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Biology of Reproduction 61, 681-686 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Identification of a Stress-Induced Protein During Human Trophoblast Differentiation by Differential Display Analysis1

Bo Xua, Lin Lina, and Neal S. Rote2,a,b

a Departments of Microbiology & Immunology and b Obstetrics & Gynecology, Wright State University School of Medicine, Dayton, Ohio 45435


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differentiation of human placental trophoblast is characterized by a process during which mononuclear villous cytotrophoblasts fuse to form a multinucleate syncytium. This event is associated with dramatic changes in gene expression. In the present study, we have applied a sensitive approach—differential display analysis—to evaluate changes in gene expression during in vitro forskolin-induced differentiation of a model of human trophoblast, the choriocarcinoma BeWo. We identified seven genes that were up-regulated; their expression and function have not previously been reported in trophoblast. Four up-regulated genes were novel upon comparison of their sequences to the GenBank database. The other three genes encode human cytochrome p450 IIC, inosine monophosphate dehydrogenase type II, and reducing agent and tunicamycin-responsive protein (RTP). Northern blot analysis revealed that RTP mRNA expression was induced to 3-fold in BeWo after 24-h incubation with forskolin and increased up to 11-fold by 72 h of forskolin treatment. The expression pattern of RTP was further investigated by in situ hybridization on second trimester and term placenta tissues. RTP mRNA was predominantly expressed in syncytiotrophoblasts in both second trimester and term placentae. The expression of RTP gene in BeWo cells was protein kinase C dependent. This is the first description of RTP gene expression in placenta and the first study elucidating the signaling pathway involved in the regulation of RTP gene expression. These results suggest that RTP may play a role in trophoblast cell proliferation and differentiation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During placental growth and development, the mononuclear cytotrophoblasts undergo progressive differentiation, resulting in their fusion to form a nonmitotic multinucleate syncytium [1–3]. This process is associated with dramatic changes of the expression of genes that play important roles in trophoblastic differentiation. Although previous studies have shown several genes whose expression level in trophoblast was altered during differentiation [4–8], the biological role of many of these genes is unclear. It is also highly possible that some of the crucial genes involved in the trophoblast differentiation remain to be identified.

The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as an in vitro model for trophoblast intercellular fusion and differentiation [9–13]. Under normal culture conditions, BeWo cells grow with cytotrophoblast-like features. Differentiation can be induced by a variety of reagents, such as growth factors, methotrexate, cyclic AMP analogues, or forskolin, an adenylate cyclase stimulator [12–15]. The system of forskolin-induced BeWo cell differentiation has been extensively used in our laboratory to study intercellular fusion, endogenous retrovirus expression, and plasma membrane phospholipid modulation [8, 16–19].

In the present study, we have applied a sensitive technique—differential display analysis—to evaluate changes in gene expression during in vitro induction of BeWo cell differentiation. We identified a set of genes that are up-regulated in this process. One of particular interest is the gene coding a new stress-induced protein termed reducing agent and tunicamycin-responsive protein (RTP) [20]. Results from the study of RTP gene regulation and its localization in placenta tissue suggest that it has a biological function in cell proliferation and differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Treatments

A human BeWo choriocarcinoma cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were maintained as a monolayer in culture flasks and induced to differentiate in vitro as described previously [8, 16, 17]. Briefly, culture medium F12 (Sigma, St. Louis, MO) was supplemented with 15% fetal bovine serum (FBS), 2 mM L-glutamine, and 0.2% glucose (Sigma). Cells were grown in a humidified incubator with 5% CO2 at 37°C. Cell differentiation was induced by 100 µM forskolin in culture medium for various times. Signal transduction pathways were evaluated using protein kinases inhibitors purchased from Calbiochem (La Jolla, CA). The optimal concentration for each inhibitor used in this study was based on its IC50 suggested by the manufacture and its concentration as used by others [21, 22]; 10 µM H-89 (protein kinase A inhibitor), 100 nM hydrochloride bisindolylmaleimide I (protein kinases C inhibitor), and 10 µM genistein (protein tyrosine kinases inhibitor). The stock solutions of forskolin (Sigma), protein kinase inhibitors, phorbol-12-myristate-13 acetate (PMA), and 4{alpha}-phorbol-12,13-didecanoate (4{alpha}PDD) (CalBiochem) were dissolved in either H2O (H-89 and hydrochloride bisindolylmaleimide I) or DMSO (genistein, PMA, and 4{alpha}PDD) at 1000-strength concentration. These solutions were added into the culture medium on monolayer cells at various concentrations. After incubation, cells were collected by trypsinization and then washed with PBS. Total cellular RNA was extracted using RNAeasy kit (Qiagen, Valencia, CA).

Differential Display Analysis and DNA Sequencing

Differential display analysis was performed using a RNAimage kit (GenHunter Corp., Nashville, TN) as described [23–25]. Total RNAs extracted from forskolin-treated BeWo cells at different time points were reverse transcribed with oligo-dT primers. This process was followed by the PCR reaction in the presence of the second 10mer arbitrary primers. Amplified cDNA subpopulations were distributed on a DNA sequencing gel. The bands of interest were cut out from the polyacrylamide gel, and cDNA was eluted by boiling in 100 µl dH2O for 10 min. The supernatant was precipitated by adding 10 µl of 3 M sodium acetate, 5 µl of glycogen (10 mg/ml), and 450 µl of pure ethanol. The cDNA fragments were reamplified using the same pair of primers. The reamplified fragments were purified from agarose gels and subcloned into pCR-TRAP vector (GenHunter Corp). DNA sequencing was carried out using Sequenase Version 2.0 kit (Amersham Life Science, Cleveland, OH). Comparison of DNA sequences to the database of GenBank was done by the Blast Service provided by NIH (Bethesda, MD)

Northern Blot Analysis

Total RNA was extracted using RNAeasy kit as described above. Ten micrograms of RNA isolated from BeWo cells after various treatment was loaded onto a 1% agarose gel containing 0.66 M formaldehyde, 40 mM MOPS-NaOH (pH 7.2), 10 mM sodium acetate, and 1 mM EDTA (Sigma). After electrophoresis at 40 volts for 4 h, RNA was transferred onto a Nytran nylon membrane using a Turbo blotter (Schleicher & Schuell, Keene, NH) overnight. The membrane was then cross linked by baking in a vacuum oven at 80°C for 1 h and hybridized with a 32P-labeled cDNA probe of RTP open reading frame using random primer labeling kit (New England Biolab, Beverly, MA) at 42°C overnight. The open reading frame of RTP (1182 base pairs [bp]) was amplified by PCR, using the RTP gene (generous gift from Dr. Kokame, National Cardiovascular Center Research Institute, Japan) as template, and subcloned in the pCR2 vector (Invitrogen, San Diego, CA). The RNA bands were visualized by autoradiography after exposure of film for 24–48 h at -80°C. The intensity of each band was quantified by densitometry using software provided by the manufacturer (IDP Inc., Huntington Station, NY). Fold induction was calculated as the ratio between each band to the zero time point after normalized by the intensity of 28S RNA. In some experiments, RNA blots were stripped with 0.1-strength SSC and 0.5% SDS at 80°C for 30 min and rehybridized with ß-hCG probe as described previously [8].

In Situ Hybridization

Human second trimester and term placentae were collected at Miami Valley Hospital in Dayton, Ohio, from patients undergoing normal delivery according to protocols approved by the Institutional Review Boards of Wright State University and Miami Valley Hospital. In vitro transcription was first performed to generate a digoxigenin (DIG)-labeled RTP cRNA probe using an RTP cDNA as a template. The RTP cDNA in the pCR2 was linearized to generate both sense and antisense strands using either HindIII or XbaI restriction enzyme digestion. The linearized cDNAs were transcribed with T7 or SP6 polymerase to produce either a sense or antisense DIG-labeled cRNA probe. The procedure for in vitro transcription was performed according to the method supplied with the Genius 4/DIG RNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN). To allow efficient hybridization and good penetration of the tissue, the synthesized cRNA probe was subjected to alkaline hydrolysis to generate 200- to 500-bp lengths before use in in situ hybridization.

The in situ hybridization protocol was based on the method of Martinez-Montero with some modification [8]. Normal human placenta tissues that were formalin-fixed and paraffin wax-embedded were cut as 5-µm-thick sections and mounted on 3-aminopropyltriethoxysilane coated slides. After dewaxing with Hemo-De (Fisher Scientific Inc., Pittsburgh, PA) and absolute alcohol, tissue sections were treated with 0.4% pepsin in 0.2 M HCl at room temperature for 15 min to unmask the target RNA. Tissue sections were covered with 100 µl of hybridization buffer containing 50% formamide, 5% dextran sulfate, double-strength SSC, 0.1 mM Tris-HCl, 100 µg/ml yeast tRNA, and a DIG-labeled cRNA probe at a concentration of 2 ng/µl. Slides were heated at 95°C for 15 min and incubated at 37°C overnight in a humidified chamber. Following hybridization, the slides were washed three times with double-strength SSC at room temperature, with double-strength SSC at 37°C, 3 times with single-strength SSC at room temperature, and with single-strength SSC at 37°C. DIG-labeled RTP probes were detected using alkaline phosphatase conjugated anti-DIG antibody (Boehringer Mannheim) and nitroblue tetrazolium containing 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP). The hybridization signal was observed under light microscopy. The dark blue/brown coloration indicated a positive hybridization signal.

To demonstrate the specificity of the hybridization signal, the following controls were utilized: RNase treatment of tissues on slides, substitution of the anti-sense probe with sense probe, and use of only hybridization buffer with no addition of cRNA probes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Differentiation-Related Genes in BeWo Cells by Differential Display Analysis

Differential display analysis allows the observation of altered expression and subsequent isolation of cDNAs that correspond to mRNAs in differentiating cell populations [26–28]. To identify genes expressed in the process of cell differentiation, we have compared mRNA expression patterns of proliferating BeWo and forskolin-induced differentiating cells. BeWo cells were treated with 100 µM forskolin in the culture medium for 4, 24, and 48 h. Total cellular RNA was extracted and subjected to differential display analysis. The band intensities of some PCR products were increased by forskolin treatment (Fig. 1). By using the combination of 8 arbitrary primers and 3 one-base anchored oligo-dT primers, we observed 18 PCR fragments that were differentially displayed (15 up-regulated and 3 down-regulated) between proliferating and forskolin-induced differentiating BeWo cells. Reamplified PCR products were sequenced and compared to the nucleotide database in GenBank; among the 15 up-regulated cDNAs, 4 had novel sequences without significant homology to the known gene sequences in the database. Several other cDNA sequences were identical to those of the following gene products: human cytochrome p450 IIC, inosine monophosphate dehydrogenase type II, and stress-induced protein RTP. Because RTP gene expression had not been reported in placental trophoblast, and its function is unknown thus far, RTP was chosen for further investigation.



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  		FIG. 1. Differential display of mRNA from proliferating and forskolin-induced differentiating BeWo cells. Total RNA extracted from proliferating and differentiating BeWo cells was subjected to differential display analysis as described in Materials and Methods. Arrows indicate the representative band pattern showing up-regulated PCR fragments in forskolin-treated cells. Cp301, RTP; Gp201, unknown gene; Gp202, inosine monophosphate dehydrogenase type II. Lane 1, time 0; lane 2, 4 h; lane 3, 24 h; lane 4, 48 h.

Confirmation of Differential Display Analysis by Northern Blot

To confirm the differential display results, Northern blot analysis was performed. Total RNA isolated from each time point after forskolin treatment was subjected to electrophoresis and transferred to the Nytran membrane. The cDNA fragment corresponding to the ORF of the RTP gene was used as probe for Northern blot. RTP gene expression was induced in BeWo cells after 24-h incubation with forskolin and continued to increase up to 72 h of treatment (Fig. 2). The size of the transcript was approximately 3 kilobases, corresponding to that in previous reports [20, 29]. The up-regulation of RTP gene during forskolin-induced BeWo cell differentiation was concurrent with ß-hCG production, one of common trophoblastic differentiation marker (Fig. 2). Quantitation analysis by densitometry revealed that the levels of RTP mRNA were increased to about 3-fold by 24 h, 5-fold by 48-h treatments, and the greatest induction (11-fold) was observed at 72-h treatment.



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  		FIG. 2. Confirmation of differential display results by Northern blot. Upper) Induction of RTP expression during BeWo cell differentiation. BeWo cells were treated with 100 µM forskolin for 24 h, 48 h, and 72 h. Total RNA (10 µg) isolated from different samples were loaded in each lane, transferred to a nylon membrane, and hybridized by a 32P-labeled ORF of RTP. Middle) RNA blot was hybridized with ß-hCG probe. Bottom) 28S and 18S RNA serve as internal control. Kb, Kilobases.

Expression of RTP Gene in Normal Placental Trophoblasts

The BeWo cell line was derived from human gestational choriocarcinoma. Our in vitro data indicated that RTP mRNA expression was increased during forskolin-induced differentiation. To determine whether the expression pattern would be also true in the process of normal trophoblast differentiation, we performed in situ hybridization in tissues from second trimester and term placentae. A positive hybridization signal was only seen in tissue sections hybridized with the antisense RTP cRNA probe (Fig. 3). Specific RTP mRNA in tissues of second trimester and term placenta was predominantly localized to the syncytiotrophoblast layer of the chorionic villi (Fig. 3, A and B). Controls using semiserial sections of tissues hybridized with the sense strand of RTP cRNA were negative (Fig. 3C). RNase pretreatment of tissue sections completely removed the positive signals in tissues hybridized with antisense RTP cRNA probes (data not shown, but identical with Fig. 3C).



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  		FIG. 3. Expression of RTP mRNA in human placenta tissues. In situ hybridization was performed using DIG-labeled RTP antisense probe in term placenta (A) and second trimester tissue sections (B), or control sense probe (C). The hybridization signal was seen as dark blue/brown coloration and observed under light microscopy. The pictures were taken at x40 magnification for A and B and x20 magnification for C (published at 76%).

Regulation of RTP Gene Expression by Signaling Pathways

Forskolin-induced BeWo cell differentiation is known to occur by activation of adenylate cyclase and subsequently increased intracellular cAMP levels [30]. To determine whether intracellular signaling pathways were involved in the regulation of RTP gene expression, BeWo cells were treated with various protein kinase inhibitors at 6 h before forskolin treatment. After 72 h, cells were harvested and total RNA was extracted for Northern blot analysis. RTP expression level was increased to 9-fold by 72-h culture with forskolin (Fig. 4A). This induction was significantly reduced to only 3-fold by PKC inhibitor, while neither PKA nor PTK inhibitor had an inhibitory effect. To further confirm that activation of the PKC pathway was required for RTP gene expression, BeWo cells were treated with PKC activator, PMA, for various time points. Northern blot analysis revealed that the induction of RTP expression by PMA was much faster than that by forskolin (Fig. 4B). RTP was induced as early as 1 h and increased to 6-fold at 4 h of PMA treatment. The induction reached the peak point (12-fold) upon 24-h incubation with PMA. On the other hand, treatment of BeWo cells with 4{alpha}PDD, a compound structurally related to PMA but lacking ability to activate PKC, did not affect RTP gene expression (Fig. 4B). These data confirm that PKC activity was indeed involved in RTP gene expression.



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  		FIG. 4. Requirement of PKC activity for RTP gene expression. The possible involvement of intracellular signaling pathways in RTP expression was investigated using different protein kinase inhibitors. A) Suppression of forskolin-induced RTP expression by PKC inhibitor. BeWo cells were cultured in absence (lanes 1 and 2) or presence of 10 µM H-89 (lane 3), 100 nM hydrochloride bisindolylmaleimide I (lane 4), and 10 µM genistein (lane 5), respectively, for 6 h followed by 100 µM forskolin (all lanes except lane 1) for 72 h. Total RNA was extracted and subjected to Northern blot. B) Induction of RTP expression by PKC activation. BeWo cells were cultured in absence (lane 1) or presence of 10 nM PMA for 1 (lane 2), 4 (lane 3), and 24 h (lane 4) and 10 nM 4{alpha}PDD for 24 h (lane 5). The effect of PMA on RTP expression was examined by Northern blot as described above. Bottom panel shows 28S and 18S as internal control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies have used a sensitive technique, differential display analysis, to identify genes that are differentially expressed in response to a variety of stimuli in various types of cells [20, 26, 29, 31]. This technique has become the most popular method of choice to identify and clone differentially expressed genes. Using the differential display analysis, we report the identification of a new stress-induced protein, RTP, that is up-regulated during trophoblast differentiation both in vitro and in vivo. Additional genes that we observed to be up-regulated in BeWo cell differentiation include human cytochrome p450 II C [32] and inosine monophosphate dehydrogenase type II [33].

Human RTP gene has been cloned independently by two other groups from vascular endothelial cells or a colon carcinoma cell line [20, 29]. In those studies, the expression level of RTP mRNA was increased either by stress conditions, such as exposure of endothelial cells to increased level of homocysteine, or during in vitro differentiation of colon carcinoma cell lines. RTP cDNA contains an open-reading frame of 1182 bp that encodes a protein with an approximate molecular weight of 43 000. The strong up-regulation of RTP during cell differentiation or exposure to stress condition suggests a specific function for the RTP gene in those processes. Our in situ hybridization studies using second trimester and term placenta tissues indicate that the RTP mRNA is predominantly expressed in postdifferentiated syncytiotrophoblasts (Fig. 3, A and B). The expression of RTP mRNA in syncytiotrophoblasts and its up-regulation during forskolin-induced BeWo cell differentiation further suggest a physiological role in trophoblast differentiation.

Because of its late expression during differentiation of colon carcinoma cells, it was proposed that RTP may regulate survival of terminal differentiated cells through protection from apoptosis [29]. This proposal may be relevant to trophoblast differentiation because apoptosis is a physiological process for normal placental and reproductive tissue development [34–37]. Apoptotic activities were reported in human fetal membrane containing chorionic trophoblasts, with highest apoptotic index in term placental tissues [38, 39]. Human trophoblasts express both Fas and its ligand (FasL), the two key components in the induction of apoptosis, throughout the entire gestation [39–41]. Additionally, trophoblasts externalize and maintain high levels of phosphatidylserine on their surface, a characteristic of plasma membrane of cells undergoing apoptosis [16–19]. After differentiation from cytotrophoblasts, the terminally differentiated syncytiotrophoblast maintains a relatively long life span in placental development. The high level of RTP expressed in syncytiotrophoblast may support a relationship to apoptotic function for RTP. The exact role of RTP in this process, however, remains to be elucidated.

In previous studies, the identification of regulatory factors for RTP genes had remained elusive. In the present study, however, we identified the intracellular signaling pathway that regulates RTP gene expression by using various protein kinase inhibitors that are effective on protein kinases in trophoblasts [22, 42]. It is known that forskolin-induced BeWo cell differentiation is achieved through activation of adenylate cyclase and subsequently increased intracellular cAMP level [30]. Most cAMP-mediated cellular effects have been considered to entail activation of PKA. Our results from the kinase inhibitor studies, however, raise the possibility that some of the physiological functions of cAMP may result from a PKA-independent pathway. This notion is supported by a recent discovery of a novel family of cAMP-binding proteins that directly activate the Ras signaling pathways in a cAMP-dependent but PKA-independent manner [43]. Data from the present study indicate that activation of PKC pathway is a crucial step for RTP gene expression in differentiating trophoblast (Fig. 4). Blockage of PKC but not PKA activity by their kinase inhibitors significantly suppressed the forskolin-induced RTP expression. Although the mechanism of PKC activation in RTP gene expression is not known, activated PKC may phosphorylate regulatory proteins that affect gene transcription. Our future study will be directed to explore the biological function of RTP by ectopic expression and/or gene knock out in trophoblasts.


   ACKNOWLEDGMENTS
 
We thank Dr. Kokame for providing the RTP cDNA clone.


   FOOTNOTES
 
1 This study was supported partially by a Research Challenge Early Start/Augmentation Grant from the Ohio Board of Regents. Back

2 Correspondence. FAX: 937 775 2012; neal.rote{at}wright.edu Back

Accepted: April 13, 1999.

Received: July 20, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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