p38αMAPK interacts with and inhibits RARα: suppression of the kinase enhances the therapeutic activity of retinoids in acute myeloid leukemia cells.

All-trans retinoic acid (ATRA) is the only clinically useful differentiating agent, being used in the treatment of acute promyelocytic leukemia (APL). The use of ATRA in other types of acute myelogenous leukemia (AML) calls for the identification of novel strategies aimed at increasing its therapeutic activity. Here, we provide evidence that pharmacological inhibition of the mitogen-activated protein kinase, p38α, or silencing of the corresponding gene sensitizes APL and AML cell lines, as well as primary cultures of AML blasts to the anti-proliferative and cyto-differentiating activity of ATRA and synthetic retinoids. P38α inhibits ligand-dependent transactivation of the nuclear retinoic acid receptor, RARα, and the derived chimeric protein expressed in the majority of APL cases, PML-RARα. Inhibition is the consequence of ligand-independent binding of p38α, which results in stabilization of RARα and PML-RARα via blockade of their constitutive degradation by the proteasome. The inhibitory effect requires a catalytically active p38α and direct physical interaction with RARα and PML-RARα. Ser-369 in the E-region of RARα is essential for the binding of p38α and the ensuing functional effects on the activity of the receptor.

A retinoic acid receptor RARα pool present in membrane lipid rafts forms complexes with G protein αQ to activate p38MAPK.

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.

New role for granulocyte colony-stimulating factor-induced extracellular signal-regulated kinase 1/2 in histone modification and retinoic acid receptor α recruitment to gene promoters: relevance to acute promyelocytic leukemia cell differentiation.

The induction of the granulocytic differentiation of leukemic cells by all-trans retinoic acid (RA) has been a major breakthrough in terms of survival for acute promyelocytic leukemia (APL) patients. Here we highlight the synergism and the underlying novel mechanism between RA and the granulocyte colony-stimulating factor (G-CSF) to restore differentiation of RA-refractory APL blasts. First, we show that in RA-refractory APL cells (UF-1 cell line), PML-RA receptor alpha (RARα) is not released from target promoters in response to RA, resulting in the maintenance of chromatin repression. Consequently, RARα cannot be recruited, and the RA target genes are not activated. We then deciphered how the combination of G-CSF and RA successfully restored the activation of RA target genes to levels achieved in RA-sensitive APL cells. We demonstrate that G-CSF restores RARα recruitment to target gene promoters through the activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the subsequent derepression of chromatin. Thus, combinatorial activation of cytokines and RARs potentiates transcriptional activity through epigenetic modifications induced by specific signaling pathways.

Dysregulation of elastin expression by fibroblasts in pulmonary emphysema: role of cellular retinoic acid binding protein 2.

BACKGROUND: All-trans retinoic acid (ATRA) stimulates elastin synthesis by lung fibroblasts and induces alveolar regeneration in animal models of pulmonary emphysema. However, ATRA treatment has had disappointing results in human emphysema. It was hypothesised that a defect in the ATRA signalling pathway contributes to the defect of alveolar repair in the human emphysematous lung. METHODS: Fibroblasts were cultured from the lung of 10 control subjects and eight patients with emphysema. Elastin and retinoic acid receptor (RAR)-beta mRNAs were measured in those cells in the presence of incremental concentrations of ATRA. RARs, retinoic X receptors (RXRs) and cellular retinoic acid binding protein (CRABP) 1 and 2 mRNAs were measured as well as CRABP2 protein content. The effect of CRABP2 silencing on elastin and RAR-beta expression in response to ATRA was measured in MRC5 lung fibroblasts. RESULTS: ATRA at 10(-9) M and 10(-8) M increased median elastin mRNA expression by 182% and 126% in control but not in emphysema fibroblasts. RAR-beta mRNA expression was induced by ATRA in control as well as emphysema fibroblasts. RARs, RXRs and CRABP1 mRNAs were similarly expressed in control and emphysema fibroblasts while CRABP2 mRNA and protein were lower in emphysema fibroblasts. CRABP2 silencing abrogated the induction of elastin but not RAR-beta expression by ATRA in MRC5 fibroblasts. CONCLUSION: Pulmonary emphysema fibroblasts fail to express elastin under ATRA stimulation. CRABP2, which is necessary for elastin induction by ATRA in MRC-5 cells, is expressed at low levels in emphysema fibroblasts. This alteration in the retinoic acid signalling pathway in lung fibroblasts may contribute to the defect of alveolar repair in human pulmonary emphysema. These results are the first demonstration of the involvement of CRABP2 in elastin expression.

The phosphoinositide 3-kinase/Akt pathway is essential for the retinoic acid-induced differentiation of F9 cells.

Retinoic acid (RA) induces cell growth arrest and differentiation through two families of nuclear receptors, the RARs and the RXRs. The phosphoinositide 3-kinase (PI3K)/Akt pathway also plays key roles in these processes, that is, cell cycle progression, cell differentiation and cell survival. We report that, in mouse embryocarcinoma cells (F9 cells), RA induces an early activation of PI3K and Akt via an increase in the expression of the p85alpha regulatory subunit. This effect is followed by an inhibition of Akt. Both effects require the integrity of the RA pathway as they are not observed in RA-resistant RARgamma null cells. We propose a model through which RA induces a biphasic regulation of Akt with an activation participating to the differentiation process, followed by an inhibition, which has been correlated to the RA-induced growth arrest.

Retinoic acid increases proliferation rate of GL-15 glioma cells, involving activation of STAT-3 transcription factor.

The molecular mechanisms underlying the heterogeneous effects of retinoic acid (RA) treatment on malignant glioma cells remain poorly understood. In this study, we present the first evidence of a functional role of the signal transduction factors (STATs) in RA-induced proliferation, in a human glioblastoma GL-15 cell line. We first observed that STAT-3 was constitutively activated and present in the GL-15 cell nuclei. We then showed that at low doses (0.01-1 microM) RA increased both the proliferation rate of GL-15 cells and the phosphotyrosine (PY) activation of STAT-3. This RA effect involved transcriptional processes and the transactivation of RA target genes, including RA receptors isoforms RARalpha2, -beta2, and -gamma2. At higher concentrations, however, RA (5-10 microM) inhibits GL-15 proliferation, induces apoptosis, and fails to activate STAT-3. An inhibitory effect on GL-15 proliferation was also observed with the synthetic retinoids CD-437 and CD-2325, two structurally related RARgamma agonists, which also fail to activate STAT-3. In addition, the phorbol ester PMA, an inducer of GL-15 differentiation, and staurosporine, a broad inhibitor of protein kinases, abrogate the stimulatory effects of RA at low concentrations. Together these observations suggest that, in GL-15 cells, activation of STAT-3 and cell proliferation share common mechanisms and that STAT transcription factors may be involved in a switch between proliferation, differentiation, and apoptosis. The proliferating effect observed at low doses of RA may be related to the failures in RA efficiency observed in clinical assays in relapsing malignant gliomas. Combining specific inhibitors of tyrosine kinases with RA might optimize the clinical outcome.

The novel co-activator CRABPII binds to RARalpha and RXRalpha via two nuclear receptor interacting domains and does not require the AF-2 'core'.

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.

PPARgamma/RXRalpha heterodimers control human trophoblast invasion.

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.

PPAR gamma/RXR alpha heterodimers are involved in human CG beta synthesis and human trophoblast differentiation.

Recent studies performed with null mice suggested a role of either RXR alpha or PPAR gamma in murine placental development. We report here that both PPAR gamma and RXR alpha are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPAR gamma (but not for PPAR alpha or PPAR delta) increase both human CG beta transcript levels and the secretion of human CG and its free beta-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPAR gamma 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, PPAR gamma/RXR alpha heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CG beta gene (beta 5) were found to bind RXR alpha and PPAR gamma from human cytotrophoblast nuclear extracts, suggesting that PPAR gamma/RXR alpha heterodimers directly regulate human CG beta transcription. Altogether, these data show that PPAR gamma/RXR alpha heterodimers play an important role in human placental development.

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

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

Effects of a novel synthetic retinoid on malignant glioma in vitro: inhibition of cell proliferation, induction of apoptosis and differentiation.

Among six synthetic retinoids tested, the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) was highly efficient in inducing growth inhibition of 8MG-BA and GL-15 human glioblastoma cell lines, with growth arrest at the S phase of the cell cycle. CD 437 also induced apoptosis in these cells, with 8MG-BA being the most sensitive. In these cells, induction of apoptosis by CD437 has been related to the downregulation of Bcl-2 expression and to CPP32 activation, but not to p53 expression. The remaining non-apoptotic cells presented a morphological pattern of astroglial differentiation with overexpression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). The mechanism of action of CD437, originally developed as a RARgamma agonist, is not yet elucidated. However, our results suggest that it acts through an increase of the expression of retinoid-inducible genes, such as RARbeta2 and/or RARalpha2.

Expression of RARalpha and RARbeta in human oral potentially malignant and neoplastic lesions.

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 RARalpha and RARbeta 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 RARalpha protein, while 10 cases did not show detectable RARbeta immunoreactivity. Immunostaining for RARalpha protein was observed in 12/31 (39%) hyperplastic lesions, 6/14 (43%) dysplastic lesions and 43/64 (67%) oral SCCs. Expression of RARalpha 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 RARalpha 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 RARbeta 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).

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.

The AF-1 and AF-2 activating domains of retinoic acid receptor-alpha (RARalpha) and their phosphorylation are differentially involved in parietal endodermal differentiation of F9 cells and retinoid-induced expression of target genes.

Retinoic acid (RA) induces the differentiation of F9 cells cultured as monolayers into primitive endodermal-like cells, whereas a combination of RA and cAMP leads to parietal endodermal differentiation. In RA receptor alpha-null F9 cells (RARalpha-/- cells), RA still efficiently triggers RARgamma-mediated primitive endodermal differentiation, but parietal endodermal differentiation is markedly delayed. To investigate the role of RARalpha1 activation functions AF-1 and AF-2 and of their phosphorylation sites during RA- and cAMP-induced parietal differentiation, cell lines reexpressing WT or mutated RARalpha1 were established in RARalpha-/- cells. We have found that the protein kinase A (PKA) phosphorylation site and the AF-2AD core (helix 12) of RARalpha1 are required for efficient parietal endodermal differentiation, whereas the AF-1 proline-directed kinase phosphorylation site is dispensible. Interestingly, deletion of the AF-1 activating domain (the A/B region), but not of the AF-2AD core, generates a dominant negative mutant that abrogates primitive endodermal differentiation when expressed in RARalpha-/- cells. We also show that the RARalpha AF-1 and AF-2 activation functions, but not their phosphorylation sites, are involved in the induction of RA-responsive genes in a differential promoter context-dependent manner.

The expression of nuclear retinoid receptors in human implantation.

Vitamin A and retinoids play an important role during development. They affect morphogenesis, cell growth and differentiation by interacting with two types of receptor, the RARs and the RXRs. Despite the well known established teratogenic effects of retinoids during human pregnancy, little is known about the effect of retinoids on human placental development. We studied the possible involvement of retinoids during the implantation process by investigating the spatial distribution of retinoid receptors in the human implantation site by in situ hybridization and immunohistochemistry. For in situ hybridization, we used digoxigenin-labelled antisense riboprobes. Immunochemical staining was performed with specific antibodies against the various retinoid receptors and a streptavidin-alkaline phosphatase immunostaining kit. We found that only two types of receptors were expressed at the implantation site: RARalpha and RXRalpha. Both types of receptors were present in the proliferative intermediate trophoblast, the invasive extravillous trophoblast and decidual cells. Both receptors were also present in the villous cytotrophoblasts. The presence of this retinoid receptor in the cytotrophoblasts suggests a key role for all-trans retinoic acid and/or 9-cis retinoic acid in the development of human placenta.

Retinoids stimulate leptin synthesis and secretion in human syncytiotrophoblast.

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 alpha and retinoid X receptor alpha (RXRalpha) 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 expression and secretion. In contrast, all-trans-RA and a RA alpha-specific ligand had no effect. These results suggest that retinoids regulate leptin expression and highlight a role for RXRalpha in this process.

The conserved amphipatic alpha-helical core motif of RARgamma and RARalpha activating domains is indispensable for RA-induced differentiation of F9 cells.

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.

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

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

TFIIH interacts with the retinoic acid receptor gamma and phosphorylates its AF-1-activating domain through cdk7.

Retinoic acid receptor gamma (RARgamma) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARgamma1 and serines 66 and 68 in RARgamma2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7.cyclin H. MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARgamma is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARgamma phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARgamma transcriptional activity in a response gene-dependent manner.