Expression of G proteins in human placentae from molar pregnancies

Placenta (1996), 17, 337-343

Expression

A. Petit,

of G Proteins

P. Geoffroy,

P. Bessette

in Human

Placentae

from

Molar

Pregnancies

and S. B61islea

Department of Obstetrics and Gynecology, University of MontrBal, 3175 Cote Ste-Catherine, MontrBal, Quebec, Canada, H3T 1 C5 Paper accepted 18 January 1996

Research

Center, Ste-Justine

Hospital,

Hydatidiform mole is a conceptus, usually devoid of an intact fetus, with variable proliferation of trophoblast and altered placental protein synthesis, including high human chorionic gonadotropin (hCG) and low human placental lactogen (hPL) production. Little is known about the control of the production of these two placental proteins in molar pregnancies. Regulatory guanine S’triphosphate (GTP)-binding proteins (G proteins) play key roles in the endocrine control of peptide production by the placenta. The present authors recently demonstrated that Gi2, Gi3 Go, and Gs u-subunits were expressed in normal human placenta throughout pregnancy. This study analysed the expression of placental G protein a-subunits in molar pregnancies. Western and Northern blot analyses were performed on membrane protein and total mRNA preparations of human placentae, respectively, from hydatidiform mole (n=5) and normal pregnancies (n=4). The levels of hPL and P-hCG mRNAs were 60 and 237 per cent respectively, of those from normal placentae. The autoradiographs for G proteins and their mRNAs showed decreased expression in molar placentae in comparison with normal tissues. Specifically, Gai2, Gai3, Goo, and Gas levels reached 39, 4,42, and 89 per cent, respectively, of those from normal placentae. In parallel with the protein levels, their mRNAs expression were 8, 3, 54 and 65 per cent of normal values for Gai2, Gui3, Gao, and Gas, respectively. The results demonstrate important changes in placental G protein expression in hydatidiform moles suggesting alterations in the signal transduction machinery within the molar trophoblast. 0 1996 W. B. Saunders Company Ltd Placenta (1996), 17, 337-343

INTRODUCTION Hydatidiform mole may be defined as a conceptus usually devoid of an intact fetus in which all, or many, of the chorionic villi show: (1) gross nodular swelling culminating in cyst formation, (2) disintegration of blood vessels, and (3) variable proliferation of trophoblast (Edmonds, 1959). Even if they are not real neoplasia, hydatidiform moles present several aspects of malignity including local proliferation, myometrium invasion and development of systemic metastasis (Szulman and Surti, 1978). Hydatidiform moles are associated with altered placental protein synthesis. The increase in P-human chorionic gonadotropin (hCG) production and the decrease in human placental lactogen (hPL) production represent two examples of this altered protein synthesis (for review see Benirschke and Kanfmann, 1990). Regulatory guanidine’5’triphosphate (GTP) binding proteins (G proteins) constitute a family of proteins involved in signal transduction across the plasma membrane, and have been shown to play a key role in the stimulusresponse coupling in many important biological systems. Our results on the effects of dopamine (DA), angiotensin II (AT II), and opioids (OP) on hPL release suggest that many G proteins, pertussis-toxin (PTX) sensitive (ex: Gai2 and Gai3) a To whom correspondenceshould be addressed. 0143S4004/96/050337+07

$12.00/O

and insensitive (ex: Gus), are important for normal placental functions (Petit et al., 1990, 1993). In agreement with the putative role of these placental G proteins, we have recently described the expression of Gus, Gao, Gai2, and Gai3 proteins during pregnancy, and showed that Gcti2 and Gai3 are expressed differently during pregnancy (Petit et al., 1995). Several disease states, including metastatic tumours and endocrine disorders, are associated with abnormalities in structure and expression of G proteins, specifically for Gus and Gcli families (Spiegel, Sherker and Weinstein, 1992). Moreover, some of them seem to be the cause of these disorders. Therefore, modifications of placental G proteins may also occur in hydatidiform moles. The present study investigates the expression of G proteins in molar pregnancies.

MATERIALS Placental

AND

METHODS

tissue

This project was approved by the Human Subject Research Committee of our institution and participating hospitals. Placentae from molar pregnancies between 11-16 weeks were obtained from the obstetrical service of the Montreal Jewish General Hospital (three cases) and the Royal Victoria Hospital 0

1996 W. B. Saunders

Company

Ltd

Placenta

338

(1996),

Vol. 15

(two cases) and normal placentae (11-16 weeks) were from Ste-Justine Hospital. The villous layers of trophoblastic tissues were dissected, frozen on dry ice and stored at - 80°C until the time of protein and RNA preparation.

cent blocking solution. The intensity of bands was quantified using LKB Ultrascan XL apparatus (Pharmacia, Dorval, Quebec, Canada). Results were normalized with placentae at the same period of pregnancy.

Western

Northern

analysis

G protein antibodies were obtained from DupontNEN (Boston, MA, USA) (Gus, # NEI-805; Gail-2, # NEI801) and Calbiochem (La Jolla, CA, USA) (Gai3, # 371729; GaO-i3, # 371726) and diluted l/1000 in Boehringer blocking solution 0.5 per cent. Diluted antibodies were kept for 4-6 weeks at 4°C. The anti-Gas antibodies may also recognize G(olf)s whereas anti-Go0 and Gai2 antibodies may also recognize Gai3 and Gail, respectively. The anti-Gai3 antibodies do not cross-react with other Go proteins (specifications of DuPont-NEN and Calbiochem).

Antibodies.

of placental microsomes.Placental microsomes were prepared as we previously described (Petit et al., 1995). Tissues were extensively washed with ice-cold 150 mM NaCl. The tissues (0.5 g) were suspended in 5 ml of 50 mM Tris-HCI (pH 7.4) with 0.25 M sucrose and 1 mM PMSF and homogenized in a Polytron appartus set at 4-5 for four lots of 10 set bursts with 2 min cooling periods between each burst. The homogenates were centrifuged at 1000 g for 15 min at 4°C. The supernatants were removed and pelleted at 100 000 g for 60 min at 4°C. The pellets were washed in homogenization buffer and recentrifuged at 100 000 g. The pellets, considered as the microsome fractions (including mitochondrial membranes), were homogenized at lo-20 mg/ml in homogenization buffer and stored at - 80°C until used. This microsome preparation did not allow enrichment of brush border versus basolateral plasma membranes. Protein concentration was measured according to Lowry et al. (Lowry et al., 1951) with bovine serum albumin as standard.

Preparation

Westem blot analysis. Placental microsome proteins (50 pg) were heated at 100°C for 2-5 min in 62.5 mM Tris-HCl (pH 6.8) with 2 per cent sodium dodecyl sulphate (SDS), 10 per cent glycerol and 5 per cent P-mercaptoethanol. Proteins were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970). Thus, as previously described (Petit et al., 1995), SDS-PAGE was performed with a 10 per cent acrylamide separating gel and a 5 per cent acrylamide stacking gel. The running buffer was 25 mM Tris-HCl (pH 8.3), including 0.1 per cent SDS and 0.2 M glycine. Electrophoresis was performed at room temperature at 35 mA for about 4 h. Electrophoretic transfers of proteins from polyacrylamide gels to nitrocellulose membranes (BioRad Laboratories, Mississauga, Ontario, Canada) were carried out for 20 h at 30 V in 25 mM Tris-HCl (pH 8.3) containing 0.2 M glycine. Immunoblotting of the placental G proteins was carried out using the BM Chemiluminescence Western blotting kit (rabbit) from Boehringer Mannheim (Laval, Quebec, Canada) using 25 ml of antisera diluted 1 : 1000 with 0.5 per

analysis

cDNA probes. Escherichia coli bacteria (HB 101 strain) were transformed by standard protocol using calcium chloride and plasmid DNA (Maniatis, Fritsch and Sambrook, 1982). pUC8 plasmids containing rat Goo and Gas cDNA inserts and pBR327 plasmids containing Gai2 and Gwi3 cDNA inserts were generously provided by Dr Robert Collu (Montreal University, Ste-Justine Hospital, Montreal, Quebec, Canada). The specificities of these cDNA probes were described previously (Jones and Reed, 1987); they were prepared from unique sequence regions of Gao (5’ end non coding sequence; 1.6 kb) and Gai3 (3’ end non coding region; 1.0 kb) the coding region of Gas (1.2 kb), and the full length cDNA of Gai2 (1.7 kb). pBR322 plasmids containing 18s rRNA were obtained from Dr Cheri Deal (Montreal University, Ste-Justine Hospital, Montreal, Quebec, Canada), those containing full-length P-LH cDNA (2.2 kb) were purchased from ATCC/NIH (Rockville, MD, USA), and those containing human full-length GH cDNA (0.8 kb) were provided by Dr Peter Cattini (Winnipeg, Manitoba, Canada). P-LH and hGH cDNAs recognized P-hCG and hPL in human placenta, respectively. DNA inserts were isolated as previously described (Petit et al., 1995) and labelled with deoxycitidine 5’-triphosphate ([a-32P]-dCTP; 3000 Ci/mmol; NEN DuPont) by random hexanucleotide primers and kleenow as DNA polymerase (Multiprime DNA labelling system, Amersham, Oakville, Ontario, Canada). The radioactive probes were separated from unicorporated [w-32P]-dCTP by an ion-exchange method on NACS prepac columns (Gibco/BRL, Burlington, Ontario, Canada). isolation. Total cellular RNA was extracted from placental villous tissues by a modification of the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987) using TRIzol Reagent (Gibco/BRL). Final RNA pellets were dissolved in diethyl pyrocarbonate treated water and stored at - 80°C. All RNA concentrations were determined by measuring absorbance at a wavelength of 260 nm. Typical extractions were 1 ug RNA/mg of tissues.

RNA

blots Total villous RNA samples (40 pg) were denatured by heating at 60°C for 15 min in buffer containing 50 per cent deionized formamide, 10 mM 4morpholinepropanesulphonic acid (MOPS) and 17 per cent formaldehyde. RNAs were separated by electrophoresis (1.2 per cent agarose), transferred to nylon membrane (Hybond-N, Amersham), fixed under UV lamp, prehybridized and hybridized as previously described (Petit et al., 1995). The membranes were then washed in 2X SSC with 0.5 per cent SDS (w/v) for 15 min at room temperature and in 1X SSC with 0.5 Northern

Petit et al.: G Proteins

in Molar

339

Placentae

(a) Mole

1

Mole

2

Mole

3

Mole

4

Normal 0.8 ~ hPL

hCG

1850

bp

Moles

Normal

Moles

Normal

18 S

Figure 1. Expression of hPL and hCG mRNAs in human placentae from hydatidiform moles. Total RNAs from trophoblastic tissues were extracted, separated by electrophoresis, and hybridized with hGH (hPL) and hLH (hCG) cDNAs as described in Materials and Methods. (a) Representative examples of blots obtained for hPL and hCG mRNAs. The 1% ribosomal RNA was used as a house keeping gene. The quantification of messengers for hPL (b) and hCG (c) by densitometry are the mean f SEM of four and five different placentae from normal and molar (mole) pregnancies, respectively. Results are expressed as the trimester to term (3rd trimester) ratio and normalized with 1% ribosomal RNA

per cent SDS for 1.5min at 60°C. Washed membranes were exposed to x-ray film (Kodak X-Omat AR5) with intensifying screens at - 80°C for l-3 days. The intensity of [32P]cDNARNA hybridization was quantified by scanning optical density of the autoradiographs (LKB Ultrascan XL). The steady state mRNA levels were expressed as arbitrary densitometric units (ADU) and standardized by comparison with hybridization results obtained with a random prime labelled 18s ribosomal RNA (rRNA) probe. Results were normalized with placentae at the same period of pregnancy.

RESULTS Expression

of hPL and hCG

The Northern analysis of hPL and hCG mRNA expression in human placentae from molar pregnancies is presented in Figure 1. Representative examples of hPL and hCG mRNA autoradiographs are shown in Figure I(a) whereas the quantitative evaluations of the Northern analysis are presented in Figure l(b) and l(c). Thus, hPL mRNA expression was lower in hydatidiform moles in comparison with normal placentae [Figure l(b)]. Contrary to that observed for hPL, hCG mRNA expression was higher in molar pregnancy than in normal pregnancy; the hCG level was around 237 per cent of normal placental at the same period of pregnancy [Figure l(c)]. Even if this pattern of gene expression is representative of molar trophoblastic tissue, and trophoblast is largely predominant in our tissue samples, a dilution of trophoblast contribution by other placental cells remains possible.

lmmunoblots The autoradiographs of the different Gu proteins were scanned with an LKB densitometer in order to obtain a semi-quantitative evaluation of placental Ga protein levels. As previously indicated (Petit et al., 1995), recombinant proteins were used as positive controls for each Go protein tested. Optical density of the immunoblots was proportional to the amount of protein applied to the gel within a range of 25-150 pg for all proteins studied with correlation coefficients 20.98 (not shown). Therefore, all studies were realized with 50 pg of proteins, and the results expressed as per cent of normal term placental membrane preparations. Each antibody recognized one major band with apparent molecular weight of 45 000 for Gas, 37 000 for Gao, 41 500 for Gai3 and 39 500 for Gai2 (Figure 2). The immunoblots presented in Figure 2 are representative of the results obtained for the different Ga protein subunits in normal and molar pregnancies. The results of the quantitative evaluations are shown in Figure 3. The relative levels of Gai3, Gai2, and Gao expressed in the human molar placenta were very low whereas Gas level was not significantly different from normal placentae. Thus, the levels of Gai3, Gai2, and Guo, reached 4, 39, and 42 per cent (PcO.05) respectively of those observed in normal pregnancies. We also observed a heterogeneity of expression, particularly for Gao and Gai2 with practically normal protein expression in some tissues and very low expression in mole 3. Moreover, a supplementary band at 47 kDa was recognized by Gai2 antibodies in mole 4.

Placenta

340 Mole

1

Mole

2

Mole

3

Mole

4

(1996),

Normal

37 700 Da

Go

39 500 Da

Gi2

41500

Gi3

Da

Figure 2. Expression of placental Ga proteins in molar pregnancy. Microsomes were extracted from trophoblastic tissues, separated by electrophoresis hvbridized with Ga orotein antibodies as described in Materials and Methods. Results are representative of blots obtained for the different Ga proteins normal and molar (mole) placentae. I

GClS

M

Gao

N

M

Gai2

N

M

Vol. 17

and with

Gai3

N

M

N

Figure 3. Quantification of placental Go proteins in molar pregnancy. The quantification of immunoblots obtained for human placental Go proteins (see Figure 2) was realized as described in Materials and Methods. Values are the mean f SEM obtained from immunoblots of Gas, Gao, Gai2, and Gai3 with four and five different placentae from normal (N) and molar (M) pregnancies, respectively. ‘WO.05 versus placental values, arbitrarily set at 100 per cent, by analysis of variance followed by Fisher’s PLSD comparison test.

Ga mRNA Northern analysis of total RNA from normal and molar human placentae were also realized. The results of these Northern blots are presented in Figures 4 and 5. A representative example of different Go mRNAs autoradiographs is shown in Figure 4 whereas the quantitative evaluation of the Northern

analysis is presented in Figure 5. Each cDNA probe recognized one band: 1.9 kb for Gas, 4.75 kb for Gao, 2.6 kb for Gai3 and 2.3 kb for Gai2 (Figure 3). The cDNA probe for Goo also recognized a minor band at 1.8 kb (not shown). As observed in immunoblot experiments, the relative levels of Gai3, Gai2, and Goo mRNA expressed in the human molar placentae were lower than normal term placenta whereas Gas

Petit et al.: G Proteins

in Molar

Placentae

341 Mole

1

Mole

2

Mole

3

Mole

4

Normal

1900 bp

4750

bp

2300

bp

2600

bp

1850

bp

Go

18 s

Figure 4. Expression of placental Ga protein mRNAs in molar pregnancy. Total RNAs from trophoblastic tissues were extracted, separated by electrophoresis and hybridized with Go protein cDNA probes as described in Materials and Methods. Results are representative of blots obtained for Gas, Gao, Gai2, and Gai3 with normal and molar (mole) placentae. The 18s ribosomal RNA was used as a house keeping gene.

Gao

Gai2

Gai3

T

r

M

N

M

N

M

N

M

N

Figure 5. Quantification of placental Gcr mRNAs in molar pregnancy. The quantification of blots obtained for human placental Gu protein mRNAs (see Figure 4) was realized as described in Materials and Methods. Values are the mean f SEM obtained from blots of Gas, Gao, Gai2 and Gai3 with four and five different placentae from normal (N) and molar (M) pregnancies, respectively. Results are expressed as the trimester to term (3rd trimester) ratio and normalized with the 18.5 ribosomal RNA. Therefore, the ratio for the third trimester has the value of 1. *SO.05 versus placental values, arbitrarily set at 100 per cent, by analysis of variance followed by Fisher’s PLSD comparison test.

levels were not significantly different (Figure 4). Thus, the level of Gai3, Gai2, and Guo in hydatidiform moles levels reached 3, 8, and 54 per cent (WO.05) respectively of normal term placentae.

DISCUSSION Our results demonstrate that Gai2, are expressed at low levels in human form moles whereas Gas proteins Moreover, a parallel in both mRNA

Gwi3, and Gclo proteins placentae from hydatidiare normally expressed. and protein expressions

was observed for all G proteins tested as previously reported for the placental ontogenesis of these proteins (Petit et al., 1995). The important changes in placental G protein expression observed in hydatidiform moles suggest significant alterations in the signal transduction machinery within the molar trophoblast. It is well known that changes in G protein expressions and G protein mutations are associated with cell transformation. Mutations of G proteins were reported, for example, in several endocrine tissues, and especially in the pituitary (for review see Gordeladze et al., 1994). Even if our study did not explore this hypothesis, it is possible that some hydatidiform moles may originate from G protein mutations.

342

Placenta

In such a may, our results suggest that the inhibitory pathways would be particularly interesting to study because the levels of inhibitory G proteins appeared relatively low in molar placentae. However, the impaired response of P-hCG and hPL to CAMP demonstrated in molar placentae (Morrish et al., 1992) suggested that stimulatory pathways may also be altered in hydatidiform mole even if Gels protein is normally expressed. Nevertheless, our results suggest that the altered placental G protein expression is associated with the modified placental functions of hydatidiform mole. However, our study does not discriminate if altered G protein expression is the causation or the effect of this pathology. Several important changes were observed in trophoblast of hydatidiform mole, and it appears clearly that the changes in G protein expression described in this study were insufficient to explain completely the cellular functions altered in these tissues. It is possible that other elements of the regulatory machinery are altered in hydatidiform mole, including G protein-coupled receptors, effecters (adenylate cyclase, phospholipases, channels) and cellular kinases. In this regard, hydatidiform mole is associated with chromosomal anomalies potentially associated with amplification of oncogene expression (Szulmann, 1988). These oncogenes, particularly c-myc, c-fms, c-ras, v-abl, v-erb A, v-fms, v-mos, v-myc, N-ras, and V-SK (Sarkar, 1986; Park et al., 1992; Cheung et al., 1993), are effectively overexpressed in molar trophoblastic tissues suggesting that cellular oncogene activation may be strongly

(1996),

Vol. 17

associated with neoplastic changes of the placenta, and may play a role in the development of molar pregnancies. The combination of the decreased cellular control by G proteins and the overexpression of oncogenes activating cellular activities could be considered as an important aspect of hydatidiform mole development. Generally, the alterations of cellular membrane structures are very important in oncology because they occur with cellular transformation and tumorigenesis (Hakomori, 1981, 1989). The membrane organization of G proteins is very complex (Neubig, 1994). The deficiency in G proteins expression in molar placenta may thus be associated with membrane defect implicated in mole pathology. Moreover, G proteins are known to participate to the cell structure by their association to cytoskeletal proteins (for review see Rasenick et al., 1985): G proteins were found associated with tubulin (Rasenick and Wang, 1988; Rasenick, Wang and Yan, 1990; Wang and Rasenick, 1991) and actin (Carlson et al., 1986; Bourguignon, Walker and Huang, 1990). Deficiency in G protein expression in molar placentae may also be associated with structure alterations affecting cellular activities. In conclusion, our results demonstrated important changes in placental G protein expression in hydatidiform moles and suggested significant alterations in the signal transduction processes within the molar trophoblast. Moreover, our results support an important role for this process in the regulation of peptide production by normal placentae.

ACKNOWLEDGEMENTS We thank

Biopedia

inc (Montreal,

Canada)

for its financial

support

and Dr Kenneth

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