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Department of Obstetrics and Gynecology (R.M., M.S., Y.O., M.H., A.I., M.K., M.O., K.T., Y.M.), Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; Department of Gynecology (T.T.), Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 594-1101, Japan; Department of Obstetrics and Gynecology (T.Y.), Sakai Municipal Hospital, Sakai 221-1700, Japan; and Department of Molecular and Cellular Biology (K.I., T.O.), Nagoya City University Graduate School of Medical Sciences, Nagoya 464-8601, Japan
Address all correspondence and requests for reprints to: Masahiro Sakata, M.D., Ph.D., Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan. E-mail: msakata{at}gyne.med.osaka-u.ac.jp.
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B that was identified by a yeast two-hybrid screen and is preferably expressed in human placenta and heart. RAI was also found to interact with Sp1 and exert an inhibitory effect against the DNA-binding activity of Sp1. We first show here that RAI mRNA expression increased as gestation proceeded and that RAI was localized mainly in the syncytiotrophoblast throughout pregnancy. The chloramphenicol acetyltransferase activity assay in Rcho-1 cells revealed that cotransfection of RAI expression vector resulted in decreased activity of the rat GLUT1 promoter but not in that of a mutated rat GLUT1 promoter lacking the Sp1 binding site. Furthermore, the protein level of RAI increased during differentiation. In addition, transfection of RAI expression vector promoted the morphological differentiation of Rcho-1 cells, and RAI knockdown using RAI-specific small interfering RNA reveals inhibitory effects on the morphological differentiation, as assessed by photomicroscopy. Taken together, these findings suggest that RAI may be involved in the regulation of trophoblast differentiation via interaction with Sp1. |
Introduction |
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Glucose is fundamental to the energy metabolism of mammalian cells (3, 4). Glucose transporter-1 (GLUT1), one of the glucose transporter protein isoforms, has been shown to function in response to insulin- and IGF-I-induced signaling (5, 6). GLUT1 is detectable in many human tissues, including colon, lung, stomach, and placenta (7, 8). Because glucose is the major energy source provided to the fetus, GLUT1 plays an essential role in the human placenta as a mediator of nutrient transfer from mother to fetus. GLUT1 can generally be detected in most parts of the placenta of both humans and rats, including the syncytiotrophoblast, cytotrophoblast cells, and fetal endothelium syncytiotrophoblast layer (9), although an earlier study indicated that in the rat placenta GLUT1 mRNA is localized in the labyrinthine syncytiotrophoblast layer and that the level of GLUT1 mRNA significantly declines from midgestation through term (10). Thus, like human chorionic gonadotropin and human placental lactogen, GLUT1 can be used as a functional indicator of trophoblast differentiation (11). We previously showed that the protein levels of GLUT1 and nuclear transcription factor specificity protein-1 (Sp1) in rat choriocarcinoma cells (Rcho-1 cells) decreased during trophoblast differentiation (12). We also showed that Sp1 was involved in the regulation of GLUT1 gene expression during this differentiation and that the Sp1 binding site located between –76 and –53 bp in the GLUT1 promoter was essential for basal GLUT1 promoter activity.
RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor B (NF-B) identified by a yeast two-hybrid screen (13). RAI also interacts with Sp1 and exerts an inhibitory effect against the DNA-binding activity of Sp1, leading to inhibition of the promoter activity of target genes (14). Although RAI mRNA is preferentially expressed in human placenta and heart, its role in the human placenta has not been investigated.
We noted the preferential expression of RAI in human placenta and investigated its functional role in the regulation of trophoblast differentiation. Therefore, in this study we examined the RAI mRNA expression and the localization of RAI in the human placenta throughout pregnancy. To examine the functional role of RAI in Rcho-1 cells, a model for trophoblast differentiation (15), we transfected the RAI expression vector and examined its role in GLUT1 transcription in Rcho-1 cells by assuring its interaction with Sp1. We also examined the change in the protein level of RAI during differentiation of trophoblasts and microscopically observed the morphological changes as well as after cotransfection of RAI expression vector or RAI gene knockdown using RAI-specific small interfering RNA (siRNA).
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Materials and Methods |
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Preparation of RNA and immunohistochemistry samples
First-trimester (7–9 wk of gestation) villus samples and midtrimester (19–21 wk of gestation) placentas were obtained after legal abortions with written informed consent of each patient as university committee approved. Full-term placentas (37–40 wk of gestation) were obtained after caesarean sections in uncomplicated pregnancies. After removal of amniochorion, decidual layer, and blood, the tissues were cut into small pieces and immediately frozen at –80 C until preparation of RNA samples. Total RNA was extracted from frozen tissues as described previously (17, 18).
Analysis of RAI RNA expression in human placentas by real-time PCR
Changes in the expression of RAI mRNA in the human placenta as gestation proceeded were examined by real-time PCR amplification. Total RNA (1 µg) was used as a template for reverse transcription by SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The PCR primer for RAI was purchased from Applied Biosystems (Foster City, CA) in the form of probe mix (Hs00198497_m1). The 50-µl PCR mix was loaded into a MicroAmp optical 96-well reaction plate (Applied Biosystems) as described previously (19). In brief, the PCR mix consisted of 5 µl cDNA, 5 µM each sense and antisense primer (5), 5 µM TaqMan probe, and 25 µl of the TaqMan universal PCR master mix (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was quantified using the Taq-Man GAPDH control reagent (Applied Biosystems) for normalization. The ABI PRISM 7700 sequence detection system (Applied Biosystems) was used for amplification and quantification. The amplification conditions were as follows: 2 min at 50 C, 10 min at 95 C, followed by a two-step cycle of 95 C for 15 sec and 60 C for 60 sec for a total of 40 cycles. The threshold cycle (Ct) value of the cycle in which the amount of product was significantly separated from the background baseline and calculated using ABI PRISM 7700 SDS software.
Immunohistochemistry
To prepare immunohistochemistry samples, the tissues were formalin fixed, paraffin embedded, cut into 5-µm slices, and placed on glass slides. After fixation, sections were incubated in 3% hydrogen peroxide to block endogenous peroxidase and incubated with blocking buffer containing 10% goat serum to minimize nonspecific binding. To detect the localization of RAI protein, we used a primary rabbit anti-RAI polyclonal antibody. In brief, an antibody against synthetic peptide (GYVPRNYFGLFPRVKPQRSK) predicted from the C terminus sequence of RAI (13) was made by immunizing a rabbit. This antibody can be used for the detection of RAI protein by Western blot analysis using human placenta (data not shown). The antiserum was diluted 1:500 with 1% BSA/Tris-buffered saline buffer and incubated with the sections at 4 C overnight, and then the sections were rinsed with PBS three times. After further incubation with biotin-labeled goat antirabbit IgG, the immunoreactivity was visualized by the avidin-biotin complex method using an LSAB-HRP universal kit (Dako Cytomation, Glostrup, Denmark). The specificity of staining was confirmed by use of nonimmunized normal goat serum instead of anti-RAI antiserum. The slides were counterstained lightly with Mayer’s hematoxylin.
Plasmids and plasmid construction
The plasmid containing the construct of rat GLUT1 (rGLUT1) promoter (–2106/+134), the plasmid containing the –76/+134 region of the rat GLUT1 genomic sequence (–76 wild), and its mutated plasmid [–76 mutant: mutation in Sp-1 binding site (–76/–53)] were used as described in a previous report (20). RAI expression vector (pFLAG-CMV2-RAI) and empty vector (pFLAG-CMV2) were used as previously reported (13).
Transient transfection and chloramphenicol acetyltransferase (CAT) assay
Rcho-1 cells were plated at 1 x 105 cells/cm2 in six-well plates. DNA transfection was performed by the Lipofectamine Plus reagent-mediated transfection procedure (Invitrogen) as recommended by the manufacturer. In each experiment, 1 µg of reporter plasmid (–2106/+134 rGLUT1 promoter CAT plasmid, –76/+134 rGLUT1 promoter CAT plasmid, or its mutant plasmid), 0.5, 1, or 2 µg of pFLAG-CMV2-RAI or 1 µg of pFLAG-CMV2 and 0.5 µg of ß-galactosidase expression plasmid (as an internal control for transfection efficiency) were used. Forty-eight hours after transfection, cell extracts were prepared as follows: the cells were washed with PBS and collected in 1 ml of PBS. After centrifugation for 5 min, the pellets were resuspended in 100 µl of 0.25 µM Tris-HCl (pH 7.8), lysed by three freeze-thaw cycles, and centrifuged for 10 min at 4 C. After the supernatants were collected, their protein concentrations were determined and they were assayed for CAT activity as described previously (12).
The CAT activities of cell extracts (equivalent of 10 µg of protein) were examined by incubation with 0.1 µCi of [14C] chloramphenicol and 10 mM acetyl CoA. The acetylated and nonacetylated forms of [14C] chloramphenicol were separated by thin-layer chromatography, autoradiographed, and quantitated using the BAS2000 image analyzer system (Fuji Photo Film Co. Ltd., Tokyo, Japan). All experiments were performed in triplicate; the mean of three replicates for each experiment was adopted as the result, and each result was expressed as the percentage of the acetylated form of [14C] chloramphenicol relative to the control (mean ± SD).
Western blotting analysis
Rcho-1 cells were cultured in 10-cm dishes in complete medium (20% FBS/NCTC), and then differentiation was induced by replacing the culture medium with differentiation medium (5% HS/NCTC) for 5 d. After this process, Rcho-1 cells in each condition (undifferentiated and differentiated) were harvested and lysed by gentle rocking at 4 C for 60 min in 100 µl of radioimmunoprecipitation assay buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing 100 µg/ml phenylmethylsulfonyl fluoride, 60 µg/ml aprotinin, and 1 mM orthovanadate (21). Cell lysates were transferred into microtubes and centrifuged at 1,5000 x g at 4 C for 20 min, and the supernatants were collected and subjected to Western blotting analysis. Subsequently protein samples were separated by 10% SDS-PAGE (25 µg/lane) and analyzed by immunoblotting with anti-RAI rabbit polyclonal antibody (1:1000) or anti-ß-actin antibody (Sigma-Aldrich, St. Louis, MO). The proteins were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) or alkaline phosphatase conjugate substrate kit (Bio-Rad, Arakawa, Tokyo, Japan). Reprobing was performed using a Reblot Western blot recycling kit (Chemicon International, Temecula, CA). The results were quantified densitometrically, and each value was expressed as relative to the value in the undifferentiated state.
Nuclear protein preparation and immunoprecipitation
In 10-cm dishes of Rcho-1 cells prepared as described above, transient transfection with RAI expression vector or control vector was performed as described above. Nuclear extracts were prepared from these cells under ice-cold conditions as follows (22): the cells were washed twice with PBS, once with PBS containing 1 mM Na3VO4 and 20 mM NaF, and once with hypotonic buffer (20 mM HEPES, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 0.125 µM okadaic acid, 1 mM EDTA, and 1 mM EGTA) containing 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 0.5 µg/ml leupeptin. Subsequently the cells were scraped from dishes into hypotonic buffer containing 0.2% Nonidet P-40. After centrifugation at 10,000 x g for 20 sec, the pellets were resuspended in high salt buffer (20% glycerol and 5 M NaCl in hypotonic buffer), followed by incubation at 4 C for 30 min with gentle rocking. After centrifugation at 15,000 x g at 4 C for 30 min, the supernatants were collected and used for Western blotting and immunoprecipitation. Nuclear extracts were subjected to Western blotting using anti-Sp1 goat polyclonal antibody (1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA) or anti-ß-actin antibody and detected as described above. Other proteins were immunoprecipitated with anti-Sp1 goat polyclonal antibody (Santa Cruz Biotechnology) using protein G Sepharose 4 fast flow (GE Healthcare, Fairfield, CT) according to the manufacturer’s instructions. Immune complexes were subjected to Western blotting analysis and detected as described above by using anti-RAI rabbit polyclonal antibody. Immunoprecipitation of the nuclear extracts with anti-FLAG antibody (Sigma-Aldrich) and Western blotting analysis using anti-Sp1 rabbit polyclonal antibody (Santa Cruz Biotechnology) were also performed as described previously. The results were quantified densitometrically, and each value was expressed relative to the value of the control.
Photomicrographic observation
Rcho-1 cells were transiently transfected with RAI expression vector or control vector as described above and observed by photomicrography using an ECLIPSE TE2000-U (Nikon, Tokyo, Japan). The experiments were done in triplicate, and a representative photomicrograph was adopted from one of three different experiments. Each result was then quantified as the percentage of giant cells (the number of giant cells per total number of cultured cells counted), compared with that of control, and expressed as mean ± SD.
RAI gene knockdown by siRNAs
Rcho-1 cells (1 x 105 cells/cm2) were grown in 6-well plates in complete medium under subconfluent conditions, and transfected with RAI-specific siRNA oligonucleotides (predesigned siRNA no. 16810; Ambion, Inc., Austin, TX) or scrambled RNA oligonucleotides as negative control (no. 4611; Ambion) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The next day, the culture medium of transfected cells were changed to either complete medium or differentiation medium and then observed by photomicrography as above.
Statistical analysis
Statistical analysis was performed by the Kruskal-Wallis test, and statistical significance was accepted at P < 0.05.
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Results |
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). Furthermore, the level of RAI mRNA showed a significant increase as the gestational age increased (P < 0.01, compared with that in the first trimester). The representative data were shown in Table 1.
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, A–C). When the control sections of full-term placental tissue were treated with a nonimmunized normal goat serum, no noticeable staining was observed (Fig. 2D).
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, filled bar) or 1 µg of control vector (Fig. 3A, gray bar) into undifferentiated Rcho-1 cells. Cotransfection of the RAI expression vector resulted in a significant suppressive effect on the rGLUT-1 promoter activity in a dose-dependent manner, compared with cotransfection of the control vector (P < 0.01). The transfection of RAI expression vector had no inhibitory effect on control ß-galactosidase expression plasmid (data not shown), which coincides with the previous report (14) that showed that gene expression from other promoters including herpes virus thymidine kinase promoters and p21 gene promoter was not inhibited by RAI.
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, filled bar) or control vector (Fig. 3B, gray bar) into undifferentiated Rcho-1 cells. When cells were transfected with the wild-type rGLUT-1 reporter plasmid (–76wild), they exhibited significantly suppressed transcriptional activity in response to RAI overexpression (P < 0.01). In contrast, RAI overexpression did not show any suppressive effect when cotransfection was performed with the mutant reporter plasmid (–76 mutant) lacking the Sp1 biding site. In addition, the promoter activity of the mutant –76/+134 rGLUT1 promoter CAT plasmid was more than 10 times higher than that of pCAT-basic when cotransfected with both pFLAG-CMV2 and pFLAG-CMV2-RAI (data not shown).
RAI protein increases in Rcho-1 cells during differentiation and interacts with Sp1
Western blotting analysis showed that the total amount of RAI protein, whose size is known to be approximately 50 kDa, significantly increased in the differentiated Rcho-1 cells, compared with the undifferentiated cells (Fig. 4A, P < 0.01). When Rcho-1 cells were transfected with RAI expression vector (Fig. 4B, lane 2) or control vector (Fig. 4B, lane 1), the introduction of the RAI expression vector caused no marked change in the basal level of Sp1 protein in the nucleus. However, when the nuclear proteins were extracted from Rcho-1 cells transfected with RAI expression vector (Fig. 4C, lane 2) or control vector (Fig. 4C, lane 1) and subsequently immunoprecipitated with anti-Sp1 antibody, the RAI protein level in the Sp1 complex from cells transfected with RAI expression vector was significantly higher, compared with that in controls (P < 0.01). Simultaneously the Sp1 protein level in FLAG complex from cells transfected with RAI expression vector also indicated a marked increase (Fig. 4D, lane 2), compared that in controls (Fig. 4D, lane 1). Taken together, these data suggested that RAI interacts with Sp1 protein in trophoblast cells.
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, right panel) or control vector (Fig. 5A, left panel) followed by quantitative analysis (Fig. 5B) of the number of giant cells, the morphological differentiation marker in cultures of Rcho-1 cells. A significant increase in the number of giant cells was observed in the cultured cells transfected with RAI expression vector, compared with the control (P < 0.01). We performed additional immunohistochemical studies using anti HA-probe (RAI expression vector epitope tag) antibody, and observed noticeable staining in the nucleus of giant cells induced by RAI overexpression (data not shown).
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, left panel) or RAI-specific siRNA oligonucleotides (Fig. 6A, right panel) and culture in the differentiation medium thereafter. A significant decrease in the number of giant cells, as shown in quantitative analysis (Fig. 6B), was observed in the cells transfected with RAI-specific siRNA, compared with the negative control (P < 0.01).
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Discussion |
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RAI was recently shown to be uniquely localized in human placenta and heart (13). Therefore, it is assumed that RAI may have a regulatory role in these organs. However, its biological roles in the human placenta have not been investigated. Here we present the first report suggesting that RAI plays a significant role in trophoblast differentiation. Immunoreactivity of RAI in the human placenta was detected mainly in the syncytiotrophoblast by immunohistochemical studies, in accordance with our previous finding that GLUT1 is localized in the plasma membrane of trophoblast cells (23).
As for the molecular mechanism by which RAI exerts its function, the result of CAT assays suggests that RAI exerts its suppressive effect on GLUT-1 transcription in a dose-dependent manner by interfering with Sp1 binding in the rGLUT-1 promoter. The basal transcription activity of the wild-type construct was significantly higher than that of the mutant construct lacking the Sp1 binding site. These findings agree with the notion put forth in our previous report that the GC box at –76/–53 bp in the rat GLUT-1 promoter is important for the basal transcriptional activity of the rGLUT-1 gene via Sp1 binding. Western blot analysis of the nuclear protein immunoprecipitated with anti-Sp-1 antibody suggested that RAI protein interacts with Sp1 protein in Rcho-1 cells. We extensively confirmed this interaction with the data from Western blot analysis of the nuclear extracts immunoprecipitated with anti-FLAG antibody. Western blot analysis of the whole-cell extracts also indicated that this interaction can occur without any effect on the total amount of Sp1 protein.
Furthermore, we observed interesting morphological changes in Rcho-1 cells transfected with RAI expression vector, compared with those transfected with control vector. In addition, we also observed the morphological changes of Rcho-1 cells into giant cells after RAI gene knockdown using RAI-specific siRNA. In this study, we confirmed the siRNA efficacy by quantifying the reduced levels of RAI mRNA by means of real-time PCR (data not shown). According to the fact that trophoblast giant cells release placental lactogen (24), which is known to the indicator of trophoblast differentiation, these findings provide evidence that RAI promotes a trophoblast cell differentiation not only functionally but also morphologically.
In this study we used not only human placental tissues patients but also Rcho-1 cells. We consider Rcho-1 cells to be an adequate in vitro model for investigation of placental gene expression because they have the ability to differentiate and produce hormones similarly to human placenta in vivo (15, 25, 26, 27). Using real-time RT-PCR, we detected RAI mRNA in the human placenta in all trimesters of pregnancy and observed that the level increased as gestation proceeded. Western blot analysis revealed that the protein level of RAI increased as well concomitantly with the differentiation of Rcho-1 cells to trophoblasts. Immunohistochemical data showed the localization of RAI in the trophoblast. These results agree with each other and suggest that RAI may promote the trophoblast and placental differentiation. However, the data from real-time PCR revealed that human GLUT1 mRNA level increased as gestation proceeded (data not shown), which was contrary to our expectations. We previously showed that the GLUT1 protein level in Rcho-1 cells decreased during trophoblast differentiation. The observation in this cell line may be representative of the events in the rat trophoblast giant cell differentiation pathway (15). It may come from different mechanism(s) in rat trophoblast differentiation compared with human trophoblast differentiation.
RAI was originally identified as a protein which inhibits the transcriptional activity of NF-B subunit p65. Although the functional role of NF-B in the trophoblast has not been fully elucidated, an increasing number of reports suggested the possible involvement of NF-B in the regulation of the expression of proinflammatory cytokines or growth factors (28, 29) by the trophoblast cells. It is possible that RAI may also play a role in the control of inflammation or apoptosis in the human placenta.
Correlations between trophoblast differentiation and major complications during pregnancy, such as preeclampsia and intrauterine growth restriction, have been demonstrated in many previous studies (30). Our findings suggest that the role of RAI may be extended to regulating appropriate placental differentiation, which can lead to better prognosis in pregnancy. Further studies are still needed to fully elucidate the role of RAI as a key factor regulating trophoblast differentiation.
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Acknowledgments |
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Footnotes |
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Disclosure Summary: The authors have nothing to disclose.
First Published Online September 13, 2007
Abbreviations: CAT, Chloramphenicol acetyltransferase; Ct, threshold cycle; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT1, glucose transporter-1; HS, horse serum; NF-B, nuclear factor B; RAI, RelA-associated inhibitor; rGLUT1, rat GLUT1; siRNA, small interfering RNA; Sp1, specificity protein-1.
Received February 1, 2007.
Accepted for publication September 4, 2007.
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B, RelA-associated inhibitor. J Biol Chem 274:15662–15670B and Sp1 actions. J Virol 76:8019–8030
ligands to inhibit the epithelial inflammatory response. J Clin Invest 104:383–389[Medline]
gene is transcriptionally activated by epidermal growth factor through cAMP response element in trophoblast cells. J Biol Chem 273:7800–7806 gene through protein kinase A pathway in trophoblast cells. Mol Cell Endocrinol 182:215–224[CrossRef][Medline]B activation. Am J Reprod Immunol 52:218–223[Medline] and interleukin-1. J Biol Chem 278:15832–15841This article has been cited by other articles:
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  F. Nishimoto, M. Sakata, R. Minekawa, Y. Okamoto, A. Miyake, A. Isobe, T. Yamamoto, T. Takeda, E. Ishida, K. Sawada, et al. Metal Transcription Factor-1 Is Involved in Hypoxia-Dependent Regulation of Placenta Growth Factor in Trophoblast-Derived Cells Endocrinology, April 1, 2009; 150(4): 1801 - 1808. [Abstract] [Full Text] [PDF]
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Ct values (GAPDH Ct-RAI Ct) in the quantitative analysis of RAI mRNA in the first (
