Skip Navigation


Mol. Hum. Reprod. Advance Access originally published online on October 25, 2007
Molecular Human Reproduction 2007 13(12):869-874; doi:10.1093/molehr/gam078
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/12/869    most recent
gam078v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liu, J.
Right arrow Articles by Maruo, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, J.
Right arrow Articles by Maruo, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. 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

The effects of progesterone on apoptosis in the human trophoblast-derived HTR-8/SV neo cells

Jin Liu1, Hiroya Matsuo2, Jovelle B. Laoag-Fernandez1, Qin Xu1 and Takeshi Maruo1,3

1Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan 2Department of Health Sciences, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan

3 Correspondence address. Tel: +81-78-382-6000; Fax: +81-78-382-6019; E-mail: maruo{at}kobe-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Progesterone (P4) is frequently used in the treatment of threatened abortion, prevention of recurrent miscarriage and threatened preterm labor. However, little is known about the molecular mechanism of P4 in the regulation of extravillous trophoblasts' (EVTs) function. This study was designed to examine the presence of progesterone receptor (PR) in the human trophoblast-derived HTR-8/SV neo cell line, which is a possible model of EVTs, and the effects of P4 on apoptosis in those cells. The HTR-8/SV neo cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. When the cell the population reached 50% confluency, the cells were stepped down to serum-free conditions in the presence or absence of graded concentrations of P4 (1, 10 and 100 ng/ml) for 48 h. The cultured cells were used for RT–PCR, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, immunocytochemistry and western blot analyses. Immunocytochemistry and western blot analyses revealed that PR was evident in HTR-8/SV neo cells. Compared with untreated cultures, treatment with P4 (10 and 100 ng/ml) resulted in significant decreases in the TUNEL-positive rate, Fas, Fas ligand (Fas-L), caspase-8, caspase-3 and poly (ADP-ribose) polymerase (PARP) expression in HTR-8/SV neo cells, and a significant increase in Bcl-2 expression in those cells. Consistently, Fas mRNA expression in those cells was significantly inhibited by the treatment with 10 ng/ml P4 compared with untreated cultures. This study suggests that PR exists in HTR-8/SV neo cells and that P4 inhibits apoptosis by down-regulating Fas, Fas-L, caspase-8, caspase-3 and PARP expression as well as up-regulating Bcl-2 expression in HTR-8/SV neo cells.

Key words: apoptosis/HTR-8/SV neo cell line/progesterone/human trophoblast cell model


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Placental tissues contain a heterogeneous population of cells, including villous cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts (EVTs). EVTs are mainly uninuclear cells comprising all the trophoblastic elements located outside the villi. EVT has two distinct phenotypes, proliferative and invasive. The development of the human fetus depends on the ability of EVTs to invade the maternal uterine tissues, then to anchor the placenta and the ability of the fetus to gain access to the maternal circulation (Genbacev et al., 1992; Irving et al., 1995; Aboagye-Mathiesen et al., 1996).

Several investigators suggest that apoptosis is an important determinant in the regulation of placental growth (Laoag-Fernandez et al., 2004). The activity of the invasive EVTs seems to be dependent on their apoptotic capacity and less on their proliferative potential because they are highly differentiated cells (Kaufmann and Castellucci, 1997). We have demonstrated that apoptosis in the invasive EVTs was more evident than its proliferative counterpart and that the extent of apoptosis was associated with augmented Fas and Fas ligand (Fas-L) expression and reduced Bcl-2 protein expression (Murakoshi et al., 2003).

Clinically, P4 is frequently used in the treatment of threatened abortion, prevention of recurrent miscarriage, threatened preterm labor and in the support of the luteal phase in assisted reproduction programs (Di Renzo et al., 2005). The physiological effects of P4 are mediated through interaction with the progesterone receptor (PR), a transcription factor and a member of a large family of structurally related gene products known as the nuclear receptor superfamily. PR is expressed in multiple tissues as two isoforms, PR-A and PR-B. These isoforms are derived from the same gene by the action of two different promoters (Giangrande and Donnell, 1999; Richer et al., 2002). Although the expression of PR on EVT populations has not been extensively analyzed, there is evidence suggesting EVT populations at the materno–fetal interface in the first-trimester pregnancy are positive for PR (Wang et al., 1996a). However, little is known about the molecular mechanism of P4 in the regulation of EVTs function.

Most experimental data regarding the molecular basis of human trophoblast functions have been obtained from primary cultures of trophoblasts. In order to make it possible to perform long-term culture of homogeneous human trophoblast cell populations, the first-trimester trophoblast cells have been transfected with the simian virus 40 large Tantigen. The resulting cell line (HTR-8/SV neo cell line) shares phenotypic properties with the progenitor cells (Graham et al., 1993), and its proliferation, migration and invasiveness are regulated by the same signaling molecules that modulate EVT cell responses in vivo (Graham et al., 1993; Lala et al., 2002). Accordingly, this cell line represents a highly useful model to study the mechanisms underlying the regulation of human trophoblast cell growth (Munir et al., 2004) as well as the control of EVT cell proliferation, migration and invasiveness operated by several modulators (Graham et al., 1993; Kilburn et al., 2000; Lala et al., 2002; Munir et al., 2004). Tarrade et al. (2002) and Oki et al. (2004) in our laboratory revealed that >99% of the primary cultured early placental trophoblasts attached to fibronectin-precoated dishes expressed CK7 (cytokeratin 7), hPL (human placental lactogen) and ErbB2 protein, but not ErbB1 protein after 48-h subculture, demonstrating the characteristics of EVT. Thus, we examined the presence of CK7, erbB1 and erbB2 in HTR-8/SV neo cell line to confirm the characteristics of the cell line.

In order to evaluate the effects of P4 on apoptosis in EVTs, we used HTR-8/SV neo cell line, a useful model, to examine the presence of PR protein and then determined the effects of P4 on the expressions of Fas, Fas-L, Bcl-2, caspase-8, caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) in those cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 Acknowledgements
 References
 
Cell culture
The HTR-8/SV neo trophoblast cells were kindly provided by Dr Benjamin K. Tsang, University of Ottawa, Ottawa, Canada. Cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies, Inc., Burlington, Canada) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were seeded into 2-well chamber slides for immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, 6-well plates for western blot analysis and 100 mm dishes for RT–PCR. Cells were incubated in a standard Sanyo CO2 incubator (5% CO2 in air, 37°C; Esbe Scientific, Markham, Canada). When the cell population reached 50% confluency, the cultured cells were stepped down to serum-free conditions in presence or absence of graded concentrations of P4 (1, 10 and 100 ng/ml) for the subsequent 48 h.

Immunocytochemical staining for PR
HTR-8/SV neo cells cultured in 2-well chamber slides were washed three times with phosphate-buffered saline (PBS), fixed in 99.9% ethanol at 4°C for 20 min, and again washed with PBS three times. The fixed cells were subjected to immunostaining by the avidin/biotin immunoperoxidase method using a polyvalent immunoperoxidase kit (Omnitags, Lipshow, MI, USA) according to the manufacturer's instructions. A mouse monoclonal antibody to human PR (AB-52) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as the primary antibody at a dilution of 1:250. To assure the specificity of the immunological reaction, cultured cells were subjected to the same immunoperoxidase method, except that the primary antibody was replaced by nonimmune murine IgG (Miles, Elkhart, IN, USA) at the same dilution as the specific antibody. The replacement of the specific primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining for PR.

In situ TUNEL assay
In situ labeling of fragmented DNA in cultured HTR-8/SV neo cells was performed with the TUNEL assay, using the ApopTag in situ apoptosis detection kit (Intergen Co., Purchase, NY, USA) according to the manufacturer's protocol for monolayer cultures. HTR-8/SV neo cells were cultured in 2-well glass chamber slides for 120 h and then cultured under serum deprivation conditions for 24 and 48 h in the absence or presence of graded concentrations of P4 (1, 10 and 100 ng/ml). At the termination of cultures, nucleotide-sized DNA fragments were tailed with digoxigenin-deoxy-UTP and then bound with peroxidase-conjugated antidigoxigenin antibodies. The nuclei were counterstained with hematoxylin (Zymed Laboratories, Inc., San Francisco, CA, USA) for determining the TUNEL-positive rate of HTR-8/SV neo cells.

Apoptosis of cultured HTR-8/SV neo cells was analyzed by two investigators in a blinded fashion without knowledge of the experimental group. All stained nuclei were scored as positive for apoptosis. The TUNEL-positive rate was determined by observing >1000 nuclei for each experimental sample.

Western blot analysis for PR, Bcl-2, Fas-l, Fas, caspase-8,caspase-3 and PARP
Proteins were extracted from cultured HTR-8/SV neo cells as described previously (Shimomura et al., 1998). At the termination of cultures, cells were lysed at 4°C for 20 min in the presence of a lysis buffer consisting of 150 mM NaCl, 2 mM phenylmethylsulfonylfluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/l aprotinin, 0.1% sodium dodecyl sulfate and 50 mM Tris–HCl, pH 7.5. The lysates were subsequently centrifuged at 13 000g for 30 min at 4°C, and the supernatants were collected. Protein content in the supernatants was determined by the Bradford assay (Bradford, 1976). Each 200 µg aliquot of the protein extracted from cultured HTR-8/SV neo cells was separated by NuPAGE Novex 4–12% Bis–Tris Gel (Invitrogen life technologis, Carisbad, CA, USA) under a reducing condition using 200 V for 50 min. The proteins were then electrophoretically transferred from gels to PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The blots were exposed overnight to a mouse monoclonal antibody to PR (AB-52) (Santa Cruz Biotechnology, Inc.), a mouse monoclonal antibody to Bcl-2 (Santa Cruz Biotechnology, Inc.), a mouse monoclonal antibody to Fas-L (Santa Cruz Biotechnology, Inc.), a mouse monoclonal antibody to Fas (Santa Cruz Biotechnology, Inc.), a mouse monoclonal antibody to caspase-8 (Santa Cruz Biotechnology, Inc.), a rabbit polyclonal antibody to caspase-3 (Santa Cruz Biotechnology, Inc.) and a rabbit polyclonal antibody to PARP (Cell Signaling Technology, Inc.) at dilutions of 1:200, 1:200, 1: 200, 1:250, 1:200, 1:200 and 1:1000, respectively. The membranes were incubated for 1 h with horse-radish peroxidase-conjugated antimouse or antirabbit secondary antibody (Amersham Biosciences, Arlington Heights, IL, USA) that was diluted at 1:1000 with blocking buffer. The antigen–antibody complexes were detected with the ECL chemiluminescence detection system (Amersham Biosciences). Membranes were visualized by exposure to X-OMAT film (Eastman Kodak Co., Rochester, NY, USA). The radioautograms were then scanned and quantified with ChemiImager 4400 (Astec Co. Ltd., Osaka, Japan).

Quantitative RT–PCR analysis
When the cell population reached 50% confluency, HTR-8/SV neo cells attached to 100 mm culture dishes were characterized by RT–PCR analysis with erbB1, erbB2 and CK7 primers. After 24 h of subsequent culture, the mRNA expression of Fas in subcultured HTR-8/SV neo cells was examined by RT–PCR analysis. Total RNA was isolated from frozen HTR-8/SV neo cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. First strand cDNA was synthesized from 4 µg total RNA using a cDNA synthesis kit (Qiagen) according to the manufacturer's protocol. PCR was performed using 0.1 µg cDNA as a template in a 25-µl reaction buffer [10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM of MgCl2 and 0.01% gelatin] containing 6.25 pM of each primer, 2.5 mM deoxy-NTPs and 0.125 U Taq DNA polymerase (all from Qiagen). Reactions were amplified by a Gene Amp PCR System 9600-R (PerkinElmer, Norwalk, CT, USA) using the following thermal profile: initial denaturation step at 94°C for 5 min, followed by 35 or 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s and a final elongation of 72°C for 5 min. Primers and antisense primer were synthesized according to the report by Tarrade et al. (2002). Separate reactions were performed using primers specific for β-actin (sense primer, 5'-CTTCTACAATGAGCTGCGTG-3'; antisense primer, 5'-TCATGAGGTAGTCAGTCA-3'). The PCR product specific for β-actin was visualized under UV light after gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide. These experiments were performed four times. The sequences of the specific oligonucleotide primers and the sizes of the respective amplification products are given. ErbB1 sense: 5'-CAGCGCTACCTTGTCATTCAG-3', erbB1 antisense: 5'-TCATACTATCCTCCGTGGTCA-3'; erbB2 sense: 5'-AGGGAAACCTGGAACTCACC-3'; erbB2 antisense: 5'-TGGATCAAGACCCCTC CTT-3'; CK7 sense: 5'-GGACATCGAGATCGCCACCT-3', CK7 antisense: 5'-ACCGCCACTGCTACTGCCA-3'; Fas sense; 5'-TGGCTTTGTCTTCTTCTTTG-3', Fas antisense: 5'-TCATCTATTTTGGCTTCATTG-3'.

Statistical analysis
The experiments were repeated at least three times with different batches of the cells. Results are presented as the mean ± SD according to the repeated experiments. Statistical significance was determined using Student t-test and one-way ANOVA. A difference with a P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 Acknowledgements
 References
 
Characterization of HTR-8/SV neo cells
RT–PCR analysis of mRNA extracted from cultured HTR-8/SV neo cells demonstrated that mRNA of erbB1, erbB2 and CK7 were present in HTR-8/SV neo cells. The expression of erbB2 was stronger than that of erbB1 (Fig. 1).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1: Expressions of erbB1, erbB2 and CK7 mRNA in culturedHTR-8/SV neo cells assessed by RT–PCR

β-actin expression was used as adjustment

 
Expression of PR in cultured HTR-8/SV neo cells
Fig. 2A shows immunocytochemical staining of PR in cultured HTR-8/SV neo cells. The immunolocalization of PR on the nuclei of cultured HTR-8/SV neo cells was noted. Replacement of the primary antibody with nonimmune murine IgG resulted in a lack of positive immunostaining for PR in those cells.


Figure 2
View larger version (41K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2: (A) Expression of PR in cultured HTR-8/SV neo cells assessed by immunocytochemical stainingThe immunolocalization of PR in the nuclei (a) and the negative immunostaining for PR with replacement of the primary antibody with nonimmune murine IgG (b). Bars, 50 mm. Original magnification, x400. (B) The expression of PR protein in cultured HTR-8/SV neo cells assessed by western blot analysis. Leiomyoma cells were considered as the positive control

 
Western blot analysis of proteins extracted from cultured HTR-8/SV neo cells revealed that HTR-8/SV neo cells contained immunoactive PR-A and PR-B with a molecular mass of 116 and 96 kDa, respectively (Fig. 2B).

Effects of P4 on the TUNEL-positive rate in culturedHTR-8/SV neo cells
Fig. 3A shows the TUNEL-positive rate in cultured HTR-8/SV neo cells in the absence or presence of graded concentrations of P4 for 24 and 48 h. Determination of the mean percentage of TUNEL-positive nuclei in the cultured HTR-8/SV neo cells demonstrated that treatment with P4 decreased the TUNEL-positive rate of the cultured HTR-8/SV neo cells compared with untreated cultures. A significant decrease in the TUNEL-positive rate of cultured HTR-8/SV neo cells was obtained by 24 and 48-h treatment with P4 at concentrations >10 ng/ml (P < 0.05).


Figure 3
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3: (A) Effects of P4 on the TUNEL-positive rate of cultured HTR-8/SV neo cells, as assessed by TUNEL assay(B) Effects of P4 on Bcl-2 protein expression in cultured HTR-8/SV neo cells at 24 h, as assessed by western blot analysis. (C) Effects of P4 on Fas-L protein expression in cultured HTR-8/SV neo cells at 24 h, as assessed by western blot analysis. β-actin expression was used as adjustment. *P < 0.05 (versus the corresponding untreated control)

 
Effects of P4 on Bcl-2 protein expression in culturedHTR-8/SV neo cells
Effects of treatment with graded concentrations of P4 on Bcl-2 protein expression in cultured HTR-8/SV neo cells at 24 h were assessed by western blot analysis (Fig. 3B). Compared with untreated cultures, treatment with P4 at concentrations >10 ng/ml significantly (P < 0.05) increased 26-kDa Bcl-2 protein expression.

Effects of P4 on Fas-L protein expression in culturedHTR-8/SV neo cells
Effects of treatment with graded concentrations of P4 on Fas-L protein expression in cultured HTR-8/SV neo cells at 24 h were assessed by western blot analysis (Fig. 3C). Compared with untreated cultures, treatment with P4 at concentrations >10 ng/ml significantly (P < 0.05) decreased 39-kDa Fas-L protein expression.

Effects of P4 on Fas protein expression and Fas mRNA expression in cultured HTR-8/SV neo cells
Effects of treatment with graded concentrations of P4 on Fas protein expression in cultured HTR-8/SV neo cells at 24 h were assessed by western blot analysis (Fig. 4A). Compared with untreated cultures, treatment with P4 at concentrations >10 ng/ml significantly (P < 0.05) decreased 51-kDa Fas protein expression. Effects of treatment with P4 on Fas mRNA expression in cultured HTR-8/SV neo cells at 24 h were assessed by RT–PCR (Fig. 4B). Treatment with P4 at concentration of 10 ng/ml significantly (P < 0.05) decreased 257 bp Fas mRNA expression compared with untreated cultures.


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4: (A) Effects of P4 on Fas protein expression in cultured HTR-8/SV neo cells at 24 h, as assessed by western blot analysis(B) Effects of P4 on Fas mRNA expression in cultured HTR-8/SV neo cells at 24 h, as assessed by RT–PCR. β-actin expression was used as adjustment. *P < 0.05 (versus the corresponding untreated control)

 
Effects of P4 on cleaved caspase-8 and caspase-3 expression in cultured HTR-8/SV neo cells
Effects of treatment with graded concentrations of P4 on cleaved caspase-8 expression in cultured HTR-8/SV neo cells at 24 h were assessed by western blot analysis (Fig. 5A). Compared with untreated cultures, treatment with P4 at concentrations >10 ng/ml significantly (P < 0.05) decreased 20-kDa cleaved caspase-8 expression. Effects of treatment with graded concentrations of P4 on cleaved caspase-3 expression in cultured HTR-8/SV neo cells at 24 h were assessed by western blot analysis (Fig. 5B). Compared with untreated cultures, treatment with P4 at concentrations >10 ng/ml significantly (P < 0.05) decreased 17-kDa cleaved caspase-3 expression.


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5: (A) Effects of P4 on cleaved caspase-8 expression in cultured HTR-8/SV neo cells at 24 h, as assessed by western blot analysis(B) Effects of P4 on cleaved caspase-3 expression in cultured HTR-8/SV neo cells at 24 h, as assessed by western blot analysis. β-actin expression was used as adjustment. *P < 0.05 (versus the corresponding untreated control)

 
Effects of P4 on cleaved PARP expression in culturedHTR-8/SV neo cells
Effects of treatment with graded concentrations of P4 on cleaved PARP expression in cultured HTR-8/SV neo cells at 24 h were assessed by western blot analysis (Fig. 6). Treatment with P4 at concentrations >10 ng/ml significantly (P < 0.05) decreased 89-kDa cleaved PARP expression.


Figure 6
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6: Effects of P4 on cleaved PARP expression in cultured HTR-8/SV neo cells at 24 h, as assessed by western blot analysisβ-actin expression was used as adjustment. *P < 0.05 (versus the corresponding untreated control)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 Acknowledgements
 References
 
In the present study, we have demonstrated that PR exists in HTR-8/SV neo cells and P4 may inhibit apoptosis in HTR-8/SV neo cells through down-regulating Fas, Fas-L, caspase-8, caspase-3 and PARP expression and through up-regulating Bcl-2 expression in those cells.

The results obtained by RT–PCR with HTR-8/SV neo cells revealed that CK7, one of the markers for trophoblast (Blaschitz et al., 2000; Leach et al., 2002), was expressed. ErbB1, a specific marker of both cytotrophoblast and syncytiotrophoblast, and ErbB2, a specific marker for EVT and syncytiotrophoblast (Tarrade et al., 2001), were also expressed in those cells. The expression of erbB2 mRNA in cultured HTR-8/SV neo cells was more abundant than that of erbB1 mRNA. The results obtained were consistent with the findings of Oki et al. (2004) that the expression of erbB2 mRNA was stable, whereas the expression of erbB1 mRNA decreassed remarkably after 24-h subculture compared with that after 1.5-h subculture. Ninety-nine per cent of the primary cultured early placental trophoblasts attached to fibronectin-precoated dishes expressed CK7, hPL and ErbB2 protein, but not ErbB1 protein after 48-h subculture. Based on these findings, most of HTR-8/SV neo cells used in the present study seemed to be EVTs, although there might be a few cytotrophoblast cells in the HTR-8/SV neo cells.

Clinically, P4 is frequently used in the treatment of threatened abortion, prevention of recurrent miscarriage, threatened preterm labor and in the support of the luteal phase in assisted reproduction programs (Di Renzo et al., 2005). However, little is known about the molecular mechanism of P4 in the regulation of EVTs function.

There have been controversies regarding the presence of PR in human placenta (McCormick et al., 1981; Rivera and Cano, 1989; Rossmanith et al., 1997; Chan et al., 2003). Several investigators have indicated the weak PR signals could be found by using immunocytochemical staining and RT–PCR in the all cell types, including the cytotrophoblast cells, syncytiotrophoblast cells and EVT, in both the first trimester and the term placenta (Wang et al., 1996a; Rossmanith et al., 1997). We have detected that PR is expressed in cultured HTR-8/SV neo cells by using immunocytochemical staining and western blot analyses. Thus, in this study, we examined the effects of P4 on apoptosis in HTR-8/SV neo cells.

Apoptosis is an essential mechanism to eliminate unwanted cells during the development and homeostasis of multicellular organisms (Jacobson et al., 1997; Raff, 1998). Unregulated cell death is implicated in a growing number of clinical disorders. There are the two major apoptotic pathways in mammalian cells, i.e. the extrinsic pathway and the intrinsic mitochondrial pathway (Riedl and Shi, 2004). In the extrinsic pathway, the apoptotic signal is initiated by direct ligand-mediated trimerization of death receptors, i.e. Fas or tumor necrosis factor-R at the cell surface. This leads to the recruitment of adaptor proteins inside the cells, to the activation of initiator caspase-8 or -10 and, subsequently, to the activation of the executioner caspase-3, -7 and possibly -6 (Markus, 2000). In the intrinsic mitochondrial pathway, caspase-8 degrades Bid, which is a member of the Bcl-2 family (Wang et al., 1996b) and the active Bid fragment localized in the mitochondria induces the release of cytochrome c from mitochondria into the cytosol and forms the apoptosome together with apoptosis-protease activating factor-1 (Riedl and Shi, 2004; Shiozaki and Shi, 2004), which recruits caspase-9 (Shi, 2004). Caspase-9 cleaves and activates the effector caspase, caspase-3 (Riedl and Shi, 2004; Shi, 2004). During the course of apoptosis, caspase-3 proteolytically cleaves the nuclear enzyme PARP (Cohen, 1997), which is implicated in DNA replication, transcription, DNA repair, apoptosis and genome stability (Bouchard et al., 2003). Thus, the cleavage of PARP is regarded as a hallmark event for the apoptotic paradigm (Pieper et al., 1999). In contrast, Bcl-2 protein that resides in mitochondrial membranes acts to prevent the release of apoptogenic proteins from mitochondria (Reed et al., 1998). Bcl-2 protein, therefore, inhibits the progress of the apoptosis cascade at the mitochondrial level between activity of initiator caspase-8 and -10 and activation of execution caspases (Huppertz et al., 2006).

Apoptosis is an important determinant in the regulation of placental growth (Laoag-Fernandez et al., 2004). It has been demonstrated that Bcl-2 and the family of caspase proteins are involved in the process of apoptosis cascade within the villous trophoblasts, and that the apoptosis cascade in EVTs may be similar to that of villous trophoblasts (Huppertz et al., 2006). However, the regulation of the apoptosis cascade in EVTs is poorly understood. Inducers of the cascade such as Fas/CD95 and its ligand Fas-L/CD95L have been detected in EVTs. Fas and Fas-L have been shown to be present on EVTs of first-trimester tissues as well as term basal plates (Huppertz et al., 2006). We have also demonstrated that Fas antibody augmented the apoptosis in the cultured EVTs (Laoag-Fernandez et al., 2004). These findings supports that the Fas/Fas-L system plays an important role in the induction of apoptosis in various cell lineages (Nagata and Golstein, 1995), including EVTs.

In this study, we found P4 inhibited the apoptosis in HTR-8/SV neo cells, at the same time, P4 suppressed the expressions of Fas and Fas-L. Thus P4 may reduce the apoptosis cascade of EVTs through reducing expressions of Fas and Fas-L. In addition, we found that P4 suppressed cleaved caspase-8, caspase-3, PARP expression and augmented Bcl-2 protein expression. These results suggest that P4 may exert antiapoptotic action in HTR-8/SV neo cells through the extrinsic apoptotic pathway by down-regulating Fas/Fas-L and the intrinsic mitochondria-mediated apoptotic pathway by up-regulating Bcl-2 protein expression, subsequently down-regulating cleaved caspase-8, caspase-3 and PARP expression.

It has been known that 3,5,3'-triiodothyronine (T3) down-regulates apoptosis of early placental EVTs through the inhibition of Fas and Fas-L expression and caspase-3 and PARP cleavage (Laoag-Fernandez et al., 2004), and T3 may play a vital role in up-regulating the invasive potential of EVTs into the deciduas (Oki et al., 2004). T3 may promote the invasion of EVTs to the deciduas by inhibiting apoptosis of EVTs in early pregnancy (Laoag-Fernandez et al., 2004). Therefore, we hypotheses that P4 may promote the invasion of HTR-8/SV neo cells to the deciduas by inhibiting apoptosis of HTR-8/SV neo cells, which may suggest the mechanism of treatment of P4 on threatened abortion.

In conclusion, we have provided evidence that PR exists in HTR-8/SV neo cells and P4 inhibits apoptosis in HTR-8/SV neo cells by down-regulating Fas, Fas-L, caspase-8, caspase-3 and PARP expression and up-regulating Bcl-2 expression in those cells. Further investigations using primary cell cultures obtained from human EVTs will be needed to shed lightly on the effects of P4 in EVTs.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
Acknowledgements
 References
 
This work was supported in part by Grants-in-Aid for Scientific Research (18659487) from the Japanese Ministry of Education, Science and Culture and by the Ogyaa-Donation Foundation of the Japan Association of Obstetricians and Gynecologists.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
References
 
The authors greatly appreciate Dr Benjamin K. Tsang, University of Ottawa, Canada for kindly providing HTR-8/SV neo trophoblast cells.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Aboagye-Mathiesen G, Laugesen J, Zdravkovic M, Ebbesen P. Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi. Clin Diagn Lab Immunol (1996) 3:14–22.[Medline]

Blaschitz A, Weiss U, Dohr G, Desoye G. Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta (2000) 21:733–741.[CrossRef][Web of Science][Medline]

Bouchard VJ, Rouleau M, Poirier GG. PARP-1, a determinant of cell survival in response to DNA damage. Exp Hematol (2003) 31:446–454.[CrossRef][Web of Science][Medline]

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal Biochem (1976) 72:248–253.[CrossRef][Web of Science][Medline]

Chan CC, Lao TT, Ho PC, Sung EO, Cheung AN. The effect of mifepristone on the expression of steroid hormone receptors in human decidua and placenta: a randomized placebo-controlled double-blind study. J Clin Endocrinol Metab (2003) 88:5846–5850.[Abstract/Free Full Text]

Cohen GM. Caspases: the executioners of apoptosis. Biochem J (1997) 326:1–16.[Web of Science][Medline]

Di Renzo GC, Mattei A, Gojnic M, Gerli S. Progesterone and pregnancy. Curr Opin Obstet Gynecol (2005) 6:598–600.

Genbacev O, Schubach S, Miller R. Villous culture of first trimester human placenta—model to study extravillous trophoblast (EVT) differentiation. Placenta (1992) 13:439–461.[Web of Science][Medline]

Giangrande PH, Donnell DP. The A and B isoform of the human progesterone receptor: two functionally different transcription factors encoded by a single gene. Recent Prog Horm Res (1999) 54:291–314.[Medline]

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

Huppertz B, Kadyrov M, Kingdom JC. Apoptosis and its role in the trophoblast. Am J Obstet Gynecol (2006) 195:29–39.[CrossRef][Web of Science][Medline]

Irving J, Lysiak J, Graham C, Hearn S, Han V, Lala P. Characteristics of trophoblast cells migrating from first trimester chorionic villous explants and propagated in culture. Placenta (1995) 16:413–433.[CrossRef][Web of Science][Medline]

Jacobson MD, Weil M, Raff MC. Programmed cell death in anima development. Cell (1997) 88:347–354.[CrossRef][Web of Science][Medline]

Kaufmann P, Castellucci M. Extravillous trophoblast in the human placenta. Tropho Res (1997) 10:21–65.

Kilburn BA, Wang J, Duniec-Dmuchkowski ZM, Leach RE, Romero R, Armant DR. Extracellular matrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line. Biol Reprod (2000) 62:739–747.[Abstract/Free Full Text]

Lala PK, Lee BP, Xu G, Chakraborty C. Human placental trophoblast as an in vitro model for tumor progression. Can J Physiol Pharmacol (2002) 80:142–149.[CrossRef][Web of Science][Medline]

Laoag-Fernandez JB, Matsuo H, Murakoshi H, Hamada AL, Tsang BK, Maruo T. 3,5,3'-Triiodothyronine down-regulates Fas and Fas ligand expression and suppresses caspase-3 and poly (adenosine 5'-diphosphate-ribose) polymerase cleavage and apoptosis in early placental extravillous trophoblasts in vitro. J Clin Endocrinol Metab (2004) 89:4069–4077.[Abstract/Free Full Text]

Leach RE, Romero R, Kim YM, Chaiworapongsa T, Kilburn B, Das SK, Johnson A, Qureshi F, Jacques S, Armant DR. Pre-eclampsia and expression of heparin-binding EGF-like growth factor. Lancet (2002) 360:1215–1219.[CrossRef][Web of Science][Medline]

Markus GG. Caspases: key players in programmed cell death. Current Curr Opin Struct Biol (2000) 10:649–655.[CrossRef]

McCormick PD, Razel AJ, Spelsberg TC, Coulam CB. Absence of high-affinity binding of progesterone (R5020) in human placenta and fetal membranes. Placenta (1981) 3(Suppl):123–132.[Medline]

Munir S, Xu G, Wu Y, Yang BB, Lala PK, Peng C. Nodal and activin receptor-like kinase (ALK) 7 inhibit proliferation and induce apoptosis in human trophoblast cells. J Biol Chem (2004) 279:31277–31286.[Abstract/Free Full Text]

Murakoshi H, Matsuo H, Laoag-Fernandez JB, Samoto T, Maruo T. Expression of Fas/Fas ligand, Bcl-2 protein and apoptosis in extravillous trophoblast along invasion to the deciduas in human term placenta. Endocr J (2003) 50:199–207.[CrossRef][Web of Science][Medline]

Nagata S, Golstein P. The Fas death factor. Science (1995) 267:1449–1456.[Abstract/Free Full Text]

Oki N, Matsuo H, Nakago S, Murakoshi H, Laoag-Fernandez JB, Maruo T. Effects of 3,5,3'-triiodothyronine on the invasive potential and the expression of integrins and matrix metalloproteinases in cultured early placental extravillous trophoblasts. J Clin Endocrinol Metab (2004) 89:5213–5221.[Abstract/Free Full Text]

Pieper AA, Verma A, Zhang J, Snyder SH. Poly (ADP-ribose) polymerase, nitric oxide and cell death. Trends Pharmacol Sci (1999) 20:171–181.[CrossRef][Medline]

Raff M. Cell suicide for beginners. Nature (1998) 396:119–122.[CrossRef][Medline]

Reed JC, Jurgensmeier JM, Matsuyama S. Bcl-2 family proteins and mitochondria. Biochim Biophys Acta (1998) 1366:127–137.[Medline]

Richer JK, Jacobson BM, Manning NG, Abel MG, Wolf DM, Horwitz KB. Differential gene regulation by the two progesterone receptor isoforms in human breast cancer cells. J Biol Chem (2002) 277:5209–5218.[Abstract/Free Full Text]

Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol (2004) 5:897–907.[CrossRef][Web of Science][Medline]

Rivera J, Cano A. Oestrogen and progesterone receptors in human term placenta. Measurement by binding assays and immunological methods. Placenta (1989) 10:579–588.[Web of Science][Medline]

Rossmanith WG, Wolfahrt S, Ecker A, Eberhardt E. The demonstration of progesterone, but not of estrogen, receptors in the developing human placenta. Horm Metab Res (1997) 29:604–610.[Web of Science][Medline]

Shi Y. Caspase activation, inhibition, and reactivation: a mechanistic view. Protein Sci (2004) 13:1979–1987.[CrossRef][Web of Science][Medline]

Shimomura Y, Matsuo H, Samoto T, Maruo T. Up-regulation by progesterone of proliferating cell nuclear antigen and epidermal growth factor expression in human uterine leiomyoma. J Clin Endocrinol Metab (1998) 83:2192–2198.[Abstract/Free Full Text]

Shiozaki EN, Shi Y. Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology. Trends Biochem Sci (2004) 39:486–494.

Tarrade A, Lai Kuen R, Malssine A, Tricottet V, Blain P, Vidaud M, Evain-Brion D. Characterization of human villous and extravillous trophoblasts isolated from first trimester placenta. Lab Invest (2001) 81:1199–1211.[Web of Science][Medline]

Tarrade A, Goffin F, Munaut C, Lai-Kuen R, Tricottet V, Foidart JM, Vidaud M, Frankenne F, Evain-Brion D. Effect of matrigel on human extravillous trophoblasts differentiation: modification of protease pattern gene expression. Biol Reprod (2002) 67:1628–1637.[Abstract/Free Full Text]

Wang JD, Zhu JB, Fu Y, Shi WL, Qiao GM, Wang YQ, Chen J, Zhu PD. Progesterone receptor immunoreactivity at the maternofetal interface of first trimester pregnancy: a study of the trophoblast population. Hum Reprod (1996) a 2:413–419.

Wang K, Yin XM, Chao DT, Milliman CL, Korsmeyer SJ. BID: a novel BH3 domain-only death agonist. Genes Dev (1996) b 10:2859–2869.[Abstract/Free Full Text]

Submitted on September 18, 2007; resubmitted on October 11, 2007; accepted on October 19, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
13/12/869    most recent
gam078v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liu, J.
Right arrow Articles by Maruo, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, J.
Right arrow Articles by Maruo, T.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?