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Endocrinology Vol. 142, No. 10 4504-4514
Copyright © 2001 by The Endocrine Society

ARTICLES

PPAR{gamma}/RXR{alpha} Heterodimers Are Involved in Human CGß Synthesis and Human Trophoblast Differentiation

Anne Tarrade, Kristina Schoonjans, Jean Guibourdenche, Jean Michel Bidart, Michel Vidaud, Johan Auwerx, Cécile Rochette-Egly and Danièle Evain-Brion

Unité Institut National de la Santé et de la Recherche Médicale 427 (A.T., J.G., D.E.-B.), Faculté des Sciences Pharmaceutiques et Biologiques de Paris, Université René Descartes, Paris V, 75006 Paris, France; Institut de Génétique et Biologie Moléculaire et Cellulaire (K.S., J.A., C.R.-E.), Université Louis Pasteur, BP163, 67404 Illkirch cedex, France; CNRS UPRES-A 8067 (J.M.B.), Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, France; and Laboratoire de Génétique Moléculaire (M.V.), Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris V, 75006 Paris, France

Address all correspondence and requests for reprints to: Danièle Evain-Brion, Institut National de la Santé et de la Recherche Médicale Unité 427, Faculté des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l’Observatoire, 75006 Paris, France. E-mail: evain{at}pharmacie.univ-paris5.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 CGß transcript levels and the secretion of human CG and its free ß-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 CGß gene (ß5) were found to bind RXR{alpha} and PPAR{gamma} from human cytotrophoblast nuclear extracts, suggesting that PPAR{gamma}/RXR{alpha} heterodimers directly regulate human CGß transcription. Altogether, these data show that PPAR{gamma}/RXR{alpha} heterodimers play an important role in human placental development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN PLACENTA is hemomonochorial. This type of placentation is specific to humans and is characterized by intense trophoblast invasion (1) and hormone secretion (2). The structural and functional units of the human placenta are the chorionic villi (3), which include a mesenchymal core and a layer of cytosyncytiotrophoblast bathing directly in maternal blood in the intervillous space. The syncytiotrophoblast arises from cytotrophoblast fusion (4, 5) and is the endocrine unit of the human placenta, producing large amounts of steroid and protein hormones such as human CG (hCG) (6), human placental lactogen (hPL) (7), human placental GH (hPGH) (8), and leptin (9, 10). HCG plays a key role. In humans, maintenance of pregnancy during the first trimester depends on the synthesis of this glycoprotein-hormone, which prevents regression of the corpus luteum (11). In addition, hCG is directly involved in stimulating cytotrophoblast differentiation into the syncytiotrophoblast, in an autocrine process (trophoblast cells express functional receptors for hCG) (12, 13).

hCG is a heterodimer containing an {alpha}-subunit (hCG{alpha}) common to several glycoprotein hormones, and a distinct ß-subunit (hCGß) responsible for the biological specificity of the hormone. While the {alpha}-subunit is encoded by a single gene located on chromosome 6 (14), there are six CGß subunit genes, located on chromosome 19q13.3. Four of these genes encode the ß-subunit of hCG (ß8, ß5, ß3, and ß7), and CGß 5 is expressed predominantly in placental and choriocarcinoma cells (15, 16). In the trophoblast, the {alpha}-subunit is expressed in relative excess compared with the ß-subunit, and hormone synthesis is thus limited by the transcription rate of the ß-subunit (17, 18).

Experiments in RXR{alpha} null mice have recently pointed out the key role of RA in placentation (19, 20). Furthermore, in vitro studies performed with human syncytiotrophoblasts demonstrated that the secretion of two pregnancy-specific hormones—hPL (21) and hCG (22)—is stimulated by retinoids. Retinoids act at the cellular level via two families of nuclear receptors: RARs (RAR{alpha}, ß, and {gamma}) activated by both all-trans and 9-cis-RA, and RXRs (RXR{alpha}, ß, and {gamma}) activated exclusively by 9-cis-RA. These receptors function as ligand-activated transcription factors and regulate gene expression by binding as heterodimers to DNA response elements present in the regulatory sequences of their target genes (23, 24, 25, 26, 27). The high levels of two retinoid receptors—RAR{alpha} and RXR{alpha}—in human placenta, demonstrated by in situ hybridization and immunohistochemistry experiments, further supports a role for RA in placentation (28, 29).

Interestingly, recent data show that PPAR{gamma} is also important for placental development, as PPAR{gamma}-deficient mice develop severe placental abnormalities (30), similar to those observed in RXR{alpha}-deficient mice. PPAR{gamma} is also a member of the nuclear hormone receptor superfamily and acts as a ligand-inducible transcription factor (31, 32, 33, 34). PPAR{gamma} forms heterodimers with RXRs, which then bind to PPAR-responsive elements (PPRE) within the promoters of PPAR{gamma} target genes (35). While RXRs have been reported to be nonpermissive partners in RAR/RXR heterodimers (36), RXRs are permissive partners in most other cases (37). This is the case of PPAR/RXR heterodimers. Furthermore, RXR-specific ligands share several activities typical of PPAR{gamma} activation (e.g. their capacity to induce adipocyte and macrophage differentiation), and often work in synergistic fashion with PPAR{gamma} ligands (38, 39, 40).

The aim of the present study was to determine whether PPAR{gamma}/RXR{alpha} heterodimers play a key role in human trophoblast function. For this purpose we used the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. Our results demonstrate that both PPAR{gamma} and RXR{alpha} are expressed in human cytotrophoblastic cells and that their cognate ligands stimulate, independently and additively, the synthesis of hCG, the key hormone of human pregnancy. We also examined whether PPAR{gamma}/RXR{alpha} heterodimers directly regulate expression of the hCGß gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthetic ligands
The RAR{alpha}-selective (BMS753) and pan-RXR-selective (BMS649) agonists were gifts from Professor P. Chambon (IGBMC, Illkirch, France). Fenofibric acid was a gift from Dr. Edgar (Laboratoires Fournier, Dijon, France). Rosiglitazone (BRL 49653) was a gift from Dr. Leibowitz (Ligand, San Diego, CA). L-165041 was a gift from Dr. Moller (Merck, Rahway).

Trophoblastic cell culture
Villous tissue was dissected free of membranes and vessels from placentas obtained near term by elective Cesarean section from healthy mothers with uncomplicated pregnancies. After rinsing and mincing in Ca2+-, Mg2+-free PBS, cytotrophoblastic cells were isolated by trypsin-DNase digestion and discontinuous Percoll gradient fractionation, as previously described (4, 5). Cytotrophoblastic cells were plated in triplicate in 60-mm culture dishes (3 x 106 cells/dish) in 3 ml of DMEM, incubated at 37 C in humidified 5% CO2/95% air, and allowed to aggregate, fuse and form syncytia. Twenty-four hours later the cells were treated with the synthetic ligands BMS649 (0.1 µM), BMS753 (0.1 µM), fenofibric acid (1 mM), L-165041 (0.1 µM) and rosiglitazone (BRL 49653, 0.1 µM) dissolved in DMSO. At the concentrations used, these compounds did not affect cell viability (as tested by trypan blue exclusion) or cell morphology. Control cultures were treated with the same volume of solvent, ethanol or DMSO (1 part per 1000).

Hormone assays
The hCG concentration was determined in culture media by using the chemiluminescence immunoassay system ACS-180SE (Bayer Diagnostics) with a detection limit of 2 mU/ml. Free ß-subunits of hCG were measured using a specific RIA as previously described (41). All values are reported as means ± SEM of triplicate determinations. Data were analyzed for variance with the Bonferroni test. Differences were considered significant at P < 0.05.

Immunocytochemical and immunohistochemical staining
Cells were cultured on sterile glass slides for 72 h, fixed for 20 min in 4% paraformaldehyde acid, and permeabilized for 30 min in 0.3% Triton X-100. After preincubation with 7% sheep serum, monoclonal antibodies against RXR{alpha} (4RX3A2 (42), diluted 1/500) and PPAR{gamma} (E-8, Santa Cruz Biotechnology, Inc., diluted 1/100) were applied overnight at 4 C. Bound antibodies were revealed after 1 h with a biotinylated antimouse antibody (Amersham Pharmacia Biotech, Les Ulis, France, diluted 1/200), followed by 45 min of incubation at room temperature in the dark with a streptavidin-fluorescein complex (Amersham Pharmacia Biotech, diluted 1/500). In all experiments cells were extensively washed with PBS containing 1% Tween 20 between each step. Finally, slides were coverslipped in a drop of fluorescent Dapimounting medium (Vector Laboratories, Inc., Burlingame, CA) and analyzed under an epifluorescence microscope. To ensure the specificity of the immunological reactions, negative controls were run by substituting the primary antibodies with nonimmune mouse serum.

For immunohistochemical staining, tissue was embedded in Tissue Tek, frozen in isopentane, and kept at -80 C. Tissue sections (10 µm) were cut, mounted on Superfrost Plus slides and fixed in 4% paraformaldehyde acid and 0.3% Triton X-100 for 30 min. Sections were processed as described above with monoclonal antibodies against PPAR{gamma} and a fluorescein-coupled antimouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, diluted 1/200).

RNA and protein analysis
Total RNA was prepared according to Chomczynski and Sacchi (43) and analyzed by Northern blotting as previously described (44) with a random-primed labeled hamster PPAR{gamma} probe (45). A human acidic ribosomal phosphoprotein 36B4 cDNA clone was used as control.

Real-time PCR (7700 Prism, Perkin-Elmer Corp. Biosystems, Foster City, CA) was performed as previously described (46, 47). The nucleotide sequences of the amplification primers and fluorescent probes are listed in Table 1Go. Each sample was analyzed in duplicate and a calibration curve was run in parallel in each analysis. To control for sample-to-sample differences in RNA concentration and quality, transcripts were normalized to the constitutive housekeeping gene pleiotropin (PO). All values are means ± SEM of triplicate determinations. Data were analyzed for variance with the Bonferroni test. Differences were considered significant at P < 0.05.


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  		Table 1. Probes and primers

 
Total protein extracts were obtained by tissue homogenization in extraction buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a standard protease-inhibitor cocktail. Proteins (50 µg) were resolved by SDS-PAGE (48) and revealed by immunoblotting and chemiluminescence according to the manufacturer’s protocol (electrochemical luminescence, Amersham Pharmacia Biotech).

Oil Red O staining of cytotrophoblasts
Cultured trophoblasts were washed in PBS, fixed in 60% isopropanol for 1 min, and incubated with 0.3% Oil Red O (Sigma) in isopropanol (wt/vol) for 10 min at room temperature. Then, after 30 sec incubation in 60% isopropanol, cells were washed in water and the nuclei were counterstained in hematoxylin for 2 min.

EMSA
EMSA was done as described in (49, 50). Recombinant PPAR{gamma} and RXR{alpha} proteins were obtained by in vitro transcription and translation using the TNT-coupled reticulocyte lysate system (T7 Quick, Promega Corp.) and the pSG5-PPAR{gamma} and pSG5-RXR{alpha} expression vectors (48, 51). Recombinant proteins or aliquots (1 µg) of cytotrophoblastic nuclear extracts (52) were mixed with an appropriate amount of poly(dI-dC) and incubated in a 20-µl reaction volume containing 40 mM Tris HCl pH 7.9, 200 mM KCl, 20 mM MgCl2, 0.4 mM EDTA, 4 mM DTT, and 4% glycerol. The DNA template (10–20 fmol, 100 x 103 cpm, of [32P]-labeled oligonucleotides, see Table 2Go) was then added and incubation was continued for 10 min at 4 C. Where indicated, 1–2 µg of monoclonal antibodies against either RXR{alpha} (4RX1D12) (42) or PPAR{gamma} 1,2 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) were preincubated with the extracts for 15 min before oligonucleotide addition. DNA-protein complexes were then separated on 5% nondenaturing polyacrylamide gel in 0.5x standard TBE buffer and detected by autoradiography.


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  		Table 2.

 
Transient transfection and luciferase assays
We used the J3-tk-Luciferase reporter construct containing three copies of the apoA-II PPRE described in (53). The (RE5)3-tk-Luc and (RE5m)3-tk-Luc heterologous reporters were generated by subcloning the double-stranded cassettes containing three copies of the responsive element RE5 (see Table 2Go): either wild-type (5'-cta gcG AAG GGT TAG TGT CGA GCT CAC CCG AAG GGT TAG TGT CGA GCT CAC CCG AAG GGT TAG TGT CGA GCT CAC Cc-3') or mutated (5'-cta gcG AAG GGT TAT TTT TTA GCT CAC CCG AAG GGT TAT TTT TTA GCT CAC CCG AAG GGT TAT TTT TTA GCT CAC Cc-3'), containing each 5' NheI- and 3' XhoI-precut sites, into the TK-pGL3 vector. All constructs were sequenced before use. In addition to the expression vectors and reporter, all transfections included the pCMX-ß-galactosidase expression vector (54) to correct for variations in transfection efficiency.

HepG2 and JEG3 cells were cultured in DMEM supplemented with 10% FCS and transiently transfected by the calcium phosphate precipitation procedure after changing to fresh medium containing 10% delipidated calf serum. After 8–16 h, cells were incubated for another 20 h with vehicle (DMSO) or ligands. Cells were harvested 48 h after transfection, and luciferase assays were performed as previously described (55).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PPAR{gamma} coexpresses with RXR{alpha} in human trophoblastic cells
In view of recent data describing a role of PPAR{gamma} in mouse placentation (30, 56), we first examined the expression of this nuclear receptor by immunohistochemistry in term human placentas. As shown in Fig. 1Go, PPAR{gamma} was strongly expressed in the nuclei of the syncytiotrophoblast forming the outer layer of the villi. PPAR{gamma} expression was restricted to the cytotrophoblast and syncytiotrophoblast cells, and was absent from the mesenchymal core of the chorionic villi. Interestingly, RXR{alpha} antibodies gave a similar immunostaining pattern (28), suggesting that PPAR{gamma} may be involved in human placentation as an heterodimerization partner of RXR{alpha}.



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  		Figure 1. Immunohistological localization of PPAR{gamma} in sections of term chorionic villi. Specific immunostaining of PPAR{gamma} was observed in the nuclei of the syncytiotrophoblast layer (left lower panel). PPAR{gamma} staining colocalized with DAPI staining (right panel) (bar, 50 µm). is, Intervillous space; m: mesenchyme; st: syncytiotrophoblast.

 
PPAR{gamma} antibodies also yielded pronounced nuclear staining in purified villous cytotrophoblasts isolated from normal term placentas (data not shown). When plated on culture dishes, these mononucleated cytotrophoblasts aggregate and fuse to form a syncytiotrophoblast within 72 h (4, 5, 57), as shown by a gathering of nuclei and a large cytoplasmic mass (Fig. 2AGo). PPAR{gamma} was also detected in multinucleated syncytiotrophoblasts (Fig. 2BGo) and colocalized with DAPI staining. Likewise, RXR{alpha} was also detected both in cytotrophoblasts (data not shown) and in syncytiotrophoblasts (Fig. 2CGo), confirming our previous report (58). Therefore, both PPAR{gamma} and RXR{alpha} are expressed during human cytotrophoblast differentiation into the syncytiotrophoblast.



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  		Figure 2. A, In vitro differentiation of human villous cytotrophoblasts. Villous cytotrophoblasts, which are mononuclear just after plating (bar, 20 µm), aggregate and fuse after 48 h (bar, 20 µm) to form a giant multinucleated syncytiotrophoblast at 72 h (bar, 50 µm). B, Immunolocalization of PPAR{gamma} in the nuclei of term trophoblastic cells after 72 h of culture. Positive cells show specific nuclear staining (lower panel), which colocalizes with DAPI counterstaining. The upper panels correspond to controls incubated with nonimmune serum (bar, 50 µm). C, Immunolocalization of RXR{alpha} in the nuclei of term trophoblast cells after 72 h of culture, as in (A) (bar, 50 µm).

 
The presence of PPAR{gamma} in cytotrophoblasts and syncytiotrophoblasts was also demonstrated by immunoblotting. Indeed, a 57-kDa protein, corresponding to PPAR{gamma}, was revealed in extracts from both cytotrophoblasts and syncytiotrophoblasts (Fig. 3AGo, lanes 2 and 3). However, PPAR{gamma} expression was not affected during trophoblast differentiation, as cytotrophoblasts and syncytiotrophoblasts expressed similar amounts of the receptor. Cytotrophoblasts and syncytiotrophoblasts also express RXR{alpha} (59, 60). Finally, we also analyzed PPAR{gamma} mRNA expression in cytotrophoblasts and cultured syncytiotrophoblasts. In keeping with the results obtained by immunoblotting and immunocytochemistry, PPAR{gamma} mRNA was strongly expressed in the cytotrophoblast and syncytiotrophoblast (Fig. 3BGo, lanes 4 and 5). It is interesting to note that PPAR{gamma} mRNA levels were higher in these cells than in differentiated 3T3-L1 cells (Fig. 3BGo, lanes 2 and 3), but somewhat lower than in white adipose tissue (Fig. 3BGo, lane 1). However, PPAR{gamma} mRNA expression was not altered during cytotrophoblast differentiation into syncytiotrophoblast, as demonstrated by Northern blotting (Fig. 3BGo) and confirmed by real-time RT-PCR (data not shown). Similar observations have been made for RXR{alpha} (60) and were confirmed in this study by real-time RT-PCR (data not shown).



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  		Figure 3. Detection of PPAR{gamma} protein and transcripts in human cytotrophoblasts. A, Immunoblot analysis of PPAR{gamma} expression in term cytotrophoblasts (lane 2) and syncytiotrophoblasts after 72 h of culture (lane 3). Extracts (10 µg) were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with a specific polyclonal antihuman-PPAR{gamma} antibody. Lane 1 corresponds to 3T3-L1 adipocyte extracts run as controls. B, Northern blot analysis of PPAR{gamma} mRNA (20 µg) in cytotrophoblasts and syncytiotrophoblasts after 72 h of culture (lanes 4 and 5). The membrane was incubated with a radioactive PPAR{gamma} cDNA and autoradiographed for 24 h. Lanes 1–3 correspond to positive controls, namely white adipose tissue (lane 1) and 3T3-L1 adipocytes (lanes 2 and 3).

 
Interestingly, staining of cultured cells with oil red O, which detects triglycerides, unsaturated cholesterids and unsaturated FFA (61), revealed more pronounced accumulation of these lipids in the syncytiotrophoblast than in the cytotrophoblast (Fig. 4Go), corroborating the role of PPAR{gamma} in the modulation of trophoblast differentiation.



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  		Figure 4. Oil Red O staining of cytotrophoblasts during in vitro differentiation. The unsaturated fatty acids and triglycerides are not detected in villous cytotrophoblasts (A). The syncytiotrophoblast starts to accumulate lipids droplets during differentiation, after 24 and 48 h of culture (B and C, respectively). At 72 h (D), large amounts of unsaturated fatty acids and triglycerides are detected in the cytoplasm of the multinucleated syncytiotrophoblast (bar, 20 µm).

 
PPAR{gamma} agonists increase hCG production in differentiating cytotrophoblasts
The morphological differentiation of cytotrophoblasts into syncytiotrophoblasts, which takes about 72 h, is associated with a gradual increase in the levels of hCG and of its free ß-subunit in the culture medium (4, 13, 62, 63). During this process, basal levels of hCG and its free ß-subunit increased from undetectable to 200 ± 12 ng/ml and 2 ± 0.2 ng/ml, respectively.

As PPAR{gamma} is expressed in human trophoblasts, we sought to determine whether its activation alters hCG production and cytotrophoblast differentiation into syncytiotrophoblast. Incubation of differentiating cytotrophoblasts with a PPAR{gamma}-specific ligand (rosiglitazone) induced a clear increase in the secretion of hCG (P < 0.05; Fig. 5AGo) and of its free ß-subunit (P < 0.05; Fig. 5BGo). This was associated with a 2-fold increase in hCGß transcript levels (Fig. 5CGo). Similarly, a pan-RXR gonist (BMS649) (64) also induced a clear increase in the secretion of both hCG and its free ß-subunit (Figs. 5Go, A and B, and 6, A and B). Combined treatment with the PPAR{gamma} and the pan-RXR agonists further increased hCGß secretion and transcript levels (Fig. 5Go, A–C). In contrast, ligands for PPAR{alpha} (fenofibrate) and PPAR{delta} (L-165,041) did not affect hCGß production, and their combination with the pan-RXR agonist did not further increase hCGß production (Fig. 5Go, D–F). Similarly, a synthetic retinoid specific for RAR{alpha} (BMS753) (64) had no effect (Fig. 6Go) and did not cooperate with pan-RXR to further increase hCGß production (Fig. 6Go). Altogether, these results suggest that PPAR{gamma}/RXR{alpha} heterodimers are preferentially involved in the modulation of hCGß synthesis.



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  		Figure 5. The PPAR{gamma} ligand, but not the PPAR{alpha} and PPAR{delta} ligands, increase the secretion of hCG (A and D) and its free ß-subunit (B and E), as well as CGß transcript levels (C and F). Human trophoblasts were cultured for 72 h in the absence (control) or presence of a PPAR{gamma} ligand (BRL 49653, 0.1 µM), a PPAR{alpha} ligand (fenofibric acid, 1 µM) or a PPAR{delta} ligand (L-165,041, 0.1 µM), either individually or in combination with the pan-RXR agonist (BMS649, 1 µM). The data are expressed as a percentage of control values (arbitrarily 100%). The results presented here correspond to the means of three different experiments. In each experiment, hCG and free ßhCG secretion were measured in the conditioned media of each culture dish (n = 3). For transcript determination, the three culture dishes were pooled and the assay was run in duplicate. The mean ± SEM of control values were, respectively, hCG: 556 ± 53; 5149 ± 434; 228 ± 18 ng/ml; free hCGß: 6 ± 0.5; 20 ± 1; 3 ± 0.5 ng/ml; hCG ßmRNA/Po: 13 ± 2; 12.6 ± 1.7; 14 ± 1.3. *, P < 0.05, significantly different from control.

 


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  		Figure 6. The pan-RXR agonist (BMS649), but not the RAR{alpha}{alpha} agonist (BMS753), increases the secretion of hCG, its free ß-subunit, and hCGß transcripts in human trophoblast cells. Human trophoblasts were cultured in the absence (control) or presence of the synthetic retinoid BMS753 (RAR{alpha} agonist, 0.1 µM) or BMS649 (pan-RXR agonist, 0.1 µM) either individually or in combination, for 72 h. The data are expressed as a percentage of control values (arbitrarily 100%). The results presented here correspond to the mean of three different experiments. A, hCG secretion (ng/ml) in the culture medium. B, Secretion of the free ß-subunit of hCG (ng/ml) in the culture medium. C, hCGß transcripts. Data are normalized according to pleiotropin (PO). The mean ± SEM of control values were, respectively, hCG 1332 ± 377; 1234 ± 81; 2023 ± 191 ng/ml; hCGß: 14 ± 4; 13 ± 5; 19 ± 0.2 ng/ml; hCGßmRNA/Po: 11.6 ± 1.2; 13 ± 1.4; 14 ± 1.3. *, P < 0.05, significantly different from control.

 
In vitro differentiation of cytotrophoblasts into syncytiotrophoblasts is not only associated with increased expression of hCG but also with increased expression of other hormones such as hPL (65), hPGH (8), and leptin (58). Therefore, the expression and secretion of these hormones were also studied after treatment with the PPAR{gamma} and pan-RXR agonists. Both individually and in combination, these ligands also stimulated the expression of hPL, hPGH, and leptin (Fig. 7).

PPAR{gamma}/RXR{alpha} heterodimers bind to responsive elements located in the hCGß promoter
The observation that the PPAR{gamma} and pan-RXR agonists increased not only hCG secretion but also hCGß mRNA levels suggested that hCGß expression might be transcriptionally controlled by PPAR{gamma}/RXR heterodimers. PPAR/RXR heterodimers bind to PPRE, which are composed of a direct repeat (DR) of the hexamer half-sites AGGTCA spaced by one nucleotide (DR1), preferentially an adenine (30). Accordingly, PPAR{gamma}/RXR{alpha} heterodimers bound efficiently to the acyl-CoA oxidase PPRE element in electromobility shift assays (Fig. 8AGo, lane 1). Extracts from cytotrophoblasts also bound to this ACO PPRE element (Fig. 8AGo, lane 2). This binding was specific, as the complex was supershifted with RXR{alpha} and PPAR{gamma} antibodies (Fig. 8AGo, lanes 3 and 4). Thus, in human cytotrophoblasts, PPAR{gamma}/RXR{alpha} heterodimers are able to bind PPREs.



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  		Figure 8. Binding of PPAR{gamma}/RXR{alpha} heterodimers to elements of the hCGß gene regulatory region. A, EMSA using a [32P]-labeled acyl-CoA oxidase PPRE element (ACO PPRE) probe and either recombinant PPAR{gamma} and RXR{alpha} proteins translated in vitro (lane 1) or extracts (1 µg) from cytotrophoblasts (lanes 2–4). The complexes were resolved on 5% polyacrylamide gels and revealed by autoradiography (arrows). In lanes 3 and 4, the complexes were incubated with anti-RXR{alpha} or anti PPAR{gamma} antibodies. B, EMSA using recombinant PPAR{gamma} and RXR{alpha} proteins translated in vitro and the [32P]-labeled probes corresponding to the sequences described in Table 2Go. C, EMSA using a [32P]-labeled RE5 probe and either recombinant PPAR{gamma} and RXR{alpha} proteins translated in vitro (lane 1) or extracts (1 µg) from cytotrophoblasts (lanes 2–5). When indicated, the complexes were incubated with a 10- to 20-fold excess of unlabeled RE5 (lanes 2 and 3) ACO PPRE (lanes 4 and 5). D, The [32P]-labeled RE5 probe was incubated with cytotrophoblast extracts in the absence or presence of antibodies against RXR{alpha} or PPAR{gamma}.

 
Only part of the regulatory region of the hCGß gene has been cloned and sequenced (66). Computer homology searches revealed six potential response elements (RE1 to RE6) for RXR heterodimers (see Table 2Go). Two of these response elements are direct repeats of the consensus AGGTCA motifs spaced by 3 (RE1) and 5 (RE2) nucleotides. The other motifs (RE3 to RE6) consist of imperfect DR1s. All these potential responsive elements were tested by EMSA for their ability to bind recombinant PPAR{gamma}/RXR heterodimers. No retarded complexes were detected, except when RE5 was used as probe (Fig. 8BGo, lane 5). This RE5 element contains 2 DRs separated by an adenine, and thus represents a favorable context for the binding of PPAR/RXR heterodimers.

To determine whether cytotrophoblasts contain proteins able to bind to this element, the RE5 probe was incubated with cytotrophoblast nuclear extracts. A retarded complex similar to that seen with recombinant PPAR{gamma}/RXR{alpha} heterodimers was observed (Fig. 8CGo, compare lanes 1 and 2). This complex disappeared in the presence of an excess of the corresponding unlabeled probe (Fig. 8CGo, lanes 4 and 5) and of an unlabeled ACO PPRE oligonucleotide (Fig. 8CGo, lane 3). Moreover, it was supershifted with RXR{alpha} and PPAR{gamma} antibodies (Fig. 8DGo, lanes 2 and 3). Altogether, these data indicate that, in the regulatory region of the hCGß gene, the RE5 sequence is able to bind PPAR{gamma}/RXR{alpha} heterodimers.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that PPAR{gamma} is expressed in human placental villous trophoblasts in vivo, and also in cultured cytotrophoblast and syncytiotrophoblast cells in vitro. Primary cultures of trophoblasts mimic some aspects of the dynamic processes occurring during cytotrophoblast differentiation into a syncytiotrophoblast. Indeed, isolated mononuclear cytotrophoblasts maintained in vitro aggregate and fuse together to form an endocrine, active, nonproliferative, multinucleated syncytiotrophoblast that secretes hCG (4, 5). This process is influenced by EGF (67, 68), CSF-1, and GM-CSF (69), hCG (12, 13), glucocorticoids (70), E2 (71), and the oxidative status of the cells (72, 73, 74). The present work shows that it is also modulated upon PPAR{gamma} activation. Indeed, in this in vitro system we found that PPAR{gamma} activation by a specific agonist (rosiglitazone) was associated with an increase in hCGß secretion and transcript levels. These observations, which are in agreement with a recent report (75), point to a potentially important role of PPAR{gamma} in the differentiation of human trophoblasts and to potentially different roles of natural and synthetic PPAR ligands.

Interestingly, this is the first study to show that PPAR{gamma} cooperates with RXR{alpha} during human trophoblast differentiation, as RXR{alpha} coexpressed with PPAR{gamma} in the nuclei of human trophoblasts cells. In addition, a pan-RXR ligand increased hCGß production (at the mRNA and protein levels) as efficiently as PPAR{gamma} ligands do. The effects of PPAR{gamma} and pan-RXR agonists were additive, not only on hCGß secretion but also on hPL, hPGH, and leptin secretion. These data are consistent with those presented by (20, 30), who reported that placental maturation is blocked in mice lacking either RXR{alpha} or PPAR{gamma}. Therefore, both PPAR{gamma} and RXR{alpha} (and thus PPAR{gamma}/RXR{alpha} heterodimers) are probably essential for trophoblast differentiation.

PPAR{gamma}/RXR{alpha} heterodimers have been shown to play an important role in the differentiation of a number of cells, including adipocytes (76), macrophages (40), and colon cells (44). In these systems, the induction of differentiation is generally associated with enhanced PPAR{gamma} expression. However, no difference was found in RXR{alpha} and PPAR{gamma} levels in isolated mononuclear cytotrophoblasts and multinucleated syncytiotrophoblasts, suggesting that enhanced expression of these nuclear receptors is not directly involved in triggering cytotrophoblast differentiation into a syncytiotrophoblast. This does not, however, rule out an important role of PPAR{gamma}/RXR{alpha} heterodimers in placental physiology. In view of the general changes in placental trophic hormone production, this heterodimer might favor placental differentiation and maintenance. Therefore, stimulation of differentiation might occur through direct induction of placental hormone production triggered by the ligand-induced activation of PPAR{gamma}/RXR{alpha} heterodimers, rather than through an increase in the levels of these receptors. Placental hormones would then induce and sustain mature placental function via a feed-forward amplification loop.

These observations raise the question: what are the potential natural ligands involved in activating PPAR{gamma}/RXR{alpha} heterodimers in the placenta? All trans- and 9-cis-RA may be synthesized de novo from placental retinol or may be obtained from the maternal circulation. However, little information is available on placental expression of enzymes involved in RA synthesis, transport, and isomerization (77). Known natural ligands of PPAR{gamma} include fatty acids and eicosanoids (fatty acid derivatives) such as 15-deoxy-{Delta}12,14-PGJ2, and endogenous constituents of oxidized LDL particles, such as 9- and 13-HODE (78, 79, 80, 81). It is interesting to note that fetal membranes, decidua, and endometrium have been reported to generate PGs (82) and that hCG itself stimulates PG synthesis in human endometrial stromal cells through the activation of cyclooxygenase-2 gene expression (83). Placental tissue also produces considerable amounts of the PGs PGD2 (84) and PGJ2 (85), including {Delta}12 PGJ2 and 15-deoxy {Delta}12,14 PGJ2 which are derived from PGD2 via a series of reactions which may proceed nonenzymatically (86, 87). In addition, given its key role in nutrient transfer between the mother and fetus, the syncytiotrophoblast contains various lipid components. Arachidonic acid metabolites such as 6-keto-PGF1{alpha}, thromboxane B2, PGF2{alpha}, leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE (88) have been identified in placental organ cultures. Moreover, the syncytiotrophoblast expresses membrane-associated and cytoplasmic fatty-acid-binding proteins such as placental membrane fatty-acid-binding protein (p-FABPpm), which permits the sequestration of arachidonic and docosahexaenoic acid. It also expresses fatty acid translocase and cytoplasmic fatty acid transport protein, which bind and transport long-chain fatty acids and eicosanoids (89, 90). As it requires large amounts of cholesterol to synthesize its membrane components and placental steroids, the syncytiotrophoblast is also very rich in LDL receptors (91, 92). Finally, it has recently been reported that placental PPAR{gamma} is activated by serum from pregnant women (93). Therefore, it is conceivable that natural ligands for PPAR{gamma} are either synthesized or taken up by the trophoblast, enabling PPAR{gamma}/RXR heterodimer activation and subsequent hCG production.

Another issue is the transcriptional control of hCG biosynthesis. hCG is a heterodimer comprising an {alpha}-subunit (hCG{alpha}) common to all glycoprotein hormones and a distinct ß-subunit (hCGß) responsible for the biological specificity of the hormone. The regulatory elements controlling hCGß expression include a trophoblast-specific element, a TATA-box sequence, and two cAMP response elements (CRE) (17, 94). Accordingly, 8-Br-cAMP has been shown to stimulate cytotrophoblast differentiation and to increase hCG secretion (57). However, hCG secretion reflects de novo biosynthesis of the ß-subunit, as synthesis of the ß-subunit is the rate-limiting step in hCG secretion and little hCGß is stored in the cell. As PPAR{gamma} and pan-RXR ligands increase hCGß transcript levels, the hCGß promoter might contain binding sites for PPAR{gamma}/RXR heterodimers. The hCGß 5 promoter has been partially cloned and sequenced (66). Our analysis of this sequence revealed multiple putative response elements for PPAR/RXR heterodimers (see Table 2Go). One of these elements, a DR1 response element (RE5), was found to bind recombinant PPAR{gamma}/RXR{alpha} heterodimers. This motif also bound proteins from cytotrophoblast nuclear extracts, which were identified as PPAR{gamma} and RXR{alpha} by supershift analysis with specific antibodies.

It remained to be determined whether hCG up-regulation by PPAR{gamma} ligands reflected PPAR{gamma}/RXR{alpha} heterodimer binding to the RE5 element present in the ß hCG promoter. Therefore, human JEG3 choriocarcinoma cells were transiently transfected with a reporter gene construct containing three RE5 copies and the tk promoter inserted upstream of the luciferase gene [(RE5)3tk-Luc reporter]. Unexpectedly, we found that PPAR{gamma} and the RXR ligands inhibited transcription of this reporter (data not shown), whether they were added alone or in combination, and whether or not PPAR{gamma} and RXR{alpha} were cotransfected. In contrast, a control PPRE-driven luciferase reporter was activated by PPAR{gamma} ligand (data not shown), confirming that JEG3 cells express this receptor (85). As these cells also express high RXR{alpha} levels (60) and respond to a pan-RXR agonist by a strong increase in hCGß secretion (22), it seems unlikely that these cells lack a coactivator required for the function of the RE5 element. The RE5 element that we identified appears to be a PPRE that works as a silencer. Finally, as the hCGß promoter has not yet been fully sequenced, the PPREs involved hCGß promoter activation by PPAR{gamma}/RXR{alpha} heterodimers may be located in another region of the promoter.

In conclusion, this study shows that both PPAR{gamma} and RXR{alpha} are expressed in human placenta, and that these two nuclear receptors cooperate to induce the synthesis of hCG, a hormone essential for human pregnancy. Although our data suggest that the hCGß gene is directly activated by PPAR{gamma}/RXR{alpha} heterodimers, conformation will require more information on the sequence and regulation of the hCGß promoter.



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  		Figure 7. The PPAR{gamma} agonist (BRL 49653) and the pan-RXR agonist (BMS649), alone and in combination, increased the expression of hPL (A), hPGH (B), and leptin (C) transcripts in human trophoblast cells. Data are normalized according to pleiotropin (PO). The data are expressed as a percentage of control values (arbitrarily 100%). The results presented here correspond to the mean of three different experiments. The mean ± SEM of control values were, respectively, hPL 11.6 ± 0.5; 10.3 ± 0.5; 12 ± 0.8; hPGH: 25 ± 3; 22.6 ± 2.6; 23 ± 1.7; leptin: 3 ± 1.5; 7.3 ± 2.5; 9 ± 2.6. *, P < 0.05, significantly different from control.

 

   Acknowledgments
 
We thank Pierre Chambon for his interest and support and Celine Haby for technical assistance.


   Footnotes
 
This work was supported by Association Recherche contre le Cancer, Comité National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, l’Hopital Universitaire de Strasbourg, Bristol-Myers Squibb Co., and Human Frontier Specific Program (Grant No. RG0041/1999-M).

Abbreviations: 5-HETE, 5(S)-hydroxyeicosatetraenoic acid; hCG, human CG; hPGH, human placental GH; hPL, human placental lactogen; PPRE, PPAR-responsive elements.

Received March 15, 2001.

Accepted for publication June 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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