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The Central Role of Human Chorionic Gonadotropin in the Formation of Human Placental Syncytium

Division of Research, Department of Obstetrics, Gynecology, and Women’s Health, University of Louisville Health Sciences Center, Louisville, Kentucky 40292

Address all correspondence and requests for reprints to: Dr. Ch. V. Rao, Department of Obstetrics, Gynecology, and Women’s Health, University of Louisville Health Sciences Center, Louisville, Kentucky 40292. E-mail: cvrao001{at}gwise.louisville.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
A number of trophoblast products, including human chorionic gonadotropin (hCG), can increase the formation of human placental syncytium through the differentiation of mononuclear cytotrophoblasts. The present study investigated the central role of hCG in this process by using antisense receptor phosphorothioate oligodeoxynucleotides (ODNs). Culturing cytotrophoblasts with the hCG/LH receptor antisense, but not sense, ODN resulted in a significant decrease in receptor protein levels and inhibited spontaneous as well as exogenous hCG induced increase in differentiation. The hCG/LH receptor antisense ODN also inhibited epidermal growth factor (EGF), TGF-, leukemia inhibitory factor (LIF), but not 8-bromo-cAMP, induced increases in differentiation, suggesting that hCG is required for EGF, TGF- and LIF, but not for the cAMP actions. Although antisense EGF receptor and LIF receptor ODNs inhibited EGF and LIF induced increase in differentiation, respectively, they were ineffective against hCG, suggesting that they use separate pathways, but they both converge on a common pathway requiring the hCG actions. Mechanism of action studies revealed that EGF treatment activates its receptors and MAPK, both of which are required for EGF to increase the differentiation, cAMP levels and activate protein kinase A. In summary, our results demonstrate that hCG is an autocrine and paracrine regulator that is required for EGF, TGF-, and LIF, but not for cAMP to increase human placental syncytium formation. Direct activation of protein kinase A seems to bypass the hCG pathway, perhaps by targeting genes associated with the differentiation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE PLACENTA IS A transient endocrine organ that mediates the physiological exchange between mother and fetus and also protects the fetus from harmful agents getting into the fetal circulation (1, 2). It consists of anchoring and free-floating villi. The free-floating villi, which bathe in maternal blood, contain the outer trophoblasts layer with multinuclear syncytium on the outside and mononuclear cytotrophoblasts on the inside followed by a connective tissue layer consisting of macrophages, fibroblasts, and fetal blood vessels (1, 2). The multinuclear syncytium is formed from mononuclear cytotrophoblasts through a process of differentiation that involves cellular aggregation followed by fusion of plasma membranes (1, 2). Each of these steps requires many biochemical and morphological changes, many of which are not well understood (3). The syncytium is a major transport, polarized, secretory epithelium whose formation and function is essential for establishment and maintenance of pregnancy. Defects in this formation were suspected to lead to complications such as preeclampsia, intrauterine growth retardation, and Down’s syndrome, etc. (1, 2, 4). It is very important for these reasons to better understand the regulation of syncytium formation. Increasing evidence suggests that the syncytium formation is an autocrine- and paracrine-regulated event involving a number of trophoblast (5, 6, 7, 8, 9, 10, 11, 12, 13, 14) and nontrophoblast (15, 16) products in placenta. Perhaps important among the trophoblast products is human chorionic gonadotropin (hCG), which is produced in large amounts by syncytiotrophoblasts and has intracrine, autocrine, paracrine, and endocrine actions during pregnancy (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). One of its autocrine and paracrine actions is increasing the syncytium formation as demonstrated by using biochemical, morphological, and biophysical techniques (7, 8, 9, 10, 11). The other trophoblast products that increase differentiation include epidermal growth factor (EGF), leukemia inhibitory factor (LIF), estradiol, and glucocorticoids (5, 10, 12, 14). Even though hCG is a differentiation marker, EGF and LIF are good stimulators of hCG production by differentiated trophoblasts (33, 34, 35, 36, 37, 38, 39, 40). This coincidental effect suggests a hypothesis that hCG may mediate their actions. If this hypothesis is correct, then preventing the hCG actions should block the differentiation induced not only by hCG but also by EGF, TGF-, and LIF. Conversely, preventing EGF/TGF- and LIF actions by inhibiting their corresponding receptor synthesis, should only block theirs, but not the hCG actions unless they happen to mediate the hCG actions. The hCG actions on differentiation are mediated by the cAMP/protein kinase A (PKA) pathway (7, 9, 11, 41). However, whether direct PKA activation by stable cAMP analogs requires hCG is not known. The present study investigated the central role of hCG in cytotrophoblast differentiation by using receptor antisense and sense oligodeoxynucleotides (ODNs).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
The following were purchased from the indicated commercial sources: recombinant human EGF from Austral Biologicals (San Ramon, CA); monoclonal antiphosphotyrosine antibody (IgG2bK) and affinity purified polyclonal antibody raised against recombinant human EGF receptor fusion protein from Upstate Biotechnology, Inc. (Lake Placid, NY); protein G-Sepharose beads from Amersham Biosciences Corp. (Piscataway, NJ); [-³²P]ATP (3000 Ci/mmol) from NEN Life Science Products (Boston, MA); enhanced chemiluminescence Western blotting detection kits from Amersham Life Sciences (Arlington Heights, IL); recombinant human TGF-, recombinant human LIF, monoclonal antibody raised against a slightly modified synthetic ß-cytoplasmic actin N-terminal peptide sequence, polyclonal antibody raised against a synthetic peptide derived from subdomain X1 of human MAPK, Percoll, normal rabbit serum, horseradish peroxidase-labeled antirabbit and antimouse IgG, deoxyribonuclease I, type III collagenase, trypsin, myelin basic protein, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, and 8-bromo-cAMP from Sigma (St. Louis, MO); peroxidase-antiperoxidase immunostaining kits from Vector Laboratories (Burlingame, CA); pre-Tag nonradioactive protein kinase A (PKA) activity measurement kits from Promega Corp. (Madison, WI); cAMP enzyme immunoassay kits from Cayman Chemical Co. (Ann Arbor, MI); PD98059 and PD153035 from Calbiochem-Novabiochem Corp. (San Diego, CA); affinity purified polyclonal antibody raised against recombinant human EGF from Oncogene Research Products (Cambridge, MA); affinity purified polyclonal antibody raised against a peptide mapping at the amino terminus of human LIF and affinity purified polyclonal antibody raised against a peptide corresponding to an amino acid sequence mapping at the carboxy terminus of the precursor form of human LIF receptor from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The following items were obtained as gifts: highly purified hCG (CR-127; 14,900 IU/mg) and hCG antiserum (lot 2) from NIDDK’s National Hormone & Pituitary Program and Dr. A. F. Parlow (Torrance, CA); 8-Cl-cAMP from the Drug Synthesis and Chemistry Branch at the National Cancer Institute (Bethesda, MD) and polyclonal hCG/LH receptor antibody raised against a synthetic N-terminal amino acid sequence of 15–38 from Dr. Patrick Roche (Mayo Clinic, Rochester, MN).

Tissues
Approximately 50 placentas were used in the entire study over a period of 2 yr. They were obtained from uncomplicated cesarean section deliveries performed at term pregnancies at the University of Louisville affiliated hospitals and transported immediately to the laboratory on ice. Their use was approved by our institutional Human Studies Committee.

Isolation of cytotrophoblasts
The procedure of Kliman et al. (42), with minor modifications, was used for cell dispersion and cytotrophoblasts isolation. Briefly, placental villi were cut and thoroughly washed to remove blood and then incubated for 30 min at 37 C with collagenase and deoxyribonuclease in a water bath with continuous shaking. The dispersed cells were filtered through a nylon mesh and centrifuged for 20 min at 1200 x g in 10–70% Percoll gradient. The middle layer (density 1.048–1.062) containing cytotrophoblasts was removed and washed once; cell viability was determined by the trypan blue exclusion test.

Cell culture
Approximately 2 x 10⁶ cytotrophoblasts per well were cultured at 37 C in DMEM containing 25 mM HEPES in humidified 5% CO₂-95% air in 12-well flat-bottom microplates. Various test agents were added to the culture medium, which was replaced every 24 h with medium of the same composition. Fetal bovine serum was excluded from the culture medium because of its potential interference with the cellular uptake of ODNs. The medium from some of the experiments were frozen at -80 C for later measurements of cAMP levels. The other details are given in the figure legends.

Design and synthesis of phosphorothioate ODNs
Fifteen to 21 base length sequences spanning the translation initiation ATG codon of hCG/LH receptor, EGF receptor, and LIF receptor were designed using designer PCR computer program (Research Genetics, Inc., Huntsville, AL). Then phosphorothioate ODN sequences were synthesized using a Pharmacia (Piscataway, NJ) LKB Gene Assembler Special automated DNA synthesizer using a standard phosphoramidate chemical procedure and desalted on NAP10 columns. The antisense sequences were effective in decreasing the corresponding receptor protein levels. The antisense as well as sense ODNs are as follows:

hCG/LH receptor antisense: 3'-GTACTTCGTCGCCAAGAGCCG-5'

hCG/LH receptor sense: 5'-CATGAAGCAGCCGTTCTCGGC-3'

EGF receptor antisense: 3'-CGCTACGCTGGGAGG-5'

EGF receptor sense: 5'-GCGATGCGACCCTCC-3'

LIF receptor antisense: 3'-TGTCTACTACCTATAA-5'

LIF receptor sense: 5'-ACAGATGATGGATATT-3'

Immunocytochemistry
Formalin-fixed cells were processed for immunostaining for hCG by an avidin-biotin-immunoperoxidase method using 1:400 dilution of hCG antibody (7). It was replaced with nonspecific IgG in the procedural control.

Quantitation of differentiation of cytotrophoblasts
The immunostaining for hCG and morphological changes were used as indexes of differentiation (10). Briefly, the cells were immunostained for hCG after the culture and then the number of different types of trophoblasts was counted among a total population of 200 under a microscope at a magnification of 40. The immunostaining intensity was not considered during the counting. The counting began at a random point near the middle of the coverslips, where the differentiation was most obvious. The mononuclear cells that did not stain for hCG were considered as cytotrophoblasts; mononuclear cells that were immunopositive for hCG were considered as transitional trophoblasts; and multinuclear syncytia that were positive for hCG immunostaining were considered as syncytiotrophoblasts. The number of syncytiotrophoblasts in cultures that were not treated with any test agent (controls) was considered 100% for the calculation of their changes in the treated cultures.

Western blotting
This procedure was performed by lysing the cells in homogenizing buffer consisting of 25 mM Tris-HCl, 1 mM EDTA, 20 mM NaCl, 1 mM dithiothreitol (DTT), 200 µM PMSF, 1 µM aprotinin and 20 µM leupeptin (43, 44). Aliquots of 30 µg protein were separated by 8% or 10% SDS-PAGE under reducing conditions and then electroblotted to Immobilon membranes. After blocking the nonspecific binding sites with 5% nonfat dry milk in 5 mM Tris-HCl (pH 7.4), 136 mM NaCl, 0.1% Tween (TBST buffer), the blots were incubated for 2 h at 22 C with 1:1500 dilution of polyclonal hCG/LH receptor antibody or 1:1000 dilution of polyclonal EGF receptor antibody. The blots were then washed three times for 10 min each with TBST buffer containing 40 mM sodium azide. The washed blots were reincubated for 1 h at 22 C with 1:2000 dilution of horseradish peroxidase-labeled antirabbit IgG or horseradish peroxidase-labeled antimouse IgG and washed. The antibody/antigen complexes were detected using the enhanced chemiluminescence Western blotting detection system. The molecular size of the proteins was determined by running standard molecular weight marker protein in an adjacent lane. To correct for protein loading differences, the membranes were washed for 30 min with TBST buffer containing sodium azide to strip the bound antibody, then rehybridized with ß-actin antibody. The relative optical densities of autoradiographic bands were determined by using a Z-gel scanning system (Zaxis Inc., Hudson, OH).

EGF receptor autophosphorylation
This procedure was performed on cultures containing only cytotrophoblasts or also those containing syncytiotrophoblasts (45, 46). In the first case, cytotrophoblasts were first allowed to attach to the culture wells by incubating for 4 h in the presence of 10% FBS. In the second case, cytotrophoblasts were cultured for 48 h in the absence of FBS. Then both cultures were treated for 10 min with 1 nM EGF and lysed in 20 mM HEPES (pH 8.0) containing 0.1% Triton X-100, 0.5% sodium deoxycholate, 5 mM NaF, 50 mM NaCl, 25 mM sodium pyrophosphate, 5 mM EDTA, 50 µM sodium vanadate, 5 µM leupeptin, 1 µM aprotinin, and 1 mM PMSF. Equal amounts of lysate protein were incubated overnight at 4 C with 1:1000 dilution of polyclonal human EGF receptor antibody to immunoprecipitate the receptors. Then the immunoprecipitates were absorbed to protein G-Sepharose beads for 1 h at 4 C with continuous mixing. The beads were washed three times with lysis buffer and resuspended in 50 µl 1x sodium dodecyl sulfate gel-loading buffer consisting of 50 mM Tris-HCl (pH 6.8) containing 100 mM DTT, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, and 10% glycerol and heated for 5 min at 100 C. The proteins in supernatants were separated by 6% SDS-PAGE under denaturing conditions and electroblotted to Immobilon membranes. The nonspecific binding sites were blocked by incubating membranes in TBST buffer containing 5% nonfat dried milk. After three washes with TBST buffer, the blots were incubated for 2 h with 1:1,000 dilution of monoclonal antihuman phosphotyrosine antibody, washed three times with TBST buffer and then incubated again for 1 h with 1:1500 horseradish peroxidase-labeled antimouse IgG. The antibody/antigen complexes were detected by using an enhanced chemiluminescence Western blotting detection kit. The molecular size of the proteins was determined by running standard molecular weight marker proteins in an adjacent lane. The relative optical densities of the bands were determined by using a Z-gel scanning system.

Measurement of MAPK activity
Cells were lysed in 20 mM Tris-HCl, pH 7.4 containing 137 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM sodium vanadate, 1 mM EGTA, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM glycerophosphate, 0.2 mM PMSF, 20 µM leupeptin, 1 µM aprotinin (TLB) (47, 48). Aliquots of 200 µg protein were incubated for 1 h at 4 C with 5 µg of polyclonal antihuman MAPK antibody to immunoprecipitate the enzyme. The immunoprecipitated MAPK was absorbed to protein G-Sepharose beads by incubating for 1 h at 4 C. The beads were then sequentially washed with TLB and TLB containing 25 mM HEPES (pH 7.4), 0.5 M NaCl, 10 mM MgCl₂, 10 mM MnCl₂, and 1 mM DTT (buffer A). The beads were suspended in 30 µl buffer A containing 3 µg myelin basic protein, 15 µM unlabeled ATP, 10 µCi [³²-P]ATP and incubated for 30 min at 30 C. Then gel-loading buffer was added to stop the reaction. The myelin basic protein in the media was electrophoresed on 12% SDS-PAGE under denaturing conditions. Then the gels were dried under reduced pressure and exposed for 2 d at -70 C to x-ray film with intensifying screens.

Measurement of cAMP levels
The cAMP levels in 50-µl media aliquots were quantified by enzyme immunoassay kits. The instructions supplied in the kits were followed in the measurement. The specificity of the cAMP antibody was 100% for acetylated cAMP, 0.3% for cAMP, 0.05% for cyclic GMP, and less than 0.01% for others. The assay was performed on batched samples. Thus, we do not have multiple assays to calculate interassay coefficient of variation. Because the original sample volumes were small, they could not be replicated several times within the same assay to calculate intraassay coefficient of variation. According to the kit manufacturer, these values were less than 10%. The variations among duplicates were well within 10% in our assay runs.

Measurement of PKA activity
The PKA activity was measured by using a pre-Tag nonradioactive detection assay kit. The instructions provided in the kit were followed. Briefly, 5 µg cell lysate protein aliquots were incubated for 30 min at 30 C with fluorescent-labeled A1 peptide. The phosphorylated and nonphosphorylated fluorescent peptides were separated on 0.8% gels and photographed. The phosphorylated peptide bands were then excised, eluted, and optical densities at 570 nm were measured using a 96-well plate reader. The PKA activities were calculated from these values. Positive (bovine heart PKA) and negative (omission of PKA) controls supplied in the kits were run in the assay at the same time as cell lysates.

Replication of experiments and statistical analysis
All the experiments were performed in duplicate and replicated three times on cells from different placentas. Six values for each data point were pooled for calculation of means and their SEs. Before transforming the values into percentages, they were analyzed by ANOVA and Duncan’s multiple range tests (48A ). The data presented in some of the figures (see Figs. 4, 6, and 13) came from a series of four experiments in which controls were run to test the effect of treatments. For example (see Fig. 4), the four series are as follows: hCG is first, EGF and TGF- are second, LIF is third, and 8-bromo-cAMP is fourth. The data sets from these experiments were separately analyzed. The data analysis did not consider time (see Fig. 3) or cell (Table 1; and see Fig. 8) variables as we knew that they would make a difference but they are not relevant to the hypothesis being tested.

View larger version (47K): [in this window] [in a new window]   Figure 4. Effect of hCG/LH receptor antisense and sense ODNs on hCG, EGF, TGF-, LIF, and 8-bromo-cAMP increased differentiation of human cytotrophoblasts. The cells were cultured for 48 h in the presence or absence of 4 µM ODNs, 500 mIU/ml hCG, 1 nM EGF, and TGF-, 10 ng/ml LIF, and 1 mM 8-bromo-cAMP. *, P < 0.05 compared with control cells cultured with any test agent.

View larger version (39K): [in this window] [in a new window]   Figure 6. Effect of EGF receptor antisense and sense ODNs on EGF, hCG, and LIF, 8-bromo-cAMP increased differentiation of human cytotrophoblasts. The cells were cultured for 48 h in the presence or absence of 4 µM ODNs, 1 nM EGF, 500 mIU/ml hCG, 10 ng/ml LIF, and 1 mM 8-bromo-cAMP. *, P < 0.05 compared with control cells cultured without any test agent. The treatments with hCG, LIF, and 8-bromo-cAMP in the absence of EGF receptor antisense ODN were performed. Because similar data were obtained as in Fig. 4, they were not repeated in this figure.

View larger version (41K): [in this window] [in a new window]   Figure 13. Effect of LIF receptor antisense and sense ODNs on LIF, hCG, EGF, and 8-bromo-cAMP induced increase in differentiation of human cytotrophoblasts. The cells were cultured for 48 h in the presence or absence of 4 µM ODNs, 10 ng/ml LIF, 500 mIU/ml hCG, 1 nM EGF, and 1 mM 8-bromo-cAMP. *, P < 0.05 compared with the control cells cultured without any test agent. The reason for not including hCG, EGF, and 8-bromo-cAMP treatments in the absence of LIF receptor antisense ODN is the same as that given in Fig. 7 legend.

View larger version (48K): [in this window] [in a new window]   Figure 3. Time- (A) and dose- (B) dependent effect of hCG/LH receptor antisense and sense ODNs on spontaneous differentiation of human cytotrophoblasts. ODNs were used at the concentration of 4 µM in the time dependency experiments and the cells were cultured for 48 h in the dose-dependency experiments. *, P < 0.05 compared with corresponding control cells cultured without the ODNs.

View larger version (35K): [in this window] [in a new window]   Figure 8. Effect of EGF on its receptor autophosphorylation in cytotrophoblasts and syncytiotrophoblasts. The cultures were treated for 10 min with or without 1 nM EGF, and then the receptor autophosphorylation was determined as described in Materials and Methods section. The phosphorylation was considered 100% in control cultures for the calculation of changes in EGF-treated cells. *, P < 0.01 compared with corresponding control cells cultured without EGF.

	Results

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Figure 1, A–C, shows that cytotrophoblasts could differentiate in the absence of FBS. Quantitation revealed a progressive decline in the number of cytotrophoblasts with a concomitant significant increase in transitional trophoblasts and syncytiotrophoblasts during 48 h of culture (Table 1). Sixty-eight percent of the cells were cytotrophoblasts, 21% were transitional trophoblasts, and 11% were syncytiotrophoblasts at the end of culture (Table 1). For the sake of simplicity, changes in syncytiotrophoblasts number, expressed as percent control, are presented in the rest of this article.

View larger version (92K): [in this window] [in a new window]   Figure 1. Spontaneous as well as hCG, EGF, TGF-, LIF, and 8-bromo-cAMP induced increases in differentiation of human cytotrophoblasts. The cells were cultured without FBS in the absence (A–C) or in the presence of 500 mIU/ml hCG (D), 1 nM EGF (E), TGF- (F), 10 ng/ml LIF (G), and 1 mM 8-bromo-cAMP (H). The cells were cultured for 12 (A), 24 (B), or 48 h (C–H). Magnification, x300.

View larger version (50K): [in this window] [in a new window]   Figure 2. The hCG/LH receptor protein levels in human cytotrophoblasts cultured for 48 h in the presence or absence of 4 µM antisense and sense hCG/LH receptor ODNs. The receptor protein levels measured by Western blotting were corrected for ß-actin signal. The levels in controls were considered 100% for the calculation of changes in the treated cells. *, P < 0.05 compared with control cells cultured without ODNs.

Figure 3B shows that hCG/LH receptor antisense ODN inhibition of spontaneous differentiation is concentration dependent. Thus, a significant inhibition occurred at the lowest concentration tested, which progressively increased up to a 10-µM concentration. Because routine use of a 10-µM concentration would be prohibitively expensive, a 4-µM concentration was used in the remaining experiments.

Figures 1D and 4 show that the addition of 500 mIU/ml hCG to the culture medium resulted in a significant increase in the differentiation of cytotrophoblasts. This increase was blocked by simultaneous addition of 4 µM antisense, but not sense, hCG/LH receptor ODN (Fig. 4).

The addition of 1 nM EGF and TGF-, which activate their common receptor (53, 54), resulted in a significant increase in the differentiation (Figs. 1, E and F, and 4). Their effects were also blocked by the simultaneous addition of antisense, but not sense, hCG/LH receptor ODN (Fig. 4).

The addition of 10 ng/ml LIF also significantly increased the differentiation, and its effect was also blocked by concomitant treatment with antisense, but not sense, hCG/LH receptor ODN (Figs. 1G and 4).

The addition of 1 mM 8-bromo-cAMP resulted in a significant increase in the differentiation (Figs. 1H and 4). This increase was unaffected by the addition of antisense or sense hCG/LH receptor ODN (Fig. 4). 8-Cl-cAMP, which is a selective inhibitor of type 1 PKA (55), had no effect on 8-bromo-cAMP action, suggesting that type II PKA was involved in mediating the cAMP actions in trophoblasts (data not shown).

The EGF receptor ODNs effect on differentiation
The human trophoblasts contain an abundance of EGF receptors (34, 37, 46, 48, 56, 57, 58, 59, 60, 61). The requirement for these receptors in the EGF action to increase the differentiation of cytotrophoblasts was tested by using EGF receptor phosphorothioate ODNs.

Figure 5 shows that the addition of 4 µM antisense, but not sense, EGF receptor ODN resulted in a significant decrease in EGF receptor protein levels.

View larger version (36K): [in this window] [in a new window]   Figure 5. The EGF receptor protein levels in human cytotrophoblasts cultured for 48 h in the presence and absence of 4 µM antisense and sense EGF receptor ODNs. The receptor protein levels measured by Western blotting were corrected for ß-actin signal. The levels in controls were considered 100% for the calculation of changes in the treated cells. *, P < 0.05 compared with control cells cultured without ODNs.

Because EGF is known to work by activating its receptors and subsequently increase the catalytic activity of MAPK (62), we tested the ability of kinase inhibitors to prevent the EGF action on differentiation. Figure 7 shows that, whereas EGF receptor kinase inhibitor (PD153035) and MAPK inhibitor (PD98059) had no effect on their own, they were able to prevent EGF induced increase in differentiation. These data led us to examine the EGF effect on its receptor activation through increased receptor autophosphorylation and catalytic activity of MAPK.

View larger version (35K): [in this window] [in a new window]   Figure 7. Effect of EGF receptor kinase inhibitor (PD153035), MAPK inhibitor (PD98059), and EGF on differentiation of human cytotrophoblasts. The cells were cultured for 48 h in the presence or absence of 1 nM PD153035, 50 nM PD98059, and 1 nM EGF. *, P < 0.01 compared with control cells cultured without any test agent.

Figure 9 shows that the addition of 1 nM EGF resulted in a significant increase in the catalytic activity of MAPK. Whereas EGF receptor kinase and MAPK inhibitors had no effect on their own, they were able to block EGF-induced activation of MAPK.

View larger version (32K): [in this window] [in a new window]   Figure 9. Effect of EGF on MAPK activation in the presence and absence of MAPK and EGF receptor kinase inhibitors. The cells were cultured for 48 h in the presence or absence of 1 nM EGF, 50 nM PD98059, and 1 nM PD153035 and then MAPK catalytic activity was determined as described in Materials and Methods. *, P < 0.05 compared with control cells cultured without any test agent.

View larger version (44K): [in this window] [in a new window]   Figure 10. Effect of EGF, MAPK inhibitor, and EGF receptor kinases inhibitor on cAMP levels in human cytotrophoblasts. The cells were cultured for 48 h in the presence or absence of 1 nM EGF, 50 nM PD98059, and 1 nM PD153035, and then cAMP levels in media were measured. *, P < 0.05 compared with control cells cultured without any test agent.

View larger version (40K): [in this window] [in a new window]   Figure 11. Effect of EGF, MAPK inhibitor, and EGF receptor kinases inhibitor on PKA activation in human cytotrophoblasts. The cells were cultured for 48 h in the presence or absence of 1 nM EGF, 50 nM PD98059, and 1 nM PD153035, and then catalytic activity of PKA was determined as described in Materials and Methods. *, P < 0.05 compared with control cells cultured without any test agent.

View larger version (98K): [in this window] [in a new window]   Figure 12. Immunostaining for LIF receptors in human cytotrophoblasts cultured for 48 h in the absence (A) and in the presence of 4 µM LIF receptor antisense (B) and sense (C) ODNs. Magnification, x300.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The formation of human placental syncytium is essential for the establishment and continuation of pregnancy (1, 2). Defects in this formation can be seen in several pregnancy complications (1, 2, 4). A growing body of evidence supports the notion that the differentiation is locally controlled by a number of trophoblast products, including hCG (5, 6, 7, 8, 9, 10, 11, 12, 13, 14). The previous studies have demonstrated that not only endogenous but also exogenous hCG can increase the morphological and functional differentiation in a dose-dependent, time-dependent, and hormone-specific manner (7, 8, 9, 10, 11).

EGF, TGF-, LIF, estradiol, and glucocorticoids are among the other trophoblast products that can increase the differentiation (5, 10, 12). Even though hCG is a differentiation marker, striking increases in hCG in production by differentiated trophoblasts in response to EGF and LIF treatment (33, 34, 35, 36, 37, 38, 39, 40) suggest a hypothesis that hCG is necessary for the EGF and LIF actions. If the hypothesis is correct, then inhibiting the hCG actions should not only block hCG but also the EGF and LIF actions on the differentiation.

hCG actions can be inhibited by its immunoneutralization or depleting its receptors from cells so hCG will not be able to act. Previous attempts to immunoneutralize hCG receptors were unsuccessful because receptor antibody mimicked the ligand (7). Therefore, in the present study we used the antisense ODN approach by designing and synthesizing phosphorothioate derivatives from human hCG/LH receptor sequences that resist degradation by nucleases (63). The sense ODN was also synthesized to determine the specificity of antisense ODN effects.

The hCG/LH receptor half-life greatly varies among cells (64, 65, 66, 67, 68). In all cases, hormone addition decreases the half-life, and this homologous receptor down-regulation is also commonly seen among other receptor systems (64, 65, 66, 67, 68). Although the half-life of trophoblast hCG/LH receptors is not known, it is likely to be much lower than in the other cell types due to high hCG production. Thus, simultaneous addition of oligos with corresponding ligands should be effective in reducing receptor protein levels. This decrease was sufficient to attenuate hCG signaling such as a cAMP increase and PKA activation (data not shown).

Culturing cytotrophoblasts with hCG/LH receptor antisense, but not sense, ODN resulted in a significant decrease in hCG/LH receptor protein levels. The decrease was not 100%, but it was sufficient to test our hypothesis.

The differentiation in culture is a spontaneous process, probably driven by endogenous regulatory molecules produced by cytotrophoblasts as well as by those that are in the process of differentiation or already completed the process. In the presence of FBS, the process is rapid and nearly complete by about 24–48 h (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 42). However, FBS could not be used in our study because of its interference with ODNs uptake. Nevertheless, cytotrophoblasts did differentiate in the absence of FBS.

Treatment of cytotrophoblasts with hCG/LH receptor antisense, but not sense, ODN resulted in a time- and dose-dependent inhibition of differentiation, suggesting that spontaneous differentiation was driven by endogenous hCG. In agreement with previous studies, treatment of cytotrophoblasts with hCG, EGF, TGF-, LIF, and 8-bromo-cAMP significantly increased their differentiation (5, 7, 8, 9, 10, 11, 41, 69, 70, 71). The effect of all these agents, except 8-bromo-cAMP, could be inhibited by cotreatment with hCG/LH receptor antisense, but not sense, ODN. These results suggest that not only the hCG’s actions, but also those of EGF, TGF- and LIF require the presence of hCG/LH receptors. The reason for hCG/LH receptor requirement is that EGF, TGF-, and LIF increase hCG production, which then activates its receptors to increase cytotrophoblasts differentiation into syncytiotrophoblasts, which results in even more hCG production. Measurement of hCG levels in the culture medium revealed that they were below the detection limit of the assay, which probably reflects low differentiation in the absence of FBS. Even though 8-bromo-cAMP also can increase hCG production (72, 73), it appears to bypass the hCG route by directly activating PKA, which may target genes associated with the differentiation.

To eliminate the possibility that EGF and TGF- could mediate the hCG actions, we again used the antisense ODN approach. The 15- and 16-oligomer antisense and sense phosphorothioate ODNs were synthesized from human EGF receptor and LIF receptor sequences. The antisense, but not sense, ODN could significantly decrease corresponding receptor protein levels. Treatment with antisense ODNs alone had no effect on spontaneous differentiation, suggesting that endogenous EGF and LIF did not play a significant role under the present culture conditions. Consistent with this possibility, cells showed very little EGF immunostaining, but did show considerable amounts of TGF- and EGF/TGF- receptor immunostaining, which did not significantly increase during culture (data not shown). Our data, in agreement with previous reports (61, 74, 75), suggest that human trophoblasts primarily produce TGF-.

EGF receptor antisense, but not sense, ODN prevented EGF from increasing the differentiation. The EGF receptor antisense ODN, on the other hand, was ineffective against hCG-, LIF-, and 8-bromo-cAMP-induced increases in differentiation. Similarly, treatment of cytotrophoblasts with LIF receptor antisense, but not sense, ODN, prevented LIF from increasing the differentiation. The LIF receptor antisense ODN, on the other hand, was ineffective against hCG, EGF, and 8-bromo-cAMP induced increases in differentiation. These findings, along with those obtained using hCG/LH receptor antisense ODN, suggest that, whereas EGF and LIF use their own pathways, they both converge on a common pathway requiring the hCG actions to increase the differentiation.

The molecular mechanisms of EGF action have been best characterized (62). However, its mechanism of action in cytotrophoblasts differentiation is unknown, but it is likely to be similar to other cases. Human trophoblasts are a rich source of EGF receptors (34, 37, 46, 48, 56, 57, 58, 59, 60, 61). Addition of EGF to cultures containing cytotrophoblasts, with or without syncytiotrophoblasts, resulted in receptor activation through increased receptor autophosphorylation in tyrosine residues. The greater response in cultures containing syncytiotrophoblasts was probably due to increased EGF receptor protein levels and/or increased responsiveness of the existing receptor protein. The latter possibility appears to be more likely because the EGF receptor immunostaining did not significantly increase during 48 h of culture (data not shown).

As expected, the activated EGF receptor stimulated MAPK catalytic activity. The inhibition of EGF receptor kinase and MAPK by synthetic compounds prevented EGF from increasing the differentiation, suggesting that activation of both kinases is required for EGF to increase the differentiation.

EGF can stimulate hCG production, which increases cAMP levels and activates PKA (Refs. 7, 21 and 22 ; and data not shown). Then, EGF should be expected to mimic hCG, which it did by increasing cAMP levels and activating PKA. If these EGF-induced changes are down stream from EGF receptor kinase and MAPK activation, then the kinase inhibitors should prevent EGF from increasing cAMP levels and activating PKA, which they did.

Figure 14 and its legend describe the proposed model of regulation of human placental syncytium formation and the mechanism of EGF action. This model predicts that trophoblast products that increase hCG production, as well as syncytium formation, require hCG. This prediction was independently verified for LIF using hCG antibody (10). The action of estradiol, which increases cytotrophoblast differentiation and hCG production, was not prevented by the addition of hCG antibody (14). This suggests that estradiol my use a different mechanism or that the antibody may not have neutralized all the hCG. Without presenting the data, Sawai et al. (41), claimed that hCG antibody could block 8-bromo-cAMP-induced increase in differentiation. Our data from using hCG/LH receptor antisense ODN, which inhibited hCG/LH receptor synthesis and blocked hCG action, contradicts this claim.

View larger version (15K): [in this window] [in a new window]   Figure 14. Proposed model of autocrine and paracrine regulation of human placental syncytium formation (A) and the mechanism of EGF action (B). In this model, hCG made by transitional and syncytiotrophoblasts acts in an autocrine and paracrine manner to increase the differentiation. In addition, EGF, TGF-, and LIF stimulate hCG production, which then acts via its receptor to stimulate the differentiation of cytotrophoblasts into syncytiotrophoblasts, which then results in more hCG being produced. Because human placenta seems to primarily produce TGF- rather than EGF, it is the former that is perhaps physiologically relevant in activation of EGF/TGF- receptors. The mechanism of action studies revealed that EGF activates its receptor and MAPKs, which are required for EGF to increase the differentiation and cAMP levels and activation of PKA. The receptor antisense ODNs and kinase inhibitors shown can block the EGF action. Even though the cAMP/PKA signaling pathway mediates the hCG actions, direct PKA activation seems to bypass the hCG pathway.

	Footnotes

 
¹ Present address: Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77030.

Abbreviations: DTT, Dithiothreitol; EGF, epidermal growth factor; hCG, human chorionic gonadotropin; LIF, leukemia inhibitory factor; ODN, oligodeoxynucleotide; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride.

Received September 3, 2002.

Accepted for publication December 3, 2002.

	References

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