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Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta M Dodeur, A Mensier, E Alsat, D Bellet, Jm Bidart, D Evain-Brion

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M Dodeur, A Mensier, E Alsat, D Bellet, Jm Bidart, et al.. Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta. Reproduction Nutrition Development, EDP Sciences, 1991, 31 (3), pp.275-285. hal-00899410

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Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta

M Dodeur A Mensier E Alsat D Bellet JM Bidart D Evain-Brion

1 CNRS-URA 1337, ENS, Laboratoire de Physiopathologie du Développement, 46 rue d’Ulm, 75230 Paris Cedex 05; 2 Institut Gustave Roussy, Département de Biology Clinique, 94805 Villejuif, France

(Received 5 December 1990; accepted 14 February 1991)

Summary ― Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10’s mol.1-1) from 265 ± 32 to 532 ± 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 105 cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10-7 moLl-1) stimulates cAMP production of these cells from 0.52 ± 0.2 to 2.58 ± 0.57 pmol.h-! per 16 O cells (mean ± SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P < 0.01 ) and 3- (P < 0.01 ) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10-3 mol.1-1) increased hCG secretion by 3-fold (P < 0.05). EGF and hPTH added separately or together significantly stimulated (P < 0.01 ) the secretion of free a subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free (3hCG. However, added together, they significantly increased (P < 0.01) the secretion of free [3 hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Tro- phoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function. human placenta / parathyroid hormone / cAMP / trophoblast secretions

Résumé ― Effet de la parathormone humaine sur la production d’AMPc et sur les fonctions endocrines des cellules trophoblastiques de placenta du 1e! trimestre. Notre précédente étude sur les cellules de tératocarcinome suggérait que la parathormone humaine (hPTH) jouait un rôle sur le développement précoce du placenta. Le but de la présente étude était d’évaluer le rôle pos-

* Correspondence and reprints sible de la hPTH sur les fonctions des cellules trophoblastiques du premier trimestre. L’activité de ladénylcyclase dans les membranes issues de villosités humaines placentaires du 1er trimestre est doublée par la hPTH (1-34) (10-6 moit-1) de 265 t 32 à 532 t 80 pmol d AMPc par mg de protéines/ 15 min. Une stimulation identique de l’adénylcyclase est observée dans le tissu de villosités de placenta humain à terme, mais pas dans 3 lignées cellulaires différentes de choriocarcinomes. Afin d’évaluer le rôle possible de la hPTH sur les fonctions des cellules humaines du trophoblaste du 1er trimestre, de telles cellules ont été isolées par de la dispase et cultivées (2.105 celluteslboîte) dans du DMEM supplémenté en sérum de veau foetal (20%) et en facteur de croissance épidermique (EGF) (i 00 nglml), sauf pour les témoins, pendant 4 j. Le 29 jour de la culture, la hPTH (1&-7 Moihl) stimule la production dAMPc de ces cellules de 0,52:t 0,2 à 2,58 t 0,07 pmoLh-! pour i06 cellules (moyenne t écart type). Par rapport à la production des cellules témoins (30 ng/ml), la production de hCG est multipliée respectivement par 1,5 (NS), 2 (P < 0,01) ou 3 (P < 0,01), par la présence d’EGF, de hPTH, ou de hPTH + EGF. Le dibutyryl AMPc (10! moit-1) multiplie le taux de sécrétion de hGC par 3 (P < 0,05). L’EGF et l’hPTH ajoutés séparément ou simultanément stimulent significativement (P < 0, 0 i) la sécrétion de la sous-unité a (x 2) (70 nglmi contre 35 nglml). En revanche, la hPTH et /’EGF ajoutées séparément ne changent pas la sécrétion de la /3hCG libre. Cependant, ajoutés ensemble, ils augmentent significativement (P < 0,01) la sécrétion de la /3hCG libre après 48 h de culture, la stimulation maximale (x2,5) étant observée au 4e jour de la culture. En conclusion les cellules trophoblastiques humaines sont des cellules cibles pour la hPTH. La hPTH agit en association avec /’EGF pour promouvoir l’expression des activités endocrines de ces cellules telle que la sécrétion de la hCG. Les cellules trophoblastiques fournissent un modèle pour l’étude de I effet coopératif entre une hormone peptidique et un facteur de croissance sur la régulation d’une fonction endocrine. placenta humain / hormone parathyroïdienne IAMPC / sécrétion trophoblastlque

INTRODUCTION the a and a subunits of the hormone (Jameson et al, 1986). In addition, activa- Human chorionic gonadotropin (hCG) is a tion of the adenylate cyclase by cholera or increases the hCG secre- placental glycoprotein hormone necessary toxin forskolin tion human from term for the maintenance of pregnancy (Simp- by cytotrophoblasts son and Mac Donald, 1981 The produc- placenta (Nulsen et al, 1988). tion of hCG is highest and of greatest sig- Epidermal growth factor (EGF) is a poly- nificance in early pregnancy, but little is peptide of 6040 Da, isolated from mouse known of the factors regulating the produc- submaxillary glands (Cohen, 1962) and hu- tion of this hormone at the present time. man urine (Cohen and Carpenter, 1975). for EGF have been described The approach to these questions has Receptors on the been facilitated by culturing human tropho- predominantly syncytiotrophoblasts in term et al, and blast cells (Kliman et al, 1986, 1987). Iso- placenta (Maruo 1987) on the membranes of lated from the vil- purified trophoblast by enzymatic digestion cells in and term et lous tissue, the mononuclear early placentas (Chen al, 1988; Mirlesse et al, 1991 ). In addition, cytotrophoblasts cultured in vitro aggre- EGF has been reported to influence the and cell fusion form a gate through syncy- differentiation of human in with functions cytotrophoblasts tiotrophoblast specifc (Kli- culture (Truman and Ford, 1986). The ef- man et al, 1986). fects of EGF on hCG secretion described The secretion of hCG is increased in vi- in mid-term or full-term organ cultures are tro by 8-bromo-cAMP (Feinman et al, conflicting; Lai and Guyda (1984) reported 1986; Ulloa-Aguirre et al, 1987). This hCG secretion stimulated by EGF, where- cAMP analogue promotes hCG synthesis as Huot et al (1981) observed no such ef- by increasing the mRNA levels encoding fect. Murine teratocarcinoma cells differen- ing 100 txg/ml gentamycin and 2.5 pglml ampho- tiate in vitro following the first step of em- tericin. bryogenesis (Martin, 1980). They offer a model to study the role of polypeptide hor- Cell characterization mones during early embryogenesis. Thus, isolation, adenylate cyclase activity of these parietal and culture endoderm cells in culture is stimulated by parathyroid hormone (PTH) (Evain Brion et Trophoblast cells were isolated by using a previ- al, 1981; Liapi et al, 1987). These previous ously described dispase dispersion method (Do- results suggested a role for this hormone deur et al, 1990). The villous tissue was incubat- in In ed with a solution of dispase (2.4 U/ml), a placental development. agreement neutral from Bacillus with this recent studies demon- protease polymyxa (EC hypothesis, 3.4.24.4) in Puck’s solution (Boerhinger- strate that the human placenta is a target Mannheim) for 3 h at 4 °C. Afterwards the dis- organ for PTH as shown by the characteri- pase solution was discarded and the tissue incu- zation of a specific receptor for PTH in pur- bated overnight in medium A: DMEM containing ified membranes from term placenta (La- 25 mM glucose, 4 mM glutamine, 25 mM fond et al, 1988). HEPES, 100 I1g/ml gentamycin, 2.5 wg/ml am- photericin (Seromed Biochrom, Lyon, France) The purpose of this investigation was and 20% heat-inactivated fetal calf serum (Bio- therefore the determination of the effect of logical industries) in humidified 5% C02 and PTH on the endocrine functions of tropho- 95% air at 37 °C. blast cells from first trimester placenta. In Trophoblast cells were spontaneously re- this study we demonstrate that human leased from villous tissue into the medium. The were at 2.5 x 105 cells/ trophoblast cells from early gestation are trophoblast cells plated specific cells for PTH. Indeed, PTH per 35 mm plates coated with human fibronectin target At 24 h of culture, stimulates of (1 mg/ml, Sigma). cytotropho- adenylate cyclase activity blast cells were positively immunostained by an- cell membrane and fails to trophoblast tibodies against cytokeratin (Pan cytokeratin, un- stimulate the adenylate cyclase activity of diluted antibody, Amersham-France) and membranes isolated from either cultured against a hCG (Mab 326-2-1, Immunotech). choriocarcinoma or placental mesenchy- Cells were treated with these antibodies at ap- mal cells. Moreover, PTH with propriate dilutions and then with biotinylated together antibodies the EGF stimulates the secretion of hCG, and secondary (Amersham-France), binding of which was visualized with an avidin- modulates the secretion of free a and p biotin-peroxidase complex detection method subunits to a different extent, as measured (Hsu and Raine, 1981). The avidin-biotin- by specific monoclonal immunoradiometric peroxidase complex detection kit was pur- assays. chased from Amersham (France). In contrast, no immunostaining was obtained using the same methodology and a monoclonal anti- vimentin Amersham- MATERIALS AND METHODS antibody (undiluted, France). At 72 h of culture, multinucleated syncytiotrophoblast was predominant as studied by electron microscopy. First-trimester placentas were obtained from vol- untary elective abortions at 6 to 12 wk of ame- Mesenchymal cells were obtained from undi- norrhea, and term placentas from spontaneous gested villous tissue by explant cultures; these full-term deliveries. Placental villous tissues cells were grown in medium A and used to their were dissected free of chorionic membranes second or third passages. and incubated at 4 °C for 1 h in phosphate- The choriocarcinoma cell lines JAR, JEG, buffered saline pH 4.7 (NaCl 137 mM, KCI 3 BEWO, were cultured as previously reported mM, Na2HP04 8 mM, KH2po4 1.5 mM) contain- (Patillo and Gey, 1968; Patillo et al, 1971). Secretion of hCG by trophoblast cells um (Steiner et al, 1972). cAMP antibody was a in culture generous gift of J Saez (Inserm U 307, Lyon, France).

Trophoblast cells were cultured during 96 h in medium A with or without EGF (100 ng/ml, Col- Determination of hCG and its free a laborative Research), human PTH (hPTH)1-34 and subunits (10-s M, a generous gift of Rorer Laboratory, f3 Levallois, France), hPTH plus EGF, or dibutyryl cAMP (10-3 M, Sigma). The medium was MAb-based specific IRMAs were used for the changed daily and stored at -80 °C until ana- determination of hCG (Bellet et al, 1986), a hCG lyzed. and p hCG (Ozturk et al, 1987) levels in cell culture media. The production of MAbs with de- fined epitope specificity, the development of Crude membrane preparation multisite IRMAs and the testing of their speci- ficty have been previously described (Bellet et al, 1986; Ozturk et al, 1987). Cultured cells were washed with ice-cold buffer (50 mM Tris-HCI, pH 7.8, 0.33 M sucrose, 1 mM MgC’2), harvested by scraping and ho- Protein concentration mogenized by 20 strokes with a Dounce homog- enizer. Homogenates were centrifuged at 12 000 g for 20 min at 4 °C to isolate crude Protein concentration was determined by the membranes. Placental villous was tissue cut in method of Bradford (1976). small pieces and homogenized by 50 strokes in the ice-cold buffer as described above. Statistical analysis Adenylate cyclase activity Statistical analysis of the data was carried out by the paired Student’s t-test to compare the ef- Adenylate cyclase activity was determined by fects of the added factors to the effect of fetal the conversion of measuring [a3zP]-ATP calf serum (FCS) alone at each time. (Amersham) to [32P]-cAMP in crude membranes (12 000 g pellet) at 37 °C as previously described (Evain Brion et al, 1981). The standard incubation mixture contained 25 mM Tris-HCI, RESULTS pH 7.8, 10 mM phosphoenolpyruvate, 4 jig of pyruvate kinase, 5 mM MgCl2, 0.2 mM ATP, 0.5 mM EGTA, 4 x 106 cpm [a32P]-ATP and 50 pg Effect of PTH on placental adenylate of membrane protein. The hormonal stimulation cyclase activity by human PTH (hPTH) 1-34 (10! M) was measured in presence of 10-5 M GTP. The effect of PTH analog (Nle8-18 Tyr34) PTH 3-34 Adenylate cyclase activity was assayed in amide (Boerhinger-Mannheim) was tested in membranes prepared from either villous 10-fold excess as to hPTH 1-34. compared tissue (crude membranes) from early gestation and term placenta, or cultured cells from first trimester placenta and from chorio- of cAMP Determination carcinoma cell lines. The results are sum- in the culture medium marized in table I. PTH (10--6 M) induced a significant increase (P < 0.025) in adenyl- After acetylation, cAMP was measured by a ate cyclase activity of crude membranes specific radioimmunoassay in the culture medi- from first trimester and full-term placenta, as compared to corresponding control val- was dose-dependent with a maximal stim- ues (GTP 10-5 M). Similar results were ob- ulation (= 5-fold) occurring at 10-7 M of served for PTH effect on membrane aden- hPTH (P < 0.01), as compared to the ylate cyclase activity of isolated cAMP production by control cells (0.50 trophoblastic cells, cultured for 2 d (P < pmol/60 min/106 cells). 0.025). The specificity of this stimulation was demonstrated using a PTH analog (Nle8-18 Tyr34) PTH (3-34) amide -able to Effect of PTH on hCG secretion compete with the PTH receptor- which by trophoblast cells in culture fails to stimulate the adenylate cyclase activity in membranes from cultured tropho- Having demonstrated the PTH stimulated blastic cells. Furthermore, in comparison adenylate cyclase activity in the mem- with control values (GTP 10-5 M), the en- branes of trophoblast cells, we next deter- zyme activity was not modified by PTH in mined whether this activation would pro- membranes from placental mesenchymal mote the expression of one endocrine cells and from 3 different choriocarcinoma activity of these cells which is stimulable cell lines (JAR, JEG and BEWO), indicat- by cAMP, the secretion of hCG. ing that PTH-induced stimulation of adeny- Trophoblast cells were cultured during late cyclase was for normal specific tropho- 96 h in the presence of PTH (10! M) or blastic cells. EGF (100 ng/ml) alone or in the presence In addition, as shown in figure 1, PTH of PTH together with EGF and their effect stimulated cAMP production by intact iso- on the secretion of hCG (fig 2) and its free lated trophoblast cells in culture. The effect a (fig 3A) and 13 (fig 3B) subunits was stud- during the first 3 d of culture and in a similar manner to the addition of dibutyryl cAMP (1Q-3 M) (fig 2, 3A). Of interest is the fact that the association of EGF and PTH which did not have any effect on the secretion of p subunit during the first 2 d of culture increased significantly (P < 0.01) by == 2-fold the output of free p subunit at 72 and 96 h of culture (fig 3B).

DISCUSSION

We have established in this study that human trophoblast cells from early gestation placenta possess an adenylate cyclase activity which is stimulated by hPTH. The specific activity of early gestational adenylate cyclase activity is in the range reported in term placenta villous homogenates (Menon and Jaffe, 1973; Milewich at al, 1982) or in term placenta trophoblast cells in culture (Nulsen et al, 1988). PTH stimulates cAMP production in intact human trophoblast cells in a dose-dependent manner similar to that reported in other fetal and adult organs (Nissenson and Ar- naud, 1979; Silve et al, 1982). Our results are in agreement with the recent report on: i), the study of adenylate ied. As compared to control (20% serum cyclase in the membranes of cultured supplemented medium), the secretion of trophoblast cells from term placenta (Nul- hCG or of its free (3 subunits was not sig- sen at al, 1988); and ii), the activity of nificantly modified by EGF at any time of adenylate cyclase stimulated by PTH in the cell culture. The secretion of free a the basal plasma syncytiotrophoblastic subunit seemed significantly enhanced by membranes of term placenta (Lafond ot al, EGF during the first 2 d of culture. Para- 1988). In addition, PTH stlmulate! phos- thyroid hormone significantly stimulated phate transport in the piaeenta (Brunette at the secretion of intact hCG and its free a al, 1989) and increases the number of subunit by p2-fold (P < 0.01) but did not EGF receptors in cultured human tropho- modify the secretion of (i subunit. blast cells (Alsat et al, 1991). Those results The addition of EGF and hPHT to the suggest that PTH stimulates ad!nyl6t! CY’ culture medium increased by = 3-fold the clase activity of syncytiotrophoblast cells secretion of hCG and of its free a subunit throughout pregnancy. Moreover, the presence of PTH receptors in membranes of sociation with EGF induced a level of se- trophoblast cells appeared to be specific creted hCG similar to that obtained by ad- for the normal cells since we failed to de- dition of dibutyryl cAMP alone during the tect any activation in the membranes of 3 mid-stages of the culture (48 and 72 h). choriocarcinoma cell lines. This result suggested that PTH in association with EGF have a role in In this study we also investigated the might specific secretion of hCG and of its free a and free the regulation of endocrine functions in [3 subunits by cultured trophoblast cells. In trophoblast cells. In contrast, PTH alone or in association with EGF se- our experimental conditions the secretion stimulated the cretion of a subunit in a similar No of hCG did not vary significantly from 24 to way. 96 h of culture. As compared to control stimulation of free a subunits was observed the first 2 of culture (20% FCS), EGF alone had no significant during days effect on the stimulation of hCG secretion. but a significant (P < 0.01) increase in free This might be related to the concentration !3 subunits secretion was observed at 72 and 96 h of culture in the of PTH of EGF already present In the fetal calf ser- presence with EGF. um. PTH alone Increased significantly (P < together 0.01 ) hCG secretion by 2rfold, maximum The apparent synergism of action be- stimulation being observed at 413 h of cul- tween PTH and EGF on hCG secretion ture. Of interest Is the fact that PTH in as- and especially on its free P subunit secretion could be related to a synergistic effect In summary, trophoblast cells are a tar- at the receptor or post-receptor levels of get for PTH. This hormone acts in associa- this growth factor and this hormone. A tion with EGF to promote expression of en- growth factor may regulate the binding of a docrine activities of these cells, such as specific hormone. For example, PTH alters hCG secretion. Trophoblast cells provide a the TGF P binding to its receptors in osteo- model for studying the cooperative effect blast in culture (Centrella et al, 1988). The between a peptide hormone and a growth interaction between hormone and growth factor in the regulation of endocrine func- factor may also be located at the coupling tion. level of adenylate cyclase (Bernier et al, 1986; Jaillard et al, 1987). It is interesting to note that EGF has been shown to pro- ACKNOWLEDGMENTS mote the phosphorylation of a protein which appears to be similar to the G protein subunit (Valentine-Braun et al, 1986). This work was supported by a grant from La Fondation pour la Recherche Medicate and La Another explanation could be that PTH Ligue Parisienne contre le Cancer. We would potentiated the role of EGF in the differen- like to thank F Lewin (H6pital Saint-Vincent-de- tiation of cytotrophoblast to syncytiotropho- Paul, Paris) and the staff of the department of blast. Therefore in presence of PTH and gynecology for their cooperation. EGF, the cytotrophoblasts which continu- ously synthesize a subunit became differentiated into syncytiotrophoblasts, more REFERENCES competent to express the p subunit of hCG first an increase in hCG secretion inducing Alsat E, Mirlesse V, Fondacci C, Dodeur M, followed by secretion of free p subunit. 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