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Mol. Hum. Reprod. Advance Access originally published online on December 19, 2005
Molecular Human Reproduction 2005 11(11):825-831; doi:10.1093/molehr/gah239
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© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Ikaros is expressed in human extravillous trophoblasts and involved in their migration and invasion

E. Yamamoto1, T. Ito1,5, A. Abe2, F. Sido1, K. Ino1, A. Itakura1, S. Mizutani3, S. Dovat4, S. Nomura1 and F. Kikkawa1

1Department of Obstetrics and Gynecology, 2Department of Hematology, 3Department of Clinical applied research of Proteases, Nagoya University Graduate School of Medicine, Nagoya, Japan and 4Pediatrics Hematology/Oncology, University of Wisconsin Medical School, Madison, WI, USA

5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: itoto{at}med.nagoya-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The transcriptional factor Ikaros was originally found to function as a key regulator of lymphocyte differentiation. In addition, we have reported that Ikaros regulates the human placental leucine aminopeptidase (P-LAP)/insulin-regulated aminopeptidase (IRAP) gene in choriocarcinoma trophoblastic cells, suggesting that Ikaros might be involved in placental development, while even its presence in human placenta remains undetermined. We therefore sought to clarify the location and roles of Ikaros in human placenta. Immunohistochemical analysis showed modest Ikaris expression in syncytium, and intense expression in extravillous trophoblasts (EVTs) in first trimester placenta. Western blot analysis showed that villous trophoblasts principally expressed Ikaros-2/3, while Ikaros-x (Ikx) was predominantly expressed in cultured EVTs. Furthermore, to investigate the functional role of Ikx in EVTs, the EVT cell line HTR-8/SVneo was infected with a retrovirus vector expressing the hemagglutinin (HA)-tagged dominant negative isoform Ikaros-6 (Ik6), which prevents the DNA-binding activity of Ikx. Antibody against HA showed successful transduction of Ik6 in HTR-8/SVneo cells on immunocytochemistry and Western blotting. Transduction of Ik6 significantly reduced the migratory and invasive abilities of HTR-8/SVneo cells. These results suggest that Ikx is involved in migration and invasion of EVTs in early placentation.

Key words: extravillous trophoblasts/human/Ikaros/placentation/transcriptional factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The transcription factor Ikaros was originally found to function as a key regulator of lymphocyte differentiation (Lo et al., 1991Go; Georgopoulos et al., 1992Go, 1994Go). Subsequent studies reported that the role of Ikaros in normal hematopoiesis extended beyond lymphoid lineages (Klug et al., 1998Go; Nichogiannopoulou et al., 1999Go; Lopez et al., 2002Go). The Ikaros gene contains seven exons and encodes at least nine alternatively spliced isoforms Ikaros-1–8 (Ik1–8) and Ikaros-x (Ikx) (Molnar et al., 1996Go; Payne et al., 2003Go). All nine Ikaros isoforms share two C-terminal Krüppel-like zinc fingers (ZnFs) that are required for hetero- or homo–dimerization and for interactions with other proteins (Georgopoulos et al., 1994Go; Hahm et al., 1994Go). Only isoforms that contain three or more N-terminal ZnFs (DNA-binding isoforms, Ik1–3 and Ikx) possess transcriptional activity by high-affinity binding to a specific sequence motif (GGGA) in the promoters of target genes. In contrast, isoforms with fewer than three N-terminal ZnFs (DNA non-binding isoforms, Ik4–8) suppress the transcriptional activity of Ikaros DNA-binding isoforms as dominant negative isoforms (Sun et al., 1996Go).

We previously demonstrated that Ikaros transactivates the expression of the human placental leucine aminopeptidase (P-LAP; EC3.4.11.3)/insulin-regulated aminopeptidase (IRAP) gene (Ito et al., 2001Go, 2002Go) in trophoblastic cells. To our knowledge, we were the first group to show the involvement of Ikx in gene regulation in non-hematopoietic cells. Yu et al. also demonstrated that Ikaros regulates the fibroblast growth factor-4 receptor gene in pituitary cells (Yu and Ezzat, 2002). P-LAP/IRAP, which is mainly expressed in syncytiotrophoblasts and extravillous trophoblasts (EVTs) in human placenta (Nagasaka et al., 1997Go; Ino et al., 2003Go), is involved in the maintenance of pregnancy homeostasis via regulating oxytocin levels (Tsujimoto et al., 1992Go; Mitchell and Wong, 1995Go). The findings that Ikaros plays a vital role in hematopoietic cell differentiation and that the Ikaros target gene P-LAP/IRAP is expressed in specific populations of trophoblasts prompted us to investigate the expression and function of Ikaros in human placenta, which remains uncertain.

In this study, we investigated (i) the localization of Ikaros in human placenta; (ii) the identification of Ikaros isoforms expressed in isolated EVTs and (iii) the effects of suppressing Ikaros transcriptional function on proliferation, migration and invasion of the EVT cell line HTR-8/SVneo. These results are the first to demonstrate Ikaros expression in placenta and its possible involvement in the migration and invasion of EVTs.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Tissue collection and processing
This study was approved by the ethics committee of Nagoya University Graduate School of Medicine, and written informed consent was obtained from each woman before clinical sampling.

Placental samples were obtained from pregnant women who met the following inclusion criteria: (i) singleton pregnancy; (ii) normal pregnancy; (iii) in good health and aged between 18 and 40 years and (iv) reliable gestational age by ultrasonographic examination in the first trimester. First trimester (6–14 weeks gestation, n = 5) and early second trimester (15–21 weeks, n = 4) placental samples were obtained from women undergoing elective pregnancy terminations. Full-term placental samples before the onset of labour (37–40 weeks, n = 4) were collected during elective Caesarean sections.

Umbilical cord blood was obtained after Caesarean sections and mononuclear cells were isolated as previously described (Hao et al., 1995Go). Cord blood cells were used as a positive control for Ikx (Payne et al., 2003Go).

Antibodies against Ikaros
Anti-Ik C-terminal sequence (CTS) antibody (Ab) (polyclonal rabbit Ab specific to the C-termini of all Ikaros isoforms) (Payne et al., 2001Go; Dovat et al., 2002Go) and the commercially available Ab Ik H-100 (polyclonal rabbit Ab specific to N-termini of all Ikaros isoforms) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for immunoblotting and immunostaining, respectively. Both Abs are able to detect all isoforms of Ikaros, including insertion forms.

Immunohistochemistry
Placental samples of various gestational ages were fixed in formalin and embedded in paraffin. Sections (thickness, 4 µm) were immunostained as previously described (Nagasaka et al., 1997Go), using Ab Ik H-100 at a dilution of 1:50 and anti-human cytokeratin7 (CK7) Ab (Ready-to-use N-series; DAKO, Carpinteria, CA, USA) at the dilution recommended by the manufacturer. Immunoreactivity was detected using the streptavidin-biotin-peroxidase complex technique (Histofine SAB-PO kit; Nichirei, Tokyo, Japan) and 3,3'-diaminobenzidine (DAB) as the choromogen substrate. Slides were counterstained with Mayer’s hematoxylin and mounted. For negative controls, the primary antibody was replaced with a non-specific IgG (Santa Cruz Biotechnology) at the same dilution.

Cell lines and culture
The EVT cell line HTR-8/SVneo (kind gift from Dr Charles H. Graham, Queen’s University, Ontario, Canada) (Graham et al., 1993Go) was grown in RPMI 1640 (Sigma Chemical Co., St. Louis, MO, USA), supplemented with 10% heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 µg/ml) and 2 mM glutamine. Two hundred and ninety-three cells (ATCC) were maintained in IMDM (Sigma Chemical Co.) with 10% FCS. Cultures were incubated at 37°C in a 5% CO2 atmosphere.

Human chorionic villous explant culture
Villous explant cultures were established using placental tissues obtained from legal abortions (6–9 weeks, n = 10) as previously reported (Sato et al., 2002Go). Briefly, placental tissues were washed with Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Burlington, ON, USA) and aseptically dissected to remove decidual tissues. After teasing apart small fragments of placental villi, villous fragments were placed in 100-mm collagen type I-coated dishes (BD Biosciences, Bedford, MA, USA) and incubated in DMEM supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml) and 5% amphotelicin B at 37°C in a 5% CO2 atmosphere for 48 h. Dishes were then gently washed with phosphate-buffered saline (PBS) and incubated with 0.05% trypsin (Gibco BRL) and 0.05% EDTA for 5 min. Detached cells were collected and filtered through a 40-µm pore Nylon Cell Strainer (BD Biosciences) to remove contaminating villous tissues and selectively obtain spreading cells from the attached villous tissues.

Western blot analysis
Preparation of cell lysates was performed using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s directions. Protein concentrations were determined using a BCA Protein Assay Kit (PIERCE, Rockford, IL, USA). Protein extracts (20 µg) were resolved on 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels and transferred onto nitrocellulose membranes (Millipore Co., Bedford, MA, USA).

Ikaros was detected using anti-Ik CTS Ab followed by anti-rabbit HRP secondary Ab (Santa Cruz Biotechnology). Signals were visualized using the ECL plus Western blot detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). An Ab against ß-actin (Abcam, Cambridge, UK) was used for standardizing the amount of sample applied. Data were obtained from at least three individual experiments performed in triplicate.

Immunocytochemistry
The villous explant was cultured on a four-well chamber slide (Nalge Nunc International, Naperville, IL, USA) coated with collagen-1 (BD Biosciences) as described above. Immunocytochemical staining was performed as previously described (Sato et al., 2002Go) using Ab Ik H-100 and anti-CK7 Ab. The chamber slides were also immunostained with anti-human CD146 Ab (Chemicon international, Temecula, CA, USA) at 1:400 dilution to examine stromal cell contamination.

Transient expression of Ik6
The hemagglutinin (HA)-tagged XhoI-NotI fragment of Ik6, which was a generous gift from Dr Tomohiro Yagi (Kyoto Prefectural University of Medicine, Kyoto, Japan) (Yagi et al., 2002Go), was subcloned into a pHBGAP retrovirus vector under the control of the GAPDH promoter to give pHBGAP/Ik6. The pCGCGP construct, which expresses vesicular stomatitis virus glycoprotein, was used to produce high-titer viral supernatants rapidly as described previously (Abe et al., 2004Go).

To generate pseudotype viruses, we co-transfected 10 µg of pHBGAP/Ik6 or pHBGAP with 10 µg of pCGCGP using calcium phosphate co-precipitation as previously reported (Abe et al., 2004Go). Culture medium was replaced with 8 ml of fresh medium 8 h after transfection, and pseudotype virus was collected 48 h after transfection. To establish HTR-8/SVneo cells transducted with Ik6, 4 x 104 HTR-8/SVneo cells were infected with 4 ml of virus supernatant in the presence of 5 µg/ml protamine. After 48 h, the Ik6-expressing cells were selected with Blasticidin S HCl (Invitrogen, San Diego, CA, USA) in standard medium, and at 3 weeks, surviving cells were analysed. We confirmed the expression of HA-Ik6 protein by Western blot analysis and immunocytochemistry using an anti-HA-peroxidase Ab (Chemicon international).

In vitro cell proliferation assay
Cells were plated in triplicate at a density of 5 x 103 cells in 100 µl volume in 96-well plates. Cell viability was determined by modified tetrazolium salt (MTS) assay using the Cell Titer 96 Aqueous One Solution Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Transwell migration and invasion assay
Invasion assay was performed after 24 h of incubation, as previously reported (Graham et al., 1993Go). The transwells (Corning Incorporated, Corning, NY, USA) with a filter of 6.5 mm diameter and 8.0 µm pore size were used. The number of cells was adjusted to 2.0 x 105/ml in serum free medium, and a 200 µl sample was added in triplicate to the upper wells, and 800 µl of complete medium was added to the lower wells. Migration assay was performed under the same conditions as those for invasion assay, except that the incubation time was 20 h and that wells were not coated with Matrigel (Collaborative Biomedical Products, Bedford, MA, USA). The number of cells was counted under a microscope at x200 magnification. Data were obtained from three individual experiments performed in triplicate.

Statistical analysis
Data are expressed as mean ± SD. As data were not normally distributed, we employed non-parametric statistics. Comparisons between groups were made by Mann–Whitney U-test for two independent samples and Boneferroni correction for multiple comparisons. Differences were considered significant when the P value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Immunohistochemical expression of Ikaros in human placenta
We first investigated the localization of Ikaros protein in human placenta by immunohistochemistry using Ab Ik H-100, which can detect all Ikaros isoforms. Simultaneous staining with anti-human CK7 Ab confirmed the identity of trophoblasts, including proliferative phenotype EVTs located in the cell columns of the anchoring villi (arrows in Figure 1A and E) and interstitial EVTs (invasive phenotype, arrow heads in Figure 1A and E).


Figure 1
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  		Figure 1. Immunohistochemical localization of Ikaros in first trimester human placenta (A, B, C and D) and in second trimester human placenta (E, F, G and H). Sections were stained with anti-CK7 antibody to distinguish extravillous trophoblasts (EVTs) from decidua cells (A and E). Ikaros protein was detected with Ab Ik H-100 (B, C, F and G). (C) and (G) are higher magnification views of the squares in (B) and (F), respectively. Arrows indicate proliferative phenotype EVTs located in the cell columns and arrow heads indicate invasive phenotype EVTs invading the decidua. Tissues showed no staining with normal rabbit IgG (D and H). C, cytotrophoblasts; S, syncytiotrophoblasts. Magnification (A, B, D–F, and H) x100, (C and G) x200; Scale bars = 50 µm.

 

In the first trimester placenta, Ikaros was mainly localized in both proliferative phenotype EVTs and interstitial EVTs (Figure 1B). In the second trimester placenta, Ikaros was intensely expressed in interstitial EVTs invading the decidua (Figure 1F). High-resolution images of extensive areas of the placental bed were examined for Ikaros, which was confirmed to be present in EVTs (Figure 1C and G). We observed no immunostaining for Ikaros in third trimester placenta (data not shown). In floating villi, Ikaros was stably expressed at moderate levels on the syncytium (Figure 1B and F). Non-immune rabbit IgG showed no immunostaining in any of placental samples (Figure 1D and H). Expression and localization of Ikaros in villous trophoblasts and EVTs are summarized in Table I. These results showed that Ikaros protein was strongly expressed in EVTs invading the decidua during the first and second trimester, which prompted us to focus our attention on EVTs.


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  		Table I. Immunohistochemistry for expression and localization of Ikaros in first, second and third trimester of placentas

 

Ikx expression in EVTs
We then isolated EVTs from cultures of human chorionic villi (6–9 weeks, n = 10) and examined Ikaros expression in the cells. Cells grown from the explanted villous tip after 5-day culture (Figure 2A) showed exclusive expression of Ikaros in the nuclei (Figure 2B). These cells also exhibited positive immunoreactivity against both CK7 and CD146 (Figure 2B), which served as EVT markers (Shih and Kurman, 1996Go). No immunostaining was observed with non-immune rabbit IgG (Figure 2B).


Figure 2
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  		Figure 2. Ikaros expression in cultured EVTs based on immunocytochemistry and Western blotting. (A) Human villous tissue (6–9weeks, n = 10) was cultured for 5 days and cells grown from the villous tip were observed. (B) Cultured EVTs were positively stained with anti-CK7 and anti-CD146Abs, which are markers of human EVTs. Ikaros was detected by Ik H-100 Ab in cultured EVTs. Cultured EVTs showed no staining with normal rabbit IgG. Scale bars = 100 µm (C) Western blotting was performed with anti-Ik CTS Ab, which can detect all Ikaros isoforms. Representative data obtained from three individual experiments is shown. CB, cord blood cells.

 

As the Ikaros family is composed of nine isoforms, we then tried to identify the predominant isoforms in cultured EVTs. Western blotting analyses using anti-Ik CTS Ab showed that the DNA-binding isoform Ikx was predominantly expressed in cultured EVTs at levels comparable to cord blood cells, which were used as a positive control for Ikx (Figure 2C). The EVT cell line HTR-8/SVneo, which is known to exhibit a natural EVT cell phenotype (Graham et al., 1993Go), also showed a pattern similar to cultured EVTs. In contrast to EVTs, normal villi of the first trimester showed Ik2/3 as predominant isoforms (Figure 2C).

Overexpression of Ik6 in HTR-8/SVneo cells
We then sought to examine the roles of Ikx in EVTs. The dominant negative isoform Ik6 is able to suppress the function of DNA-binding isoforms of Ikaros (Sun et al., 1996Go). We therefore established HTR-8/SVneo/Ik6 cells by transfection with pHBGAP/Ik6 and selection in blasticidin-containing culture medium.

Immunocytochemical analyses using Ab Ik H-100 showed that both parental HTR-8/SVneo wild-type (WT) and HTR-8/SVneo/Ik6 cells showed immunoreactivity against Ikaros, whereas only HTR-8/SVneo/Ik6 cells were immunostained by HA Ab (Figure 3A). To further confirm the expression of Ik6 in HTR-8/SVneo/Ik6 cells, we performed Western blotting (Figure 3B). Ab Ik CTS gave a band corresponding to Ik6 only in HTR-8/SVneo/Ik6 cells. In addition, we observed a band for HA-Ik6 only in HTR-8/SVneo/Ik6 cells. Transduction of the Ik6 gene did not alter cell morphology (Figure 3A).


Figure 3
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  		Figure 3. Ik6 transfection of HTR-8/SVneo cells. WT, parental HTR-8/SVneo cells; mock-transfected, HTR-8/SVneo cells with infection of pHBGAP retrovirus vector; and HTR-8/SVneo/Ik6, HTR-8/SVneo cells with infection of pHBGAP/Ik6 retrovirus vector. (A) Immunocytochemistry of WT and HTR8/SVneo/Ik6 cells for Ikaros and HA. Magnification, x400; Scale bars = 25 µm. (B) Western blot analysis of Ik6 and HA in WT, mock-transfected and HTR-8/SVneo/Ik6 cells with anti-Ik CTS, anti-HA Abs and ß-actin.

 

Effect of Ik6 transduction on cell proliferation
We assessed the effect of Ik6 transduction on cell proliferation by MTS proliferation assay. The number of HTR-8/SVneo/Ik6 cells increased for 96 h of incubation, and this was not significantly different from WT and mock-transfected cells (Figure 4).


Figure 4
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  		Figure 4. Effects of Ik6 on cell proliferation. Cell proliferation of WT, mock-transfected and HTR-8/SVneo/Ik6 cells was determined by modified tetrazolium salt (MTS) assay. Wild-type (WT) parental HTR-8/SVneo cells. Data were obtained from three independent experiments with at least four samples. Results are expressed as a means ± SD.

 

Effect of Ik6 transduction on migration and invasion
The most representative feature of EVTs is their invasion to maternal decidua. We therefore examined whether suppression of Ikx activity affects this invasion. As shown in Figure 5A, WT and mock-transfected cells displayed high potential to penetrate filters, whereas the number of migratory HTR-8/SVneo/Ik6 cells was significantly reduced to 64.1 ± 10.0% (mean ± SD; P < 0.05), as compared with WT. We observed no significant difference between WT and mock-transfected cells. Similarly, the number of cells penetrating to the matrigel was significantly smaller for HTR-8/SVneo/Ik6 cells than for WT and mock-transfected cells (66.1 ± 5.9%, P < 0.05) (Figure 5B).


Figure 5
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  		Figure 5. Effects of Ik6 on cell migration (A) and invasion (B) of WT, mock-transfected and HTR-8/SVneo/Ik6 cells. Data are plotted as percentage of migratory and invasive WT cells and were obtained from at least three individual experiments performed in triplicate. Values represent mean ± SD. *P < 0.05. Images show representative migratory (A) and invasive (B) cells at x200 magnification. Wild-type (WT) parental HTR-8/SVneo cells.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We provide here the first evidence for the localization of Ikaros in human placenta and its involvement in EVT function.

In human placenta, cytotrophoblast stem cells at the basement of villi differentiate in two distinct directions: syncytiotrophoblasts and EVTs. EVTs are further divided into the proliferative phenotype and the invasive phenotype. Syncytiotrophoblasts form the syncytial layer in floating villi, which primarily manage transport and endocrine functions. On the other hand, EVTs located in anchoring villi (proliferative phenotype) develop to invade the maternal decidua and uterine vasculature (invasive phenotype). Our finding that Ikaros was strongly present in EVTs, moderately present in syncytiotrophoblasts and scarcely present in cytotrophoblasts suggests that Ikaros may be involved in the differentiation of trophoblasts, similarly to its role in lymphoid lineages. However, in contrast to the evidence that Ikaros-null mice lack fetal and adult B lymphocytes as well as fetal T lymphocytes (Wang et al., 1996Go), distinct defects in placental development in Ikaros-null mice have not been reported. In addition to detailed analyses of placenta in mice, studies using human trophoblast stem cells, which have not been isolated, are required to investigate this point.

In this study, because Ikaros was predominantly expressed in EVTs, we focused on EVTs and examined the role of Ikaros in these cells. Immunocytochemistry using EVTs in explant cultures of human chorionic villi confirmed the presence of Ikaros in EVTs. We then carried out Western blotting to identify Ikaros isoforms, as we were unable to determine this by immunochemistry due to the lack of isoform-specific Abs. Predominance of Ikx in EVTs would be significant because normal villi, possibly syncytiotrophoblasts, showed strong Ik2/3 expression and faint Ikx expression. Ikx is a novel Ikaros isoform that was recently reported to be expressed during normal myeloid differentiation (Payne et al., 2003Go). To investigate the function of Ikx in EVTs, we used the EVT cell line HTR-8/SVneo. HTR-8/SVneo cells expressed Ikx as a predominant isoform, and this result was similar to that in cultured EVTs, indicating that HTR-8/SVneo cells could be used as a model in this study. Cultured cytotrophoblasts are sometimes used in the study of trophoblasts, but we did not utilize them due to our focus on EVTs and the low transduction efficiency with adenovirus vectors. We transducted the dominant negative Ik6 gene into HTR-8/SVneo cells to suppress the DNA-binding activity of Ikx, which affected the transcription of target genes (Tucker et al., 2002Go; Yagi et al., 2002Go). As no Ik6-specific Ab is available, we transfected HA-tagged Ik6 to confirm the expression of Ik6 in HTR-8/SVneo cells. As expected, the Ik H-100 Ab, which recognizes all Ikaros isoforms, showed immunoreactivity in both parental and transducted cells, whereas the Ab against the HA-tag only showed expression in transducted cells. Western blotting revealed a band corresponding to Ik6 only in HTR-8/SVneo/Ik6, not in HTR-8/SVneo or mock-transfected cells, thus suggesting the successful transduction of Ik6 in HTR-8/SVneo cells.

In HTR-8/SVneo/Ik6 cells, we first evaluated the changes in cell growth. We may speculate that transduction of Ik6 favours EVT cell proliferation, because overexpression of Ik6 is implicated in up-regulation of anti-apoptotic proteins such as Bcl-2 and Bcl-XL in lymphoid lineage (Sezaki et al., 2003Go). However, no effect on HTR-8/SVneo cell proliferation was noted after Ik6 transduction. The influence of Ik6 transduction on cell growth may depend on cell types, although we need to clarify whether these anti-apoptotic proteins are involved in the growth of HTR-8/SVneo/Ik6 cells.

Ik6 overexpression significantly reduced migration and invasion of HTR-8/SVneo cells. We believe that these findings were not due to chance because mock-transfected cells showed no migratory or invasive changes and because proliferation was not affected by overexpression of Ik6. Ikx was the predominantly expressed Ikaros DNA-binding isoform in EVTs, and it is thus possible that Ikx induces migratory and invasive ability in EVTs. However, it is also possible that other Ikaros DNA-binding isoforms, in addition to other Ikaros family members (Helios and Aiolos) (Hahm et al., 1998Go), might be associated with these functions. Ikaros family proteins interact with one another to form heterodimers, which are involved in gene regulation and nucleosome remodelling (Morgan et al., 1997Go; Schmitt et al., 2002Go). Tight binding of Ik6 to Aiolos has been shown in lymphocytes that can bind DNA (Morgan et al., 1997Go). Moreover, all DNA-binding isoforms are thought to bind the same DNA sequence, and thus differences in their function remain undetermined. Specifically preventing the function of Ikx is essential to confirm the involvement of Ikx in migration and invasion of EVTs, while the finding that Ikaros isoforms are produced by alternative splicing complicates the application of RNA silence techniques to investigate the function of each isoform.

These are the first data to suggest that Ikaros plays an important role in cell migration and invasion. Ikaros may transcriptionally regulate genes that function in these processes in EVTs. Although the identity of factors regulated by Ikaros remains uncertain, the report that fibroblast growth factor-4 receptor is regulated by Ikaros (Yu et al., 2002Go) indicates that one possible candidate is fibroblast growth factor-4 receptor, which is known to be involved in the invasive activity of EVTs (Anteby et al., 2004Go). We are now attempting to identify the factors regulated by Ikaros.

Hematopoietic cells and trophoblasts share several common features. They are differentiated from stem cells and progenitors. Interestingly, many cluster differentiation (CD) antigens from hematopoietic cells have emerged as membrane-associated peptidases, which are abundantly present in placentas (Shipp and Look, 1993Go). Although speculative, Ikaros, which was thought to be specific to hematopoietic cells, may be associated with these features. We observed predominant expression of Ikx, which is known to participate in myeloid differentiation (Payne et al., 2003Go), in EVTs. Syncytiotrophoblasts mainly expressed Ik2/3, which resembles Ik1- and Ik2-regulated lymphoid and erythroid development (Molnar and Georgopoulos, 1994Go; Payne et al., 2003Go). One striking contrast is the finding that extra-villous and villous trophoblasts showed little Ik1 expression. Attempts to identify the target genes for Ikaros would pave the way to elucidate the common mechanisms of lineage differentiation between hematopoietic cells and trophoblasts.

In conclusion, this is the first report showing the location and possible function of Ikaros in human placenta, with particular focus on EVTs. Ikx is the preferentially expressed isoform in EVTs and may have an important role in regulating EVT migration and invasion. Further studies are required to clarify the mechanisms underlying Ikaros function in EVTs.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We thank the laboratory of Dr Charles H. Graham (Queen’s University, Ontario, Canada) for generous gift of HTR-8/SVneo cells and Dr Tomohiro Yagi (Kyoto Prefectural University of Medicine, Kyoto, Japan) for providing the construct HA-Ik6. This research was partly supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, from the Ministry of Public management, Home affairs, Posts and Telecommunications of Japan for specific medical research (in collaboration with Nagoya Teishin Hospital) and from the Ogya fund.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Abe A, Emi N, Kanie T, Imagama S, Kuno Y, Takahashi M, Saito H and Naoe T (2004) Expression cloning of oligomerization-activated genes with cell-proliferating potency by pseudotype retrovirus vector. Biochem Biophys Res Commun 320,920–926.[CrossRef][ISI][Medline]

Anteby EY, Greenfield C, Natanson-Yaron S, Goldman-Wohl D, Hamani Y, Khudyak V, Ariel I and Yagel S (2004) Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and –10 stimulate trophoblast plasminogen activator system and metalloproteinase-9. Mol Hum Reprod 10,229–235.[Abstract/Free Full Text]

Dovat S, Ronni T, Russell D, Ferrini R, Cobb BS and Smale ST (2002) A common mechanism for mitotic inactivation of C2H2 zinc finger DNA-binding domains. Genes Dev 16,2985–2990.[Abstract/Free Full Text]

Georgopoulos K, Moore DD and Derfler B (1992) Ikaros, an early lymphoid-specific transcription factor and a putative mediator for T cell commitment. Science 258,808–812.[Abstract/Free Full Text]

Georgopoulos K, Bigby M, Wang JH, Molnar A, Wu P, Winandy S and Sharpe A (1994) The Ikaros gene is required for the development of all lymphoid lineages. Cell 79,143–156.[CrossRef][ISI][Medline]

Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N and Lala PK (1993) Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 206,204–211.[CrossRef][ISI][Medline]

Hahm K, Ernst P, Lo K, Kim GS, Turck C and Smale ST (1994) The lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced mRNAs derived from the Ikaros gene. Mol Cell Biol 14,7111–7123.[Abstract/Free Full Text]

Hahm K, Cobb BS, McCarty AS, Brown KE, Klug CA, Lee R, Akashi K, Weissman IL, Fisher AG and Smale AT (1998) Helios, a T cell-restricted Ikaros family member that quantitatively associates with Ikaros at centromeric heterochromatin. Genes Dev 12,782–796.[Abstract/Free Full Text]

Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM and Crooks GM (1995) A functional comparison of CD34 + CD38-cells in cord blood and bone marrow. Blood 86,3745–3753.[Abstract/Free Full Text]

Ino K, Kikkawa F, Suzuki T, Kajiyama H, Shibata K, Nomura S, Itakura A, Ito M, Nagasaka T, Hattori A et al. (2003) Expression of placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in human normal and malignant invasive trophoblastic cells. Lab Invest 83,1799–1809.[CrossRef][ISI][Medline]

Ito T, Nomura S, Okada M, Katsumata Y, Iwase A, Kikkawa F, Tsujimoto M and Mizutani S (2001) Transcriptional regulation of human placental leucine aminopeptidase/oxytocinase gene. Mol Hum Reprod 7,887–894.[Abstract/Free Full Text]

Ito T, Nomura S, Okada M, Katsumata Y, Kikkawa F, Rogi T, Tsujimoto M and Mizutani S (2002) Ap-2 and Ikaros regulate transcription of human placental leucine aminopeptidase/oxytocinase gene. Biochem Biophys Res Commun 290,1048–1053.[CrossRef][ISI][Medline]

Klug CA, Morrison SJ, Masek M, Hahm K, Smale ST and Weissman IL (1998) Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes. Proc Natl Acad Sci USA 95,657–662.[Abstract/Free Full Text]

Lo K, Landau NR and Smale ST (1991) LyF-1, a transcriptional regulator that interacts with a novel class of promoters for lymphocyte-specific genes. Mol Cell Biol 11,5229–5243.[Abstract/Free Full Text]

Lopez RA, Schoetz S, DeAngelis K, O’Neill D and Bank A (2002) Multiple hematopoietic defects and delayed globin switching in Ikaros null mice. Proc Natl Acad Sci USA 99,602–607.[Abstract/Free Full Text]

Mitchell BF and Wong S (1995) Metabolism of oxytocin in human decidua, chorion, and placenta. J Clin Endocrinol Metab 80,2729–2733.[Abstract]

Molnar A and Georgopoulos K (1994) The Ikaros gene encodes a family of functionally diverse zinc finger DNA-binding proteins. Mol Cell Biol 14,8292–8303.[Abstract/Free Full Text]

Molnar A, Wu P, Largespada DA, Vortkamp A, Scherer S, Copeland NG, Jenkins NA, Bruns G and Georgopoulos K (1996) The Ikaros gene encodes a family of lymphocyte-restricted zinc finger DNA binding proteins, highly conserved in human and mouse. J Immunol 156,585–592.[Abstract]

Morgan B, Sun L, Avitahl N, Andrikopoulos K, Ikeda T, Gonzales E, Wu P, Neben S and Georgopoulos K (1997) Aiolos, a lymphoid restricted transcriptional factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J 16,2004–2013.[CrossRef][ISI][Medline]

Nagasaka T, Nomura S, Okamura M, Tsujimoto M, Nakazato H, Oiso Y, Nakashima N and Mizutani S (1997) Immunohistochemical localization of placental leucine aminopeptidase/oxytocinase in normal human placental, fetal and adult tissues. Reprod Fertil Dev 9,747–753.[CrossRef][Medline]

Nichogiannopoulou A, Trevisan M, Neben S, Friedrich C and Georgopoulos K (1999) Defects in hemopoietic stem cell activity in Ikaros mutant mice. J Exp Med 190,1201–1214.[Abstract/Free Full Text]

Payne KJ, Nicolas JH, Zhu JY, Barsky LW and Crooks GM (2001) Cutting edge: predominant expression of a novel Ikaros isoform in normal human hemopoiesis. J Immunol 167,1867–1870.[Abstract/Free Full Text]

Payne KJ, Huang G, Sahakian E, Zhu JY, Barteneva NS, Barsky LW, Payne MA and Crooks GM (2003) Ikaros isoform x is selectively expressed in myeloid differentiation. J Immunol 170,3091–3098.[Abstract/Free Full Text]

Sato Y, Fujiwara H, Higuchi T, Yoshioka S, Tatsumi K, Maeda M and Fujii S (2002) Involvement of dipeptidyl peptidase IV in extravillous trophoblast invasion and differentiation. J Clin Endocrinol Metab 87,4287–4296.[Abstract/Free Full Text]

Schmitt C, Tonnelle C, Dalloul A, Chabannon C, Debré P and Rebollo A (2002) Aiolos and Ikaros: regulators of lymphocyte development, homeostasis and lymphoproliferation. Apoptosis 7,277–284.[CrossRef][ISI][Medline]

Sezaki N, Ishimaru F, Takata M, Tabayashi T, Nakase K, Kozuka T, Fujii K, Nakayama H, Teshima T, Harada M et al. (2003) Over-expression of the dominant-negative isoform of Ikaros confers resistance to dexamethasone-induced and anti-IgM-induced apoptosis. Br J Haematol 121, 165–169.[CrossRef][ISI][Medline]

Shih IM and Kurman RJ (1996) Expression of melanoma cell adhesion molecule in intermediate trophoblast. Lab Invest 75,377–388.[ISI][Medline]

Shipp MA and Look AT (1993) Hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key!. Blood 82, 1052–1070.[Free Full Text]

Sun L, Liu A and Georgopoulos K (1996) Zinc finger-mediated protein interactions modulate Ikaros activity, a molecular control of lymphocyte development. EMBO J 15,5358–5369.[ISI][Medline]

Tsujimoto M, Mizutani S, Adachi H, Kimura M, Nakazato H and Tomoda Y (1992) Identification of human placental leucine aminopeptidase as oxytocinase. Arch Biochem Biophys 292,388–392.[CrossRef][ISI][Medline]

Tucker SN, Jessup HK, Fujii H and Wilson CB (2002) Enforced expression of the Ikaros isoform IK5 decreases the numbers of extrathymic intraepithelial lymphocytes and natural killer 1.1 + T cells. Blood 99,513–519.[Abstract/Free Full Text]

Wang JH, Nichogiannopoulou A, Wu L, Sun L, Sharpe AH, Bigby M and Georgopoulos K (1996) Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5,537–549.[CrossRef][ISI][Medline]

Yagi T, Hibi S, Takanashi M, Kano G, Tabata Y, Imamura T, Inaba T, Morimoto A, Todo S and Imashuku S (2002) High frequency of Ikaros isoform 6 expression in acute myelomonocytic and monocytic leukemias: implications for up-regulation of the antiapoptotic protein Bcl-XL in leukemogenesis. Blood 99,1350–1355.[Abstract/Free Full Text]

Yu S, Asa SL and Ezzat S (2002) Fibroblast growth factor receptor 4 is a target for the zinc-finger transcription factor Ikaros in the pituitary. Mol Endocrinol 16,1069–1078.[Abstract/Free Full Text]

Submitted on June 17, 2005; accepted on October 20, 2005.


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