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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 7 2550-2555
Copyright © 2000 by The Endocrine Society

Original Studies

Retinoids Stimulate Leptin Synthesis and Secretion in Human Syncytiotrophoblast1

J. Guibourdenche, A. Tarrade, I. Laurendeau, C. Rochette-Egly, P. Chambon, M. Vidaud and D. Evain-Brion

INSERM U-427 (J.G., A.T., D.E.-B.) and Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes Paris V (I.L., M.V.), 75006 Paris; and Centre National de la Recherche Scientifique/INSERM/Université Louis Pasteur (C.R.-E., P.C.), 67404 Illkirch, France

Address all correspondence and requests for reprints to: Dr. Danièle Evain-Brion, INSERM U-427, Faculté des Sciences Pharmaceutiques et Biologiques de Paris, Université René Descartes, 4 avenue de l’Observatoire, 75006 Paris, France. E-mail: u427{at}pharmacie.univ-paris5.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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} (RXR{alpha}) 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 RXR{alpha} in this process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE product of the ob gene, is expressed essentially in adipose tissue, and its role in the control of food intake and body weight via its hypothalamic cognate receptors is clearly established (1). However, in humans, leptin is also expressed at lower levels in gastric epithelium and placenta (2, 3). In addition, leptin has been recently shown to be released from human brain (4).

In the placenta, leptin immunoreactivity is localized in the cytosyncytiotrophoblast layer at the surface of the chorionic villi (3, 5). The syncytiotrophoblast arises from fusion of the cytotrophoblasts (6, 7). It bathes directly in the maternal blood that arrives in the intervillous space through the uterine spiral arteries (8). The highly polarized syncytiotrophoblast secretes its hormonal products into the maternal circulation with almost no storage capacity. Therefore, leptin is secreted by the syncytiotrophoblast in the maternal circulation (9, 10, 11). Placental leptin production is increased in pregnancy with severe preeclampsia (12) or with diabetes mellitus (13). Plasma leptin concentrations are also elevated in patients with trophoblastic diseases such as hydatidiform mole or choriocarcinoma (3, 14).

Experiments conducted in transgenic mice have recently pointed out the key role of retinoic acid (RA) in placentation (15, 16). Indeed, according to data obtained from in vitro studies performed in humans, the syncytiotrophoblast production of two pregnancy-specific hormones, human placental lactogen (17), and hCG (18), is stimulated by all-trans-RA or 9-cis-RA, respectively, pointing out a regulatory role for retinoids in placental function and development. Retinoids act at the cellular level via two families of nuclear receptors: the RA receptors (RAR{alpha}, -ß, and -{gamma}), which are activated by either all-trans-RA or 9-cis-RA, and the retinoid X receptors (RXR{alpha}, -ß, and -{gamma}), which are activated only by 9-cis-RA. These receptors function as ligand-activated transcription factors and regulate gene expression by binding as dimers to DNA response elements associated with their target genes. RXRs not only form homodimers, but can also heterodimerize with RARs and a variety of other nuclear receptors (19, 20, 21). We previously demonstrated by in situ hybridization and immunohistochemistry, that syncytiotrophoblast from human term placenta express the two RAR{alpha} and RXR{alpha} isotypes (18). In the present work we therefore used the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast to study the effect of physiological and synthetic retinoids on leptin synthesis and secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Placentas

Term placentas were obtained after elective cesarean section from healthy mothers near term with uncomplicated pregnancies. Villous tissue was dissected free of membranes and vessels, rinsed, and minced in Ca2+- and Mg2+-free phosphate-buffered saline for cytotrophoblast cell isolation and culture.

Cell culture

Cytotrophoblast cells were prepared as previously described (22, 23). The cells were plated on 60-mm culture dishes (3 x 106 cells/dish) in 3 mL DMEM. They were incubated at 37 C in a humid atmosphere containing 5% CO2-95% air and were allowed to aggregate, fuse, and form syncytia. The cells were treated in triplicate with 0.1 µmol/L all-trans-RA, 9-cis-RA, BMS 649 (pan-RXR-selective agonist), or BMS 753 (RAR{alpha}-selective agonist) or were left untreated. BMS 649 and 753 were gifts from Prof. P. Chambon (Institut de Génétique et de Biologie Moleculaire et Cellulaire, Strasbourg, France). Stock solution of retinoids were made up in ethanol. Control cultures were treated with the same volume of ethanol (1:1000). At the dose used, retinoids did not affect cell viability (using blue trypan exclusion) or cell morphology. The culture media were collected and stored at -20 C. Cells were scrapped in lysis buffer to prepare total ribonucleic acid (RNA) following the procedure developed by QIAGEN (Courtabeuf, France) and were stored at -80 C.

Immunocytochemical staining

Cells cultured on sterile glass slides for 24 or 72 h were fixed for 20 min in 4% paraformaldehyde and permeabilized in 0.3% Triton X-100 for 30 min. Nonspecific binding was reduced using 7% sheep serum. Monoclonal antibodies against RAR{alpha} [Ab9{alpha} (F)]and RXR{alpha} (4RX3A2) (24, 25), diluted at 1:500 or 1:200 in 1% BSA, were applied overnight at 4 C. An antimouse, biotinylated, species-specific, whole antibody was used as the second antibody (1:500 for RAR{alpha} and 1:200 for RXR{alpha}, 1 h at room temperature), and revelation was performed using the streptavidin-fluorescein complex (1:400 for RAR{alpha} and 1:1000 for RXR{alpha}, 45 min at room temperature in the dark; Amersham Pharmacia Biotech, Les Ulis, France). Slides were coverslipped in a drop of fluorescent Dapi mounting medium (Vector Laboratories, Inc., Burlingame, CA) and analyzed under an epifluorescence microscope. Each step was followed by several extensive washes in phosphate-buffered saline-0.1% Tween. To ensure the specificity of the immunological reactions, adjacent control sections were subjected to the same method, except that the primary antibodies were replaced by nonspecific mouse IgG. No positive staining was observed in these controls.

Real-time PCR (7700 Prism, PE Biosystems, Foster City, CA)

Theoretical basis of the method have been previously described (26, 27). The Leptin TaqMan system consisted of the amplification primers LEP-F (5'-ACATTTCACACACGCAGTCAGT-3'), LEP-R (5'-CCATCTTGGATAAGGTCAGGAT-3'), and a dual labeled fluorescent TaqMan probe LEP-P (5'-TGGAGCCCAGGAATGAAGTCCAAA-3'). In addition, the level of transcripts for cyclophylin A was measured in each sample to control for sample to sample differences in RNA concentration. In each case, 1 µg total RNA extracted from cultured cells following the procedure of QIAGEN was denatured. RT was then performed for 30 min at 42 C with 1.5 mmol/L random hexamers (Pharmacia), 3 mmol/L MgCl2, 75 mmol/L KCl, 50 mmol/L Tris buffer (pH 8.3), 500 mmol/L deoxy (d)-NTP, 10 mmol/L dithiothreitol, 10 U RNasin ribonuclease inhibitor (Promega Corp., Madison, WI), and 50 U Moloney virus reverse transcriptase (Superscript II, Life Technologies, Inc., Gaithersburg, MD) in a total of 20 µL. The amplification reactions were set up in a reaction volume of 50 µL by use of components (except primers and probes) supplied in a TaqMan PCR Core Reagent Kit (PE Biosystems). One microliter of the RT reaction was used for quantitative two-step PCR (a 10-min step at 95 C, followed by 40 cycles of a 15-s step at 95 C and a 1-min step at 65 C) in the presence of 200 nmol/L specific forward and reverse primers; 100 nmol/L specific fluorogenic probe; 5 mmol/L MgCl2; 50 mmol/L KCl; 10 mmol/L Tris buffer (pH 8.3); 200 mmol/L each of dATP, dGTP, and dCTP; 400 mmol/L dUTP; and 1.25 U AmpliTaq Gold. Each sample was analyzed in duplicate, and a calibration curve constructed with a 10-fold serial dilution of total RNA extracted from first trimester placenta was run in parallel with each analysis. For each sample, the amounts of leptin messenger RNA (mRNA) and cyclophylin A mRNA were determined from the standard curves. Then the amount of leptin was divided by the amount of cyclophylin A to obtain a normalized leptin value.

Hormone assays

Leptin determination was performed using a human leptin-sensitive RIA kit (Linco Research, Inc., St. Charles, MO) in 4-fold concentrated conditioned medium. The sensitivity of the assay was 0.05 ng/mL, and the within- and between-assay variations were below 9%.

Data analyses

Hormonal secretion data are expressed as the mean ± SEM of triplicate determinations. Data were analyzed for variance with the Bonferroni test. Differences were considered significant at P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunolocalization of retinoid receptors in syncytiotrophoblast

Isolated cytotrophoblasts from term placenta aggregate and fuse together in vitro to form within 72 h a syncytiotrophoblast (22, 28). This is indicated by a gathering of nuclei in a large cytoplasmic mass. Mononuclear cytotrophoblasts and multinucleated syncytiotrophoblasts were immunostained with monoclonal anti-RAR{alpha} and anti-RXR{alpha} antibodies. As shown in Fig. 1Go, both antibodies gave a pronounced nuclear staining, which colocalized with the Dapi staining, in cytotrophoblasts and syncytiotrophoblasts. These results confirm the presence of RAR{alpha} and RXR{alpha} during the in vitro differentiation of cytotrophoblasts into syncytiotrophoblast (18).



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  		Figure 1. Immunocytochemical localization of RAR{alpha} and RXR{alpha} in the nuclei of trophoblast cells at 24 and 72 h of culture. After fixation in 4% paraformaldehyde and permeabilization in 0.3% Triton X-100, the mononuclear cytotrophoblasts and the multinucleated syncytiotrophoblasts were incubated with monoclonal antibodies against RAR{alpha} (top) or RXR{alpha} (bottom) as described in Materials and Methods. No staining was observed in controls. The right panels show the labeling of DNA with Dapi. Bar, 50 µm.

 
Leptin mRNA and protein expression during in vitro human cytotrophoblast differentiation

The morphological differentiation of cytotrophoblasts into syncytiotrophoblast was associated with a progressive increase in leptin mRNA expression (Fig. 2Go, top). Indeed, in 96-h cultured syncytiotrophoblast, the leptin mRNA level was 50-fold higher than in cytotrophoblasts at 24 h of culture. Leptin secretion in the culture medium, which was undetectable at 24 h (Fig. 2Go, bottom), began to increase at 48 h and reached a 5-fold higher level at 96 h (0.24 ± 0.03 ng/mL; P < 0.01).



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  		Figure 2. Expression of leptin transcripts (top) and leptin secretion in the culture medium (bottom) during cytotrophoblast differentiation. Top, Data are reported as the number of transcripts per number of transcripts for the constitutive housekeeping gene product, cyclophylin A. Three culture dishes were pooled for each determination, and leptin transcript levels were assayed in duplicate. Bottom, Leptin secretion in the culture medium, expressed as nanograms per mL. The results are expressed as the mean ± SEM of these three culture dishes. ***, P < 0.001. The figure corresponds to a representative experiment of three performed.

 
Effects of all trans-RA and 9-cis-RA on leptin synthesis and secretion

The effects of all-trans-RA and its 9-cis isomer (Fig. 3Go) on leptin synthesis and secretion by cultured human trophoblast cells were studied. 9-cis-RA (0.1 µmol/L) slightly increased leptin mRNA expression at 72 and 96 h of culture (Fig. 3Go, top). Similarly, leptin secretion was significantly (P < 0.01) increased in 9-cis-RA-treated cells compared to that in untreated cells (Fig. 3Go, bottom). In contrast, all-trans-RA was without effect on either leptin mRNA expression or leptin secretion during the 4 days of culture.



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  		Figure 3. Effect of physiological all-trans-RA and 9-cis-RA on leptin transcript expression (top) and leptin secretion (bottom). Human trophoblast cells were cultured in the absence (control) or presence of 0.1 µmol/L all-trans-RA and 9-cis-RA for 3 days. Top, Data are reported as the number of transcripts per number of transcripts for the constitutive housekeeping gene product, cyclophylin A. Three culture dishes were pooled for each determination. Leptin transcript levels were assayed in duplicate. Bottom, Leptin secretion in the culture medium, expressed as nanograms per mL. The results are expressed as the mean ± SEM of these three culture dishes. ***, P < 0.001. The figure corresponds to a representative experiment of three performed.

 
Effects of synthetic RAR and RXR agonists on leptin synthesis and secretion

The effects of synthetic retinoids that act as specific agonists of RAR{alpha} (BMS 753) or all RXRs (BMS 649) were then tested (Fig. 4Go). The pan-RXR ligand, BMS 649, induced a 3-fold increase in leptin mRNA expression at 96 h of culture (Fig. 4Go, top). Similarly, BMS649 increased leptin secretion significantly by 2-fold. Note that the effect of the synthetic pan-RXR ligand on leptin secretion was significantly (P < 0.01) greater than that observed with 9-cis-RA (Fig. 4Go, bottom). In contrast, the synthetic RAR{alpha} agonist (BMS 753) was without effect on leptin mRNA expression and leptin secretion.



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  		Figure 4. Effect of the synthetic RAR{alpha} (BMS 753) and pan-RXRs (BMS 649) agonists on leptin transcripts expression (top) and leptin secretion (bottom). Human trophoblast cells were cultured in the absence (control) or presence of 0.1 µmol/L synthetic retinoids for 3 days. Top, Data are reported as the number of transcripts per number of transcripts for the constitutive housekeeping gene product, cyclophylin A. Three culture dishes were pooled for each determination. Leptin transcript levels were assayed in duplicate. Bottom, Leptin secretion in the culture medium expressed in nanograms per mL. The results are expressed as the mean ± SEM of these three culture dishes. ***, P< 0.001. The figure represents one of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The syncytiotrophoblast is bathed in maternal blood and is the endocrine unit of the human placenta. It is a highly polarized cell that secretes steroids and polypeptide hormones (29). Some of these polypeptides, such as hCG, are specific and indispensable for pregnancy, whereas others are involved in the maternal metabolic adaptive changes that occur during pregnancy. This is the case for human placental GH, which replaces pituitary GH in the maternal circulation during pregnancy and regulates maternal insulin-like growth factor I (30).

The syncytiotrophoblast also secretes leptin (3, 5). As the syncytiotrophoblast discharges more than 99% of its polypeptide secretion into the maternal circulation (31), maternal leptin levels reflect in part placental leptin production. In that respect, serum leptin concentrations rise during pregnancy and fall during delivery (3, 32, 33). As leptin was recently reported to have angiogenic effects (34), a role for leptin in syncytiotrophoblast development may be suggested. Such a hypothesis is corroborated by the fact that syncytiotrophoblasts, which are in contact with the maternal blood, share common properties with endothelial cells. Indeed, these cells express some markers of endothelial cells, such as endothelial nitric oxide synthase, endothelin, and thrombomodulin (35, 36, 37). However, the physiological role of leptin during pregnancy remained unknown. It has been suggested that leptin may be involved in the control of parturition and lactation (38) as well as in the metabolic adaptive changes that occur during pregnancy, such as the insulin resistance state (1) A paracrine or an autocrine effect of leptin on syncytiotrophoblast development and function has been also suggested due to the presence of leptin receptors at their plasma membrane (39, 40, 41). Finally, although leptin synthesis and secretion have been shown to be increased by agents stimulating protein kinases A and C (42) and by hypoxia (12), the mechanism of regulation of leptin expression during pregnancy was also unknown.

Thus, the present work was undertaken to study the regulation of leptin expression and secretion in human placenta. For this study we used the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblasts. We have shown that both leptin expression and secretion are increased during syncytiotrophoblast formation, as previously reported for other hormones, such as human placental lactogen and hCG (18, 43), which are specifically synthesized by the syncytiotrophoblast. Interestingly, we also observed that physiological 9-cis-RA slightly, but significantly, increased leptin expression and secretion. Moreover, the synthetic pan-RXR agonist enhanced markedly leptin expression and secretion. This effect was similar to that observed upon addition of 8-bromo-cAMP to cell culture (data not shown). In contrast, all-trans-RA and the synthetic retinoid specific for RAR{alpha} had no effect.

Together, these results indicate that leptin expression and secretion increase during placental development and are modulated by retinoids that specifically bind and activate RXRs. RXRs bind as homodimers or heterodimers to a variety of response elements that consist of a direct repeat of two core motifs (5'-PuGG/TTCA-3' or a closely related sequence) separated by 1–5 bp (14). We have confirmed by immunocytochemistry with specific antibodies that syncytiotrophoblasts express RXR{alpha} and RAR{alpha}. However, analysis of the actually known promoter region of the leptin gene (43, 44, 45) did not reveal the presence of any binding sites for RXRs, suggesting that the mechanism of regulation of leptin expression does not rely on classical interaction of RXR homo- or heterodimers with a responsive element. The same conclusion has been proposed for the induction of leptin by glucocorticoids (46). In fact, multiple transcription factors have been shown to interact with the leptin promoter (36, 47). Therefore, it can be suggested that during the differentiation of cytotrophoblasts into syncytiotrophoblasts, leptin is up-regulated by transcription factors that remain to be identified. Then, due to the absence of variation in the level of expression of RXR{alpha} during the differentiation process and to the delayed increase in leptin secretion in response to RXR agonists, an indirect mechanism involving the activation of a cascade of transcription factors, which, in turn, modulate the transcription of leptin, can be proposed. The cloning of the proteins involved in the trophoblast-specific transcription of the human leptin gene would provide some new insights into the mechanism of regulation of placental leptin expression. It must be stressed that in the placenta an enhancer sequence of the leptin gene has the ability to bind specific transcript factors (47). The binding of these placenta-specific factors might contribute to the placental expression of leptin and the increased leptin levels during human pregnancy and subsequently to retinoid treatment. Conversely, the absence of these transcription factors in other tissues might explain the absence of leptin expression or the decrease observed upon RA treatment (48, 49).

In conclusion, the present study indicates that RXR agonists modulate the expression of placental leptin. The same observation has been previously made for hCG (18), which is involved in the induction of cytotrophoblast differentiation into syncytiotrophoblast via an autocrine process (50, 51). Therefore, as both leptin and hCG expressions are regulated by synthetic RXR agonists (18), our data highlight the role of RXR in the regulation of expression of the placental polypeptide hormones. Subsequently, a possible modulation of human cytotrophoblast differentiation by RXR-specific ligands cannot be ruled out.

Finally, studies of RXRs expression and functions in placenta from mothers with preeclampsia will help to elucidate the possible involvement of retinoids in the pathological increase in leptin secretion observed in this disease (12).


   Acknowledgments
 
This study was approved by the local ethical committee. We thank the staff of Saint Vincent de Paul Obstetrics Department for providing us with placentas. We thank E. Alsat for cell cultures and constructive criticisms of the manuscript, and N. Burky for typing it. We are grateful to Dr. P. Reczek for the gifts of the synthetic retinoids BMS753 and BMS649.


   Footnotes
 
1 This work was supported by a grant from La Fondation de France, Contract de recherche cliniqiue Grant 95266, and funds from the Centre National de la Recherche Scientifique, INSERM, the Hôpital Universitaire de Strasbourg, and Bristol-Myers Squibb Co. Back

Received November 19, 1999.

Revised February 26, 2000.

Revised April 1, 2000.

Accepted April 10, 2000.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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