Retinoic acid receptor gamma: specific immunodetection and phosphorylation.

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma 1 (hRAR-gamma 1 and mRAR-gamma 1, respectively) were used to generate anti-RAR-gamma 1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1 gamma (A1)), region F (Ab2 gamma (mF) and Ab4 gamma (hF)) and region D2 (Ab5 gamma (D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma 1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma 1 cDNAs. They all reacted with both human and mouse RAR-gamma 1 proteins, except Ab4 gamma (hF) that was specific for hRAR-gamma 1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma 1 F region were also obtained (RP gamma (mF)) and found to be specific for mouse RAR-gamma 1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma 2 isoform which differs from RAR-gamma 1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma 1 (Ab1 gamma (A1)) only reacted with the RAR-gamma 1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma 1 and gamma 2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma 1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.

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.

Physical and functional interactions between cellular retinoic acid binding protein II and the retinoic acid-dependent nuclear complex.

Two sorts of proteins bind to, and mediate the developmental and homeostatic effects of, retinoic acid (RA): the RAR and RXR nuclear receptors, which act as ligand-dependent transcriptional regulators, and the cellular RA binding proteins (CRABPI and CRABPII). CRABPs are generally known to be implicated in the synthesis, degradation, and control of steady-state levels of RA, yet previous and recent data have indicated that they could play a role in the control of gene expression. Here we show for the first time that, both in vitro and in vivo, CRABPII is associated with RARalpha and RXRalpha in a ligand-independent manner in mammalian cells (HL-60, NB-4, and MCF-7). In the nucleus, this protein complex binds the RXR-RAR-specific response element of an RA target gene (RARE-DR5). Moreover, in the presence of retinoids that bind both the nuclear receptors and CRABPII, enhancement of transactivation by RXRalpha-RARalpha heterodimers is observed in the presence of CRABPII. Thus, CRABPII appears to be a novel transcriptional regulator involved in RA signaling.

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.

Expression of retinoid receptor genes and proteins in non-small-cell lung cancer.

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<.0001). Decreased expression of RARgamma was observed by RT-PCR in 41% of the tumors (P<.0001). LOH at 3p24 was observed in 41% of the tumor specimens from informative patients and in 20% of the non-neoplastic lesions. CONCLUSIONS: Expression of RARalpha and RXRalpha is either normal or elevated in NSCLC. In contrast, a large percentage of tumors show a marked decrease in the expression of RARbeta, RARgamma, and RXRbeta as well as a high frequency of LOH at 3p24, which was also observed in non-neoplastic lesions. These data suggest that altered retinoid receptor expression may play a role in lung carcinogenesis.

Modulation of HT-29 human colonic cancer cell differentiation with calmidazolium and 12-O-tetradecanoylphorbol-13-acetate.

The effects of a protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and of a calmodulin antagonist calmidazolium (CZ), on a human colonic cancer cell line HT-29 were analyzed. HT-29 cells are undifferentiated in standard culture conditions (HT-29 G+) and display an enterocytic differentiation when cultured in glucose-deprived medium (HT-29 G-). Early effects of TPA and CZ on the localization of cytoskeletal proteins (caldesmon, alpha-actinin and vinculin) and on cell proliferation were examined. Differentiation of the cells was assessed after 4 weeks on the basis of ultrastructural and functional characteristics of enterocytic polarity, presence of apical brush borders, expression of brush border membrane antigens (Caco 5/50 and sucrase-isomaltase), and segregation of calmodulin to the brush border cytoskeleton. TPA treatment of HT-29 G+ or G- cells induced early morphological and cytoskeletal alterations: the cells rounded up and lost their stress fibers with the associated caldesmon, alpha-actinin, and vinculin. TPA did not modify the differentiation of G- cells, but induced in G+ cells the expression, although limited, of enterocytic differentiation characteristics. Addition of CZ to HT-29 G- cells enhanced their differentiation state but did not provoke any early morphological or cytoskeletal alterations. No effects of CZ on HT-29 G+ cells were obvious. The results suggest that protein kinase C, the TPA receptor, is involved in the triggering of HT-29 G+ cell differentiation whereas calmodulin-dependent functions would be implicated in HT-29 G- cell maturation.

Distinct sensitivity of neuroblastoma cells for retinoid receptor agonists: evidence for functional receptor heterodimers.

Retinoic acid (RA) plays a major role in embryogenesis of the nervous system and has been reported to induce differentiation in neuroblastoma cell lines. To identify RA signaling pathways involved in such differentiation processes, two RA-sensitive neuroblastoma cell lines (LA-N-5 and SH-SY5Y) were extensively studied. Northern blot experiments determined that of the three RAR mRNAs, only RARalpha was significantly expressed, with respectively weak or undetectable levels of RARgamma and RARbeta. RXRs (alpha and beta) receptors were weakly expressed. Western blotting analysis confirmed the constitutive expression of RARalpha and absence of RARbeta and weak levels of RXRalpha. Treatment with all-trans-RA up-regulated RARalpha and induced a drastic increase of RARbeta (both at the RNA and protein level). To further characterize the function of RARalpha, RARbeta and RXRalpha in NB cells, nuclear extracts from LA-N-5 cells were analysed by EMSA studies. Three specific retarded complexes were observed which were significantly decreased or shifted in the presence of monoclonal antibodies to RARalpha, RARbeta and RXRalpha. RA treatment dramatically induced a DR5-binding RXRalpha-RARbeta heterodimer. Treatment with combinations of RARalpha or RARbeta agonists with a RXRalpha agonist or with a RARalpha agonist alone, induced neurite-outgrowth supporting the probability that both RXRalpha-RARalpha or RXRalpha-RARbeta heterodimers are involved in RA-mediated differentiation of NB cells. The availability of novel synthetic RA-specific receptor ligands should provide the possibility of tissue specific therapeutic regimes.

Implication of retinoic acid receptor gamma in squamous differentiation and response to retinoic acid in head and neck SqCC/Y1 squamous carcinoma cells.

Nuclear retinoic acid receptors are considered to be the mediators of most of the effects of retinoic acid (RA) on gene expression. To explore the role of RA receptor gamma (RARgamma) in the growth and differentiation of SqCC/Y1 head and neck squamous carcinoma cells, they were transfected with RARgamma sense and antisense expression vectors and stable clones in which RARgamma expression was either increased or blocked were isolated. The growth inhibitory effect of RA in monolayer culture was enhanced in the sense transfectants and decreased in the antisense ones. The ability to form colonies in semisolid medium was abolished by RA in the sense transfectants, while the antisense transfected clones exhibited heterogeneous responses. The expression the squamous differentiation markers cytokeratin K1 transglutaminase type I, and involucrin was increased in the absence of exogenous retinoid in a sense transfected clone and decreased in an antisense transfected clone. RA suppressed squamous differentiation in both types of transfectant. The expression of epidermal growth factor receptor (EGFR) was higher in the antisense and lower in the sense transfectant than in the parental cells and RA decreased EGFR mRNA level in the parental and the sense transfectant but not in the antisense transfectant. In addition activator protein-1 (AP-1) binding activity was decreased by the RA treatment in the sense clones, but not in the antisense ones. These results suggest that RARgamma mediates the effects of RA on the cell growth both in monolayer culture and in semisolid medium possibly through AP-1 suppression.

Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis.

The results reported here indicate that retinoic acid (RA) induces growth arrest and differentiation only in MyoD-expressing muscle cells. Transient transfection assays reveal a functional interaction between MyoD, a key myogenic regulator and RA-receptors, principal mediators of RA actions. Interestingly, we demonstrate that RXR-MyoD-containing complexes are recruited at specific MyoD DNA-binding sites in muscle cells. Furthermore, we also demonstrate that RA-receptors and the muscle basic helix-loop-helix (b-HLH) proteins interact physically. Mutational analysis suggests that this interaction occurs via the basic region of muscle b-HLH proteins and the DNA-binding domain of RA-receptors and is important for functional interactions between these two families of transcription factors. In conclusion, these results highlight novel interactions between two distinct groups of regulatory proteins that influence cell growth and differentiation.

RXR alpha is essential for mediating the all-trans retinoic acid-induced growth arrest of C2 myogenic cells.

In C2 muscle cells, retinoic acid (RA) induces growth arrest associated with terminal differentiation. These RA actions are presumed to be mediated through nuclear receptors (RARs and RXRs) that belong to the superfamily of ligand-dependent transcription factors. In this study, we have characterized a myogenic C2 subclone, that unlike parental cells, is resistant to growth inhibition and differentiation by RA. Examination of these RA-sensitive and resistant C2 cells for the expression of retinoid acid receptors revealed a lack of RXR alpha expression at the myoblast stage in resistant C2 cells. To determine the functions of RXR alpha, we introduced an RXR alpha expression vector into RA-resistant C2 cells by transient or stable transfections. Our results show that RXR alpha restores the response to RA in this subclone with respect to AP1 inhibition and growth arrest. These observations indicate that RXR alpha plays a crucial role in mediating RA induced growth arrest of C2 myogenic cells.

Evidence for a role of sulfhydryl groups in catalytic activity and subunit interaction of the cyclic GMP-dependent protein kinase from silkworm.

Guanosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from silkworm pupal fat body (Bombyx mori) which is devoid of any adenosine 3':5'-monophosphate-dependent protein kinase. The enzyme displayed catalytic properties which were roughly similar to those described for adenosine 3':5'-monophosphate-dependent protein kinase. This similarity has been found in substrate specificity, optimal Mg2+ dependency, polyamines effects and the lack of dependency upon sulfhydryl compound for activation by cyclic GMP. Treatment of the enzyme with sulfhydryl reagents, N-ethylmaleimide or p-chloromercuribenzoic acid, inhibited the catalytic activity as well as the dissociation of the binding and catalytic activities as shown by means of sucrose-density gradient ultracentrifugation. In the presence of cyclic GMP or histone, the disulfide-linked structure did not dissociate into separate subunits nor did it migrate as the holoenzyme but sedimented as an intermediate form carrying both binding and catalytic activities.

Potentiation of VD-induced monocytic leukemia cell differentiation by retinoids involves both RAR and RXR signaling pathways.

Retinoids and vitamin D (VD) cooperate to induce the differentiation and inhibit the proliferation of human myelomonocytic leukemia cells. Two classes of retinoids receptors, the RARs and RXRs, respectively, can mediate these effects. RXR forms heterodimers with a variety of nuclear receptors, including RAR and the VD receptor. We have previously found that VD treatment increases RXR alpha levels in myelomonocytic leukemia cells. By immunoanalysis, we observed in the present work that the RAR alpha protein is expressed in proliferating U937, HL-60 and THP-1 human leukemia cells and that VD treatment induces alterations of its electrophoretic pattern, although with large differences between cell lines. In the three cell lines, 9-cis RA, an agonist of both RARs and RXRs, cooperated with VD more efficiently than all-trans RA and RAR-specific synthetic ligands, thus suggesting an involvement of both RAR and RXR pathways in cell differentiation. Using U937 cells as a model, we delineated the relative contributions of RAR and RXR by assessing the effects of receptor-selective synthetic retinoids. The synergy between VD and all-trans RA or RAR-specific agonists (TTNPB and Ro 40-6055) was abrogated by a RAR alpha-specific antagonist (Ro 41-5253), confirming an involvement of RAR alpha. However, the cooperation between VD and 9-cis RA, although reduced, was not suppressed by the antagonist, suggesting also an involvement of the RXR pathway. The role of RXR as a ligand-activated receptor was confirmed using RXR-specific agonists (CD2608 and LGD1069), which also proved able to cooperate with VD. Finally, while each synthetic agonist alone was significantly less potent than 9-cis RA, combinations of the RAR and RXR selective agonists TTNPB and LGD1069 appeared to be as effective as the pan agonist 9-cis-RA. These results confirm that various retinoids can cooperate with VD and demonstrate that, at a whole cell level, optimal effects require the activation of both RAR and RXR receptors.

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.

Retinoic acid receptor-beta: immunodetection and phosphorylation on tyrosine residues.

Polyclonal (RP) and monoclonal (Ab) antibodies were raised against synthetic peptides (or fusion proteins) corresponding to amino acid sequences unique to human and mouse retinoic acid receptor-beta (RAR beta) isoforms. Antibodies directed against the A2 region [Ab6 beta 2(A2), Ab7 beta 2(A2), and RP beta 2(A2)], the D2 region [RP beta(D2)], or the F region [Ab8 beta(F)2, RP beta(F)1, and RP beta(F)2] were selected. The monoclonal and polyclonal antibodies directed against the D2 and F regions specifically immunoprecipitated and recognized by Western blotting all human and mouse RAR beta isoforms (mRAR beta 1, -beta 2, -beta 3, and -beta 4), produced in COS-1 cells transfected with expression vectors containing the corresponding RAR beta cDNA. Furthermore, in gel retardation assays, the monoclonal antibodies supershifted RAR beta protein-RA response element oligonucleotide complexes. Antibodies directed against the A2 region were specific for the RAR beta 2 isoform. The above antibodies allowed us to detect the presence of mRAR beta 2 proteins in mouse embryos and to show that their presence in embryonal carcinoma cells (F9 and P19 cell lines) is dependent upon RA treatment. The antibodies were also used to demonstrate that RAR beta proteins produced by transfection in COS-1 cells are phosphorylated. RAR beta 2 phosphorylation was not affected by RA treatment, whereas the phosphorylation of RAR beta 1 and RAR beta 3 isoforms was greatly enhanced by RA. We also show that, in contrast to RAR alpha 1 and RAR gamma 1, RAR beta 2 proteins contain phosphotyrosine residues and are only weakly phosphorylated in vitro by cAMP-dependent protein kinase. These results support our previous proposal that the various receptors have distinct functions in the RA-signaling pathway.

Phosphorylation of the retinoic acid receptor-alpha by protein kinase A.

The phosphorylation of retinoic acid receptor-alpha 1 (RAR alpha 1) by PKA was investigated both in vitro and in vivo. We show that bacterially expressed RAR alpha 1 is phosphorylated in vitro by protein kinase A (PKA) at the unique serine residue 369 located in the C-terminal end of the E region. We also show that RAR alpha 1 overexpressed in COS-1 cells is phosphorylated on multiple serine residues and that phosphorylation at serine 369 occurs only when COS-1 cells are cotransfected with PKA or treated with forskolin. RAR alpha 1 mutants were constructed in which serine 369 was replaced by an alanine (S369A) or a glutamic acid (S369E) residue. Comparison of the tryptic phosphopeptide patterns of wild type and mutated RAR alpha 1 overexpressed in COS-1 cells allowed us to confirm that serine 369 is the unique phosphorylation site for PKA in cultured cells. The DNA-binding efficiency of RAR alpha/retinoid X receptor-alpha (RXR alpha) heterodimers was enhanced in vitro by the S369E mutation. However, in transfected RAC65 cells, the same S369E mutation did not affect the ligand-dependent transcriptional activation by RAR alpha 1 of reporter genes containing a retinoic acid (RA)-response element. In contrast, the S369A mutation slightly decreased both DNA binding and the efficiency of PKA to enhance RA-induced transactivation by RAR alpha 1. Finally, we show that endogenous RAR alpha is also phosphorylated in vivo at serine 369 in forskolin-treated F9 cells, supporting the idea that phosphorylation of RARs at this site is involved in the modulation of the RA-induced differentiation of F9 cells by (Bu)2cAMP.

Role of the retinoic acid receptor beta (RARbeta) during mouse development.

Homozygous RAR beta mutants are growth-deficient, but are fertile and have a normal longevity. They display homeotic transformations and malformations of cervical vertebrae and a retrolenticular membrane. This latter abnormality arises from the persistence and hyperplasia of the primary vitreous body. In contrast, we found that abnormalities of cranial nerves IX and X which were previously proposed to be specific features of the RAR beta mutant phenotype (Luo et al., Mech. Dev. 53: 61-71, 1995) occur with the same low penetrance in wildtype littermates. Although the RAR beta protein is expressed at high levels in the striatum and interdigital mesenchyme, the brain and limbs of RAR beta mutants appear morphologically normal. RAR alpha/RAR beta double mutants display numerous visceral abnormalities, most of which are incompatible with post-natal life. The majority of these abnormalities was previously detected in RAR alpha/RAR beta2 mutants with the notable exceptions of agenesis of the stapedial (2nd aortic arch-derived) artery, thymic and spleen agenesis and abnormal inferior vena cava. RAR beta/RAR gamma double mutants show major ocular defects including a shortening of the ventral retina and pre-natal retinal dysplasia, both of which represent the only abnormalities of the fetal vitamin-A deficiency (VAD) syndrome not previously detected in RAR beta2/RAR gamma compound mutants. In addition, RAR beta is apparently functionally redundant with either RAR alpha or RAR gamma for the formation of a small subset of craniofacial skeletal elements, as well as for eyelid development and digit separation. We also provide evidence that, at least in some instances, this phenomenon of functional redundancy between RARs may be an artifactual consequence of gene knock-out.

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.

Retinoid receptors expression in human term placenta: involvement of RXR alpha in retinoid induced-hCG secretion.

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.

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.