Molecular and Cellular Endocrinology 10.5 (1994) 165-173

Nuclear retinoic acid receptor characterization in cultured human trophoblast cells: effect of retinoic acid on epidermal

growth factor receptor expression

Sylvie Roulie@ *, CXcile Rochette-Eglyb, Charlyne Rebut-Bonnetona, Dominique Porquetc, DaniHe Evain-Briona

aLaboratoire de Physiopathologie du DPveloppement, CNRS URA 1337, ENS, 46 rue d’Ulm, 75230 Paris cedex 05, France

bUnitJ de Biologie Mol&ulaire du D&eloppement et de G&tie GMtique, INSERT V 184, II rue Humann, 67085 Strasbourg cedex, France ‘Service de Biochemie-Hormonologie, Hfipital Robert Deb& 48 bd Serurier, 75019 Paris, France

Received 9 May 1994; accepted 10 August 1994

Abstract

Vitamin A is an important factor during gestation and its metabolite, retinoic acid (RA), is a potent teratogen. However, RA action on the placenta is still poorly understood. In this study we analysed the presence of RARs and RXRs in human trophoblastic cells. We determined that RARa was the more expressed form in term placenta, and that RAW was induced by RA treatment. Then we analysed RA effects on endocrine activities and on epidermal growth factor (EGF) receptor expression. We found that RA decreased 1251-labeled EGF binding and EGF-dependent phosphorylation. Furthermore, RA treatment led to a concentration-dependent decrease in the amount of EGFR protein expression. This treatment also decreased EGF receptor mRNA levels, suggesting transcriptional regulation of the EGF receptor. Thus we demonstrated that RA could interact with fete-placental development by modulating trophoblast EGF receptors expression, probably via its nuclear receptors.

Keywonis: Human trophoblast cells differentiation; Retinoic acid nuclear receptor; hCG secretion. Epide~~ growth factor receptor; Placenta; Retinoic acid

1. Introduction

Vitamin A (retinol) plays an important role during im- plantation and feto-placental development. In the rat, reti- no1 is essential for normal placental growth and differen- tiation (Howell et al., 1964; Moore, 1967). Retinoic acid (RA), the physiological acid metabolite of vitamin A, is nought to be a natural mo~hogen and is among the most potent known teratogens in humans (Sulik and Alles, 1991). Although RA was shown to increase placental hor- mone secretion by choriocarcinoma cell lines (Kate and Braunstein, 1991), its action on the placenta is still poorly understood.

The pleiotropic effects of RA are likely to be mediated by two types of proteins: the cellular retinoic acid binding proteins CRABP I and II (Eshubeita et al., 1987; Gigubre et

*Corresponding author. Tel. 33 144 32 37 32. Fax 33 1 44 32 39 17.

al., 1990) and the nuclear retinoic acid receptors (RARs) (Chambon et al., 1991). RARs belong to the steroid/thyroid hormone receptor superfamily, whose members act as li- gand-inducible transcriptional factors modulating the tran- scription of target genes (for reviews see Green and Cham- bon, 1988; Leid et al., 1992a; Chambon, 1993; Giguare, 1994). In addition, a distinct family of nuclear receptors, referred to as retinoid X receptors (RXRs), has recently been identified (Leid et al., 1992b; M~gelsdo~ et al., 1992). The RAR group includes three genes designated RARa, B and y. The RXR group similarly encompasses three genes. The diversity of receptors is further increased by alternative splicing which can generate isoforms of each receptor (Chambon et al., 1991; Gigubre, 1994).

In order to gain a better understanding of the effect of RA in placental growth and development, we have investi- gated the presence of RARs and RXRs in human placenta and their possible modulation by RA.

0303-72~~4/$~.# 0 1994 Elsevier Science Ireland Ltd. AI1 rights reserved SSDI 0303-7207(94)03376-5

For this purpose, we used human trophoblastic cells in culture. In vitro, isolated mononuclear cytotrophoblasts ag~egate and fuse to form syn~y~al statures ~Klim~ et al., 1986; Ringler and Strauss, 1990). During this process of morphologic differentiation, functional differentiation of these cells with respect to their endocrine activities also occurs; human chorianic gonadotropin (hCG) and placental lactogen (PL) are only expressed by syncytiotrophoblast ~Mo~isb et al., 1987; Ringler and S~auss, 19901, reciting the ~i~ere~tia~on state of these cultured cells.

RA has been shown to modulate cell growth and differ- entiation by interacting with growth factors and their recep- tors at the cell surface (Evain-Brion et al., 1991). Depend- ing an the epithelial cell line, RA either increases (Jetten, 1984; Komura et al., 1986; Thorn son and Rosner, 1989) or decreases (Abbot and Pratt, 1988; Zheng and Goldsmi~, 1990; Zheng and Goldsmith, 1992) epidermal growth fac- tor EGF receptor expression. EGF was shown to induce differentiation and hCG secretion in human term placenta ~Mo~ish et al., 1987) and in first trimester placenta (Barnes et al., 1990). EGF receptors are ab~ndantIy ex- pressed at the surface of the syncyti~~ophobl~t (Magid et al., 1985). Both EGF receptor numbers and the correspond- ing messenger RNA levels in syncytiotrophoblast mem- branes increase during gestation, as well as during tro- phobl~t cell diffe~ntiation in vitro (Chen et al., 1988; Mirlesse et al., 1990; ~e~u~-Bonneto~ et al., 1993; Alsat et al., 1993a).

We have demonstrated the presence of RA nuclear re- ceptors in human trophoblastic cells. It was therefore of interest to study the effect of RA on hCG secretion by those cells and the possible infraction with EGF receptors. In this study, we could not show any effect of RA on hCG secretion. However, RA was found to decrease EGF bind- ing, tyrosine kinase activity and protein expression of EGF receptors in human trophoblastic cells in culture. This treatment also decreased ECF receptor m gesting ~ans~r~ptio~al reg~Iation of the RA.

2. Materials and methods

Carrier-free Na t*sI for protein i~d~~ation was o~tain~ from CEA (Commissariat & 1’Energie Atomique, France). EGF was iodinated in a chloramine-T procedure to a spe- cific activity of 2100-2300 Ci/mmol. [J2P]yATP (3000 Ci/ mmol) was purchased from Amersham International ~~~~ki~gbarnshire, UK). A pulyclon~ a~ti~dy produced in rabbits, directed against a C-terminal peptide of EGF receptor was a generous gift from 3. Schlessinger (Medical Center, NY University, USA). Antibodies against RAR CI, #J and y and RXR Q and /? were polyclonal antibodies pro- duced in rabbits and were a generous gift from P. Chambon ~~S~~~ W 184, S~asbourg~ Francis. EGF r~eptor cDNA probe (64-Z fragments was k~~~~y provided by Dr.

F. Caulin and Dr. F. Bellot from Rorer Biotechnology (King of Prussia, PA}. The other reagents were from the fullowi~~ sources: bovine serum albums (BSA, macros V> was from CaIbi~hem (La Jolla, CA, USA); DNase I, receptor grade EGF, all-trans RA and protease inhibitors were from Sigma (St Louis, MO, USA); Percoll was from Pharmacia (Uppsala, Sweden); fetal calf serum (FCS) was from Biological Industries (Israel); trypsin 1:250 was from Difeo Laborato~es (permit, MI, USA); Hanks’ balance salt solution (HBSS), Ca2+, Mg2*-free Dulbecco’s modifi Eagle’s medium (DMEM), HEPES, L-glutamine and anti- biotics were from Gibco (Paisley, UK); all culture materials were from Costar. All other chemicals were of analytical grade.

Term placentas were obtained after elective cesarean section from women with uncomplicated pregnancies near term. The study was approved by the local ethics commit- tee of CHU-C~hi~-Pod-Royal (Paris, France).

Villous tissue was dissected free of mernbr~~ and ves- sels, rinsed and minced in PBS medium and submitted to trypsin-DNase digestions as previously described (Kliman et al., 1986). The resulting cell suspension was carefully layered over a discontinuous Percoll gradient (570%) and Gen~~ged. The homo~~~euus population af mononu~l~ cells, Fontaine in the middle layer ~de~sity 1.04&- 1.062 g/l), consisted of more than 90% cytotrophoblast cells (Malassinb et al., 1990).

For primary culture, the cells were plated in 35 mm or 60 mm Costar culture dishes (lo6 or 3 X 106 cells per dish, res~~tively~ with supplement with 10% heat- inactivated FCS, HEPES, 100 IUlml ~enici~Ij~, 100 mg/ml streptomycin, 2.5 pug/ml fungizone and 2 mM glutamine. Cultures were incubated in humidified 5% CO,/95% air at 37°C.

Stock solutions of lC@ M all-trans RA were made up in ethanol, stored at -2OOC and used for up to 1 week. Control cultures were treated with the same volume of ethanol (1 per 1000). Trophoblastic cells were cultured in DMEM supplemented with 10% FCS for 24 h, then in fresh DM~~l% FCS in the absence ~~0~~~1~ or presence of RA for 24 h. The RA concentration is indicated for each ex- periment, RA up to lo_6 M did not alter cell morphology or cell viability using blue trypan exclusion.

Trophobi~~~ cells were plated in 60 mm culture dishes. Culture media were collected at different time of culture, hCG was quantitated by using an immunoradiometric assay kit (Amersham, Buckinghamshire, UK) with a specific labeled monoelonai antibody directed against the@ subunit of hCG. After acetylation, cAMP was measured by a specific RIA (Steiner et al., 1972). The eAMP a~t~~y

S. Roulier et al. I Molecular and Cellular Endocrinology IO5 (1994) 165-173 167

was a generous gift of J. Saez (INSERM, U 307, Lyon, France).

2 5 ‘2SI-EGF binding experiments . . Treated and control cells were rinsed twice with DMEM

to eliminate the EGF contained in FCS. Binding studies were performed at equilibrium (90 min at room tempera- ture) with 80 pM 12sI-labcled EGF in 1 ml of PBS contain-

ing Ca2+ and Mg =+. At the end of the incubation period,

cells were chilled on ice and rinsed rapidly with ice-cold PBS. Cell-associated radioactivity was then measured using a gamma-spectrometer after cell lysis with 0.5 M NaOH.

Internalization was slight under the experimental condi- tions used (Alsat et al., 1991).

Specific binding was calculated from triplicate determi-

nations by subtracting from the total binding, the non- specific binding determined in the presence of an excess of unlabelled EGF (40 nM). Non-specific binding was always

less than 1% of the total added radioactivity. The protein concentration was measured after cell detachment by

means of a calorimetric assay using BSA as standard

(Bradford, 1976). The binding data presented are the mean of at least three

different placental cell preparations. The characteristics of EGF binding (affinity (&) and maximal binding capacity (B,)) were assessed using Scatchard plots (Scatchard, 1947) of bound and free EGF at equilibrium, from the EGF displacement data. The binding parameters from a two-site binding model were calculated by graphic analysis accord- ing to Rosenthal (1967). Statistical significance was de- fined using Dunnett’s test. The threshold was P 50.05.

2.6. Phosphorylation of membrane proteins After 1 day of culture, cytotrophoblast cells were cul-

tured for 24 h in the presence or absence of RA. The cells were washed twice with ice-cold PBS (pH 7.4), scraped off into 1 ml of homogenization buffer (0.25 M sucrose, 10mM Tris-HCl, pH 7.8, 1 mM MgC12, and IO~g/ml leupeptin, antipain, aprotinin, soybean trypsin inhibitor, and benzamidine as protease inhibitors), homogenized in a Dounce homogenizer and centrifuged at 800 X g for

10 min. The supernatant was then centrifuged at 12 000 X

g for 15 min and the resulting pellet (crude membranes) was resuspended in buffer containing 20 mM Tris-HCI, 50 mM NaCI, 1 mM CaC12, and 2 mM MgC12 (pH 7.4) to give IOO~g protein/IO@. Crude membrane proteins (IOpg) were incubated in the presence and absence of 100 ng EGF (0.15pM) with 5pM [32P]yATP in a final volume of 100~1 of 16 mM HEPES buffer (pH 7.4), con- taining 0.1 mM AMP-PNP, 1 mM MnC12, and 0.1% BSA. After 10 min at 4°C the reaction was terminated by adding 20~1 of stop solution (9% SDS, 15% glycerol, 3 mM EDTA, and 30 mM Tris buffer, pH 8) and heating at 100°C for 10 min (Alsat et al., 1991). Two microliters of /I-

mercaptoethanol and 5 ~1 of 0.1% bromophenol blue in

50% glycerol was added and aliquots (100~ 1) of each re- action mixture were then subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.

2.7. Preparation of cell extracts Whole cell extracts from trophoblastic cells were pre-

pared as described by Rochette-Egly et al. (1991) for trans- fected COS-1 cells. Briefly, cells were washed with chilled PBS, scrapped and centrifuged. The pellet was homoge-

nized at 4°C with a glass Dounce homogenizer (20 pestle strokes) in 2 ~01s. of 10 mM Tris-HCl (pH 8), containing

20 mM sodium molybdate, 0.6 M KCl, 1.5 mM EDTA, I mM PMSF and protease inhibitors. After centrifugation for 1 h at 105 000 X g and 4°C the supernatant was con- centrated by ultrafiltration through Centricon 30 microcon-

centrators (Amicon Corp., Danvers, MA). The extracts thus obtained were used for RARs and RXRs immunodetection.

For EGF receptor immunodetection, cells were lysed in 0.5 ml of RIPA buffer (10 mM Tris, 0.15 M NaCI, 1% so- dium deoxycholate, 1% Triton X-100 and protease inhibi- tors, centrifuged at 800 X g for 15 min and the super- natants were kept at -80°C until further use.

2.8. Immunoblotting Proteins (2Opg) were denatured in Laemmli sample

buffer containing 5% mercaptoethanol, boiled for 3 min, fractionated on SDS/IO% polyacrylamide gels and blotted onto nitrocellulose membranes. The nitrocellulose filters were blocked in PBS/l % non-fat powdered milk for 30 min at room temperature and then incubated overnight at 4°C with rabbit polyclonal antibodies at the required dilution. After extensive washing in PBS containing 1% Tween 20,

the filters were incubated with peroxidase labeled anti- rabbit immunoglobulins and specific complexes were re- vealed by chemiluminescence according to the manufac-

turer’s protocol (Amersham).

2.9. RNA isolation and Northern blotting Poly(A)+ RNA from cultured human cytotrophoblasts

was isolated by MicroFasttrack mRNA isolation kit (Invitrogen, San Diego, CA, USA). RNA samples, dena-

tured at 55°C were then separated on 1% agarose/formal- dehyde gel, transferred to nitrocellulose membrane in 20x SSC, fixed by 3 min in UV radiation and hybridized by using randomly primed 32P-labeled probes for detection of mRNAs specific for EGF receptor and glyceraldehyde phosphate dehydrogenase (GAPDH). A 64-2 fragment of EGF receptor was used as the probe. The hybridization was assessed in 5~ SSC, 2~ Denhardt’s solution, 0.1% SDS, 50% formamide, lOO~g/ml herring sperm DNA at 42°C. The membranes were then washed twice with 2~ SSC, 0.1% SDS at room temperature and once with 0.5 X SSC, 0.1% SDS at 55°C. The filters were then placed with X-ray film (Kodak X-Omat, Eastman Kodak, Rochester, NY) for

autoradiography at -80°C.

168 S. Roulier et af. I Molecuku and Cefiular Etuiocri~logy 105 (1994) 165-l 73

3. RemIts

3.1. Specific detection of RAR a, p and y and RXR a andp proteins by immunoblotting in human cultured trophoblastic cells

We examined the distribution of nuclear RA receptors in isolated cytotrophoblasts during their differentiation into syncytia. Equal amounts of whole cell extracts from tro- phoblastic cells, cultured during 24 h, 48 h and 96 h, were analysed by immunoblotting with rabbit polyclonal anti- bodies directed against RARa, /l and y and ma and /I. Whole cell extracts of COS-1 cells transfected with vectors expressing the corresponding RAR or RXR protein were analysed in the same experiments.

In whole cell extracts of trophoblastic cells, a doublet (51-47 kDa) was detected with the RPa(F) antibody di- rected against region F of either human or mouse RARa (Fig. lA, lanes 2-4). This signal was present with a con- stant intensity during the differentiation of cytotropho- blasts. Furthermore these species were in keeping with the predicted molecular weight of RAR-al (Gaub et al., 1992), and were similar to that detected in human RARal trans- fected COS-1 cells (COS-al) (Fig. IA, lane 1).

The RPRXa(A) rabbit polyclonal antibody directed against the A region of FZRa revealed in extracts of tro- phoblastic cells a unique signal with an apparent molecular mass of -54 kDa (Fig. lB, lanes 2-4) corresponding to the RXRa species present in transfected COS-1 cells (COSX- a) (Fig. lB, lane 1). No modulation of RXRa was observed during the differentiation into syncytiotrophoblast (Fig. lB, lanes 2-4).

0 A

RPa( F) kDa 71

106 -

32 -

1 2 3 4

; 24 48 96

hours

The 47,51 and 54 kDa signals co~esponding to RARa and RXRa respectively were specific since they disap- peared when immunoblotting was performed with depleted antibodies.

With the RP&F)2 antibody directed against the F region of human or mouse RAR-/32 (Rochette-Egly et al., 1992) and with the RPy(F) antibody directed against the human F region of RARy (Rochette-Egly et al., 1991), no signal could be detected in trophoblastic cells up to 96 h of cul- ture, whereas a specific signal was revealed in extracts of RAR-@2 and RARyl transfected COS-1 cells (Fig. 2, lane 4 and data not shown).

Similarly, we failed to find any signal in whole cell ex- tracts of trophoblastic cells with the RPRX&A) rabbit polyclonal antibody directed against the A region of RXR#l (data not shown), while a specific major band of -65 kDa, in keeping with the predicted size of hRXR#l (Leid et al., 1992), was detected in human RXR-bl transfected cell ex- tracts.

Overall we found that RARa was the more qualitatively and quantitatively expressed form and that RXRa was ex- pressed at very weak levels in the different placentas stud- ied and in our conditions.

3.2, E$%ct of R9 on RAR and RXR expression Cultured trophoblastic cells were treated during 24 h

between day 1 and day 2 of culture with 10” M RA, as described in Section 2.

RARa and RXRa were not modulated by RA treatment as shown in Fig. 2 (lanes 2, 3 and 8, 9). However RARJ?, which w ndetectable in trophoblastic cells during their

0 B

RPRX~(A) I 1

Fig. 1, Characterization of RARa (A) and RXRa (B) in human trophoblastic cells in culture. Whole cell extracts (20,~g) from trophoblastic cells after 24, 48 and 96 h of culture were fractionated by SDS-PACE, electrotransferred to nitrocellulose tiltem, and immunoprobed with rabbit polyclonal antibodies. (A) RPa(F) directed against the F region of RARa-F region; (B) RPRXa(A) directed against the A region of RXRa. The specific complexes were revealed by chemiiuminescence. As positive control, whole cell extracts (S-IOpg protein) of COS-1 cells transfected with vectors expressing the corresponding RAR or RXR isoform were run in parallel. The arrows indicate the position of the specific band detected in transfected COS-I cells. Arrowheads and thin arrows indicate the position of the specific bands detected in trophoblastic cells.

S. Roufier et al. I ~~lecu~~ and CelluIaai Endocrinology 105 (1994) 165473 169

RPa( F)

RA - _ - kDa

106- 80 -

i

RPP(W I _ _

4 5 6

RPRX~~F)

I _ _

7 8 9

F%g. 2. Effect of RA on RARa, RAW and RXRCZ expression as defined by means of imm~~io~ng. Whole ceil extracts Born trophoblastic cells cultured in the absence (lanes 2,s and 8) or presence (lanes 3,6 and 9) of RA (tOA M) were fractionated by SDS-PAGE, ekctrotransferred to NC! fikers and immunoprobed with rabbit polyclonal antibodies: RPa(F) directed against the F region of RARa-F region (lanes l-3); RP&F)2 directed against the F region of RAR-PZ (lanes 4-6); RPRXa(A) directed against the A region of RXRa (lanes 7-9). As positive control, whole cell extracts (WOpg protein) of COS-1 cells transfected with vectors expressing the corresponding RAR or RXR isofom were run in parallel (lanes 1,4 and 7).

in vitro differentiation, was induced by RA, as previously described in other cell types (Sucov et al., 1990; Vivanco Ruiz et al., 1991). As shown in Fig. 2 (lanes 5 and 6), the RF/?(F)2 antibody revealed a band with an apparent mo- lecular weight of -51 kDa in RA treated cells. This signal was similar to the one observed in extracts of RAM2 trans- fected COS-1 cells (COS-fi2) (Fig. 2, lane 4). The addi- tional bands of higher molecular weight detected were not specific as they did not disappear when immuno- blotting was performed with a depleted RP/?(F)2 antibody (Rochette-Egly et al., 1992). RARy and RXRg remained undetectable even after RA treatment (data not shown).

3.3. Effect of RA on endocrine activities As shown in Fig. 3A,B, treatment by lo4 M RA did not

modulate hCG secretion by trophoblastic cells, and CAMP concentration, at different days of culture.

3.4. Effect ofRA on EGF biting As RA nuclear receptors were expressed in human cyto-

trophoblastic cells in culture, we investigated the effect of RA on the EGF receptor expression in cytotrophoblasts in culture.

Competitive binding experiments were performed with 24-h RA-treated and control cells isolated from term pla- centas and cultured in vitro for up to 2 days. In primary cytotrophoblast cultures from term placentas, the average 12SI-labeled EGF binding capacity was 34.4 zl: 7.0 fmol

EGF/mg protein at 48 h of culture. Treatment for 24 h with lo4 M RA reduced 1251-labeled EGF binding capacity by 57 + 6% (mean f SEM).

Scatchard analyses of the data revealed curvilinear plots compatible with two classes of EGF binding sites, with both control and RA-treated cells. The K* values for both high-Annie ((1.35 -+ 0.08 )X 10-i@ M) and low-unit binding sites ((3.51 + 0.30) X lO+’ M) were not modified by RA treatment of cells. A 50% decrease in high-affinity

JLU values was observed with RA-treated cells (0.19+: 0.06 versus 0.44 f 0.07 pmol/mg protein in con- trols). The mean B,, values for low-affinity sites were not altered by RA treatment of trophoblastic cells. These re- sults indicate that the effect of RA on EGF binding activity occurs via a decrease in the number of high-affinity sites.

3.5. Efect of RA on tyrosine kinase activity of EGF re~e~t5r~~orn tro~~b~asti~ cells

As binding of EGF to its receptor leads to an increase in tyrosine kinase activity, which induces autophosphoryla- tion of the receptor itself and phosphorylation of other cell proteins, we looked at the effect of RA on kinase activity. In isolated membranes from trophoblastic cells, EGF stimulated the active phosphorylation of two major proteins with apparent molecular weights of 170 kDa and 35 kDa (Fig. 4). The 170 kDa band corresponds to EGF receptor, while the 35 kDa band may correspond to lipocortin I, the phosphorylation of which is EGF-dependent in placental

S. Roulier et al. I Molecular und Cellular EndmzGwtogy 105 (1994) 165-t 73

’ B:cAMP

4-

3-

3 4 5 6 7

Days

2

1

I OI.s*r*‘-l.l“-l

0 1 2 3 4 5 6 7

Days Fig, 3. RA effect on hCG and CAMP suctions by ~phobl~tic cells. Control (solid line) and IOdM RA-treated (dotted line) trophoblastic cells wem cultured during 6 days. Culture media was collected at differ- ent days of cultme, then hCG secretion (A) and CAMP (B) concentration were determined respectively by specific immunoradiometric assays and RIA. Each value is the mean f SEM of one representative culture over 7 concording experiments.

membrane preparations (Sheets et al., 1987). EGF- dependent phosphorylation of the 170 kDa band (corres- ponding to the EGF receptor) was decreased by 30% in membranes from RA-treated cells.

We confirmed these results in immunoprecipitation ex- periments with an antibody directed against EGF receptor after EGF-dependent phosphorylation in vitro. We obtained one 170 kDa band, the intensity of which was decreased 44% by RA treatment (data not shown).

3.6. Immunoblotting of EGF receptors Total cell proteins were extracted from trophoblastic

cells cultured in the absence or presence of RA, as de- scribed in Section 2, and analysed by means of immuno-

blotting. The blots were probed with a specific antibody directed against EGF receptor. As shown in Fig. 5, a band with an apparent molecular weight of 170 kDa correspond- ing to EGF receptor emerged, and the amount of EGF re- ceptor was significantly decreased by RA treatment. This decrease was concentration-dependent with a maximal ef- fect at lo6 M RA.

3.7. Effect of RA on EGF receptor mRNA expression

In order to assess the possible effect of RA on EGF re- ceptor synthesis, the specific expression of EGF receptor mRNA was analysed by means of nor~em blotting in tro- phobl~tic cells treated with RA lo-6 M. As shown in Fig. 6, two prominent EGF receptor transcripts of 10.5 and 5.8 kb were identified. RA treatment decreased the level of EGF receptor mRNA by 47%, as shown by densitometric analysis.

4. Discussion

In the present study, we report for the first time the presence of nuclear RA receptors in human trophoblastic cells. RARa was the more expressed form while RXRcx depicted a low level of expression. We did not detect any

-RA +RA

ECFR )

L> aId

-205

-116

-80

-49.5

**c

Fig. 4. EGF-dependent phosphorylation of membrane proteins from tro- phoblastic cells. Cells were cultured for 24 h in the absence (control) or presence of IO+ M RA. Membrane proteins were phoaphorylnted in the presence (+) and absence (-) of 100 ng EGF, as described in Section 2. The samples were subjected to SDS-PAGE and the gels nutom- diogmphed. (EGFR, EGF receptor; L. Iipocortin).

S, Roulier et al. /Molecular and Cellular Endocrinology 105 (1994) 165-173 171

EGFR, A B C D kDa

Fig. 5. Effect of RA on EGF receptors, as determined by means of im- munoblotting. Cells from term placentas were cultured for 24 h in DMEM containing 1% FCS in the absence (lane A) or presence of vari- ous RA concentrations (lane B. lo-* M; lane C, lo-’ M; lane D, IO- 6 M). Total cellular extract was subjected to SDS-PAGE, transferred and immunoblotted as described in Section 2.

variations in the level of expression of RARa and RXRa either during the in vitro differentiation of the cytotro- phoblasts into syncytiotrophoblasts or after RA treatment. However, RAR@ expression was induced by R4 treatment in trophoblast cells as has previously been described in other cell types (Sucov et al., 1990; Vivanco Ruiz et al., 1991; Rochette-Egly et al., 1992). Green and Ford (1986) demonstrated the presence of cellular RA binding proteins CRABPs in human term placenta, and in this paper we have shown the presence of nuclear RA receptors; therefore the two types of proteins which belong to the retinoid sig- nalling pathway are present in human placenta.

Since RA nuclear receptors were expressed in tropho- blastic cells, we studied the effect of RA on growth and differentiation of these cells. First, as hCG secretion re- flects the differentiation process of trophoblastic cells and as CAMP induces hCG secretion (Morrish et al., 1987; Ringler and Strauss, 1990; Jameson and Hollenberg, 1993) we analysed RA effect on hCG secretion and CAMP con- centration. Obviously, we did not find any differences in RA treated cells, suggesting that RA did not modulate en- docrine activities of trophoblastic cells in culture, thus normal trophoblastic cells differ in their response to RA from choriocarcinoma cells (Kato and Braunstein, 1991). Then, because of the important role of EGF during differ- entiation, we analysed the RA effect on EGF receptors. In a previous study, we found that the number of EGF receptor spontaneously increase twofold during the first 2 days of culture of human trophoblast cells (Mirlesse et al., 1990; Alsat et al., 1993a), the time at which these cells differen-

tiate by aggregation and fusion into multinucleated syn- cytiotrophoblasts in vitro (Kliman et al., 1986; Mirlesse et al., 1990; Alsat et al., 1991). In addition, we reported that the effect of parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) on EGF receptors is al- ready maximal at 48 h of culture (Alsat et al., 1991, 1992, 1993b).

Thus in the present study, RA was added to trophoblas- tic cells between 24 and 48 h of culture, which corresponds to the period of in vitro cyto-syncytiotrophoblast differen- tiation. Such a RA treatment decreased the EGF binding capacity of trophoblastic cells at concentrations greater than 10e8 M. The physiological circulating concentration of RA is around 1Om8 M (Silverman et al., 1987) and concen- trations of lO-‘M (Brazzell and Colbum, 1982) are ob- served in patients on retinoid therapy. The concentrations of RA producing a decrease in EGF receptor numbers in human trophoblastic cells are in the range of those effective in numerous cell lines (Evain-Brion et al., 1991). In fact, the decrease in EGF binding reflected a reduction in the number of high-affmity sites since their apparent affinity was not altered by RA.

EGF-dependent phosphorylation of membrane proteins was also decreased in RA-treated trophoblastic cells. By specific immunoprecipitation, the phosphorylated protein

RA - +

EGFR

10.5 kb

5.8 kb

Fig. 6. Northern blot analysis of poly(A)+ RNA from human cytotro- phoblasts cultured in the absence or presence of IO4 M RA. Poly(A)+ RNA was denatured, blotted, and hybridized with the 32P-labeled ECF mceptor-cDNA as described in Section 2. The size of EGF receptor spe- cies was estimated from the migration of molecular weight markers (0.24-9.5 kb RNA ladder from Gibco BRL, Cergy Pontoise. France) in the same run.

172 S. Roulier et al. I Molecular and Cellular Endocrinology IO5 (1994) 165-173

was identified as EGF receptor. This result confirmed the conclusion drawn from the Scatchard analysis and im- munoblotting experiments, that RA decreased the number of biologically active EGF receptors. Furthermore, north- ern blot analysis showed a decrease in EGF receptor tran- script levels in trophoblast cells after incubation with RA, suggesting that the decrease in receptor transcript levels could fully account for the decreased number of biologi- cally active receptors. Moreover, the RA-responsive ele- ment has been shown to exist in the enhancer sequence of the EGF receptor gene (Hudson et al., 1990), RA may therefore act in trophoblast cells by reducing EGF receptor synthesis, as seen in other cell lines such as ME180 (Zheng and Goldsmith, 1990).

Trophoblast cell EGF receptors are modulated by differ- ent agents such as PTH (Alsat et al., 1991), or benzopy- rene, a component of cigarette smoke, which decreases EGF receptor numbers only in cultures from early placen- tas (Guyda et al., 1991). A decrease in EGF receptor ex- pression has also been described in intrauterine growth retardation (Fujita et al., 1991; Fondacci et al., 1994), sug- gesting that EGF plays an important role in fetoplacental development. Thus, the IL4 effect on EGF receptors ob- served in our experiments suggests that RA is likely to negatively modulate trophoblast differentiation via its nu- clear receptors, thereby regulating fetoplacental develop- ment.

In conclusion, trophoblastic cells offer a useful tool to study the interactions (cross-talk) of growth factors and RA pathways, in modulating cellular endocrine functions.

Acknowledgements

We gratefully acknowledge Professor P. Chambon for his generous gift of RA nuclear receptors polyclonal anti- bodies. We wish to thank the staff of the Department of Obstetrics and Gynecology (Hapita Saint Vincent de Paul, Dr Chavinit, Paris) for the placental tissue contributions, and Dr. M. Donnadieu for help in statistical analyses. This paper was supported by a grant from la Fondation de France and the European Community (Science III-TL 910004).

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