RA-induced adipogenesis, they might be acting through a Wnt pathway-independent mechanism.

Retinoic Acid Receptor Isotype Specificity in F9 Teratocarcinoma Stem Cells Results from the Differential Recruitment of Coregulators to Retinoic Acid Response Elements

The retinoic acid receptor (RAR) alpha, beta(2), and gamma isotypes each regulate specific subsets of target genes in F9 teratocarcinoma stem cells. We used chromatin immunoprecipitation assays to monitor the association of RARgamma, retinoic X receptor (RXR) alpha, and coregulators with the RARbeta(2), Hoxa1, and Cyp26A1 retinoic acid response elements (RAREs) in F9 wild type and RARalpha, -beta(2), and -gamma null cells. Additionally we quantitatively monitored expression of the corresponding mRNAs. We demonstrated that the association of RARgamma and/or RXRalpha with a RARE was not sufficient for retinoic acid (RA)-mediated transcription of the corresponding target gene. However, the ability of RARgamma and/or RXRalpha to recruit pCIP (AIB1/ACTR/RAC-3/TRAM-1/SRC-3) and p300 to a RARE did correlate with RA-associated transcription of target mRNAs. Therefore, the specific functions of the RAR isotypes do not manifest at the level of their DNA binding but rather from a differential ability to recruit specific components of the transcriptional machinery. We also demonstrated that RA-mediated displacement of the polycomb group protein SUZ12 from a RARE was inhibited in the absence of RARgamma. Thus, transcriptional components of the RAR signaling pathway are specifically required for displacement of SUZ12 from RAREs during RA-mediated differentiation of F9 cells.

Requirement of Retinoic Acid Receptor Isotypes α, β, and γ 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.

Synthesis and Biological Activity of Novel Retinamide and Retinoate Derivatives.

Retinoic acid and its amide derivative, N-(4-hydroxyphenyl)retinamide (4-HPR), have been proposed as chemopreventative and chemotherapeutic agents. However, their low cytotoxic activity and water solubility limit their clinical use. In this study, we synthesized novel retinoid derivatives with improved cytotoxicity against cancer cells and increased hygroscopicity. Our syntheses were preceded by selective O-acylation and N-acylation, which led to the production of retinoate and retinamide derivatives, respectively, in one pot directly from aminophenol derivatives and retinoic acid without protection. Transcription assays in COS-1 cells indicated that the N-acylated derivatives (2A-5A) and 4-HPR (1A) were much weaker ligands for all three subtypes of retinoic acid receptor (RAR) than all-trans retinoic acid (ATRA), although they showed some selectivity for RARbeta and RARgamma. In contrast, the O-acylated retinoate derivatives (1B-5B) activated all three RAR isotypes without specificity to an extent similar to ATRA. The cytotoxicity was determined using an MTT assay with HCT116 colon cancer cells, and the IC(50) of N-acylated retinamide derivative 4A and O-acylated retinoate derivative 5B was 1.67 microM and 0.65 microM, respectively, which are about five and 13-fold better than that of 4-HPR (8.21 microM), a prototype N-acylated derivative. When retinoate derivative 5B was coupled to organic acid salts, the resulting salt derivatives 5C and 5D had RAR activation and cytotoxicity similar to those of 5B. These data may delineate the relationship between the structure and function of retinoate and retinamide derivatives.

Synthesis and biological activity of novel retinamide and retinoate derivatives.

Retinoic acid and its amide derivative, N-(4-hydroxyphenyl)retinamide (4-HPR), have been proposed as chemopreventative and chemotherapeutic agents. However, their low cytotoxic activity and water solubility limit their clinical use. In this study, we synthesized novel retinoid derivatives with improved cytotoxicity against cancer cells and increased hygroscopicity. Our syntheses were preceded by selective O-acylation and N-acylation, which led to the production of retinoate and retinamide derivatives, respectively, in one pot directly from aminophenol derivatives and retinoic acid without protection. Transcription assays in COS-1 cells indicated that the N-acylated derivatives (2A-5A) and 4-HPR (1A) were much weaker ligands for all three subtypes of retinoic acid receptor (RAR) than all-trans retinoic acid (ATRA), although they showed some selectivity for RARbeta and RARgamma. In contrast, the O-acylated retinoate derivatives (1B-5B) activated all three RAR isotypes without specificity to an extent similar to ATRA. The cytotoxicity was determined using an MTT assay with HCT116 colon cancer cells, and the IC(50) of N-acylated retinamide derivative 4A and O-acylated retinoate derivative 5B was 1.67 microM and 0.65 microM, respectively, which are about five and 13-fold better than that of 4-HPR (8.21 microM), a prototype N-acylated derivative. When retinoate derivative 5B was coupled to organic acid salts, the resulting salt derivatives 5C and 5D had RAR activation and cytotoxicity similar to those of 5B. These data may delineate the relationship between the structure and function of retinoate and retinamide derivatives.

Retinoic acid receptor antagonism in vivo expands the numbers of precursor cells during granulopoiesis.

The role/s of retinoids in granulopoiesis has been recognised for many years, being powerful differentiation inducers. The physiological role/s of retinoic acid receptor (RAR)-mediated signalling during adult haemopoiesis has by contrast proved more elusive. The recent generation of highly specific pan-RAR antagonists has now made possible an assessment of the specific physiological role/s of RAR signalling, allowing the separation for the first time of the RAR and RXR pathways. Mice were treated with AGN194310, a synthetic retinoid that antagonises the physiological function of the three RAR isotypes (alpha, beta, gamma) but does not interact with RXRs. Analyses of the granulocytic lineage using Gr-1, c-Kit and CD11b antibodies, demonstrated that granulocyte numbers were strikingly increased across haemopoietic compartments in all AGN194310-treated mice. A significant increase in the frequency of progenitor cells containing granulocytes was observed in the bone marrow of mice following treatment with AGN194310. In contrast we were not able to detect any differences in cell death of either mature granulocytes or granulocytic progenitors from AGN194310-treated mice compared with control animals. These data demonstrate an essential role for RAR signalling in regulating the numbers of granulocytic precursors in vivo.

Retinoic acid receptor alpha1 variants, RARalpha1DeltaB and RARalpha1DeltaBC, define a new class of nuclear receptor isoforms.

Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (alpha, beta and gamma) are comprised of six regions designated A-F. Two isoforms of RARalpha, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RARalpha1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RARalpha1DeltaB, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RARalpha1DeltaBC, retains most of the functional domains of RARalpha1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RARalpha1DeltaBC interacts with RXRalpha and, as an RXRalpha/RARalpha1DeltaBC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXRalpha/RARalpha1 heterodimer is mediated only by RAR ligands, transactivation through the RXRalpha/RARalpha1DeltaBC heterodimer is mediated by RAR and RXR ligands. Whilst RARalpha1 has a broad tissue distribution, RARalpha1DeltaBC has a more heterogeneous distribution, but with significant expression in myeloid cells. RARalpha1DeltaBC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain.

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 RARβ, RXRα, RXRβ, and RXRγ. In addition, a previously unidentified mouse RXRβ isoform containing a 12-nucleotide insertion in exon 7 was detected. Furthermore,in vitrometabolic 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.

Specific and redundant functions of retinoid X Receptor/Retinoic acid receptor heterodimers in differentiation, proliferation, and apoptosis of F9 embryonal carcinoma cells.

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)alpha and retinoic acid receptor (RAR)alpha genes or the RXRalpha and RARgamma genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRalpha-/-, RARalpha-/-, and RARgamma-/- cells. RXRalpha-/-/ RARalpha-/- cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRalpha-/- cells. In contrast, RXRalpha-/-/RARgamma-/- cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRalpha and RARgamma represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype- and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.

Distinct retinoid X receptor-retinoic acid receptor heterodimers are differentially involved in the control of expression of retinoid target genes in F9 embryonal carcinoma cells.

The F9 murine embryonal carcinoma cell line represents a well-established system for the study of retinoid signaling in vivo. We have investigated the functional specificity of different retinoid X receptor (RXR)-retinoic acid (RA) receptor (RAR) isotype pairs for the control of expression of endogenous RA-responsive genes, by using wild-type (WT), RXR alpha(-/-), RAR alpha(-/-), RAR gamma(-/-), RXR alpha(-/-)-RAR alpha(-/-), and RXR alpha(-/-)-RAR gamma(-/-) F9 cells, as well as panRXR and RAR isotype (alpha, beta, and gamma)-selective retinoids. We show that in these cells the control of expression of different sets of RA-responsive genes is preferentially mediated by distinct RXR-RAR isotype combinations. Our data support the conclusion that RXR-RAR heterodimers are the functional units transducing the retinoid signal and indicate in addition that these heterodimers exert both specific and redundant functions on the expression of particular sets of RA-responsive genes. We also show that the presence of a given receptor isotype can hinder the activity of another isotype and therefore that functional redundancy between retinoid receptor isotypes can be artifactually generated by gene knockouts.