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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 8 4069-4077
Copyright © 2004 by The Endocrine Society

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

Jovelle B. Laoag-Fernandez, Hiroya Matsuo, Homare Murakoshi, Anna Lissa Hamada, Benjamin K. Tsang and Takeshi Maruo

Department of Obstetrics and Gynecology (J.B.L.-F., H.Ma., H.Mu., A.L.H., T.M.), Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; and Reproductive Biology Unit (B.K.T.), Departments of Obstetrics and Gynecology and Cellular and Molecular Medicine, University of Ottawa, and Hormones, Growth, and Development Program, Ottawa Health Research Institute, Ottawa, Canada K1Y 4E9

Address all correspondence and requests for reprints to: Takeshi Maruo, M.D., Ph.D., Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan., E-mail: maruo{at}kobe-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was conducted to determine whether T3 receptor exists in early placental extravillous trophoblasts (EVTs) and evaluate the influence of T3 on Fas/Fas ligand expression, caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage and apoptosis in cultured early placental EVTs. EVTs with invasive phenotype, isolated from normal placental explants from early pregnancy through preincubation on human fibronectin-coated dishes and exhibited cytokeratin 7 and human placental lactogen immunopositive staining, were cultured in the absence or presence of T3 (10–7 to 10–9 M). The presence of T3 receptor in cultured EVTs was examined by immunocytochemistry, RT-PCR, and Southern blot analysis. Fas sensitivity was determined by treating the cells with an agonistic Fas antibody. Apoptosis was assessed by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, flow cytometry, and Hoechst nuclear staining. Fas and Fas ligand expression and caspase-3 and PARP cleavage were evaluated by immunocytochemistry. Early placental EVTs expressed a 212-bp c-erb Aß1 transcript and the T3 receptor protein and exhibited significant levels of apoptosis in culture. Treatment with T3 reduced the expression of Fas and Fas ligand as well as cleavage of caspase-3 and PARP and suppressed apoptosis in cultured EVTs. Although addition of agonistic Fas antibody increased apoptosis in these cells, this response was markedly attenuated by the presence of T3. These results demonstrate that T3 receptor is present in early placental EVTs and that T3 suppresses apoptosis by down-regulating the expression of Fas and Fas ligand. These findings are consistent with the hypothesis that T3 promotes EVT invasion to the decidua by suppressing apoptosis in early pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXTRAVILLOUS TROPHOBLASTS (EVTs) are mainly uninuclear cells comprising all the trophoblastic elements located outside the villi. EVT has two distinct phenotypes, proliferative and invasive. EVTs located proximal to the basal lamina are proliferative stem cells, whereas the invasive EVTs are those located distally (1). Although these cells have the ability to invade into endometrium and maternal vasculature (2, 3, 4, 5, 6, 7, 8) and play essential roles in implantation and subsequent placentation, the cellular mechanisms involved in EVT invasion to the decidua is poorly understood.

Considerable evidence suggests that apoptosis, or programmed cell death, is an important determinant in the regulation of placental growth (9, 10, 11, 12, 13, 14). The activity of the invasive EVTs seems to be dependent on its apoptotic capacity and less on its proliferative potential because they are highly differentiated cells (1). We recently noted that apoptosis in the invasive EVT was more evident than its proliferative counterpart and that the extent of apoptosis was associated with increased Fas and Fas ligand expression and suppressed Bcl-2 protein expression (15).

Thyroid hormone is vital for the maintenance of early pregnancy and exerts its action through amplifying trophoblast endocrine function and by inducing the production of an epidermal growth factor-like factor by the early placental villous trophoblasts (16). Maternal thyroid hormone deficiency has been implicated in early pregnancy loss (17, 18). Although early placental villous trophoblasts have been reported to be rich in T3 receptor (19, 20), whether T3 receptors are indeed present in EVTs and whether T3 has a physiological role in EVT function are not known. Using primary cultures of EVT developed from early placental explants (21), we have demonstrated in the present study the presence of T3 receptor in early placental EVTs and examined the role of T3 in the regulation of EVT survival. We have shown that T3 is an important antiapoptotic factor and exerts its effects by suppressing the expression and signaling of the Fas-Fas ligand system in early placental EVTs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Placental tissue explants

Normal early placental tissues were obtained from 28 patients who underwent elective abortion at 7–11 wk gestation for psychosocial reasons. The gestational age of the placentas was determined by estimating the duration of pregnancy from the date of the patients’ last menstrual period and by ultrasound examination. All women gave informed consent for the use of the placental tissues for the present study. The study was approved by the Kobe University Institutional Review Board.

Early placental tissues were collected directly in cold PBS, transported to the laboratory on ice, and processed within 2 h from the termination. The tissues (approximately 20 g) were washed thoroughly with cold 150 mM PBS (pH 7.2–7.4) to remove adhering blood cells. Areas rich in chorionic villi were dissected from the membranes and cut into small explants (~2–3 mm3). Light microscopic examination of selected placental tissue sections consistently confirmed the presence of placental villi and absence of endometrial tissue.

Primary cultures of EVTs

After a 1-h preincubation period at 37 C in 5% CO2, 0.5 ml purified human fibronectin (FN; 10 µg/ml, ICN Biomedicals, Aurora, OH) was placed into 6-well 35-mm culture dishes (Becton Dickinson, Oxnard, CA) and 2-well chamber glass slides (Nalge Nunc International, Naperville, IL). A piece of placental tissue explant was then placed on the FN-coated dish, incubated for 1 h to allow the tissues to adhere to the FN, and then carefully flooded with culture medium consisting of bicarbonate-buffered DMEM (Gibco BRL, Grand Island, NY) supplemented with fetal bovine serum (FBS; 10%) streptomycin (50 mg/liter), penicillin (105 U/liter), and Fungizone (0.50 µg/ml) (BioWhittaker, Walkersville, MD). All nonadherent villi were removed after 1-h incubation, and the adherent ones were kept in culture for an additional 72 h at 37 C in 5% CO2 in DMEM (supplemented with 4% FBS) containing various concentrations of T3 (10–7 to 10–9 M) and/or agonistic monoclonal anti-Fas antibody (2 µg/ml) (CH-11, Upstate Biotechnology, Lake Placid, NY). Because cell number reached maximum after 24 h of culture, the duration of all studies were limited to 24 h. Southern blot analyses were carried out after 24 h of culture. T3 (Sigma Chemical Co., St. Louis, MO) was dissolved in approximately 50 µl of 1 M NaOH/absolute ethanol, brought to a final concentration of 10–4 M with warm distilled water and filtered with a 0.22 µm filter (22). This T3 stock solution was diluted in culture medium immediately before use.

Characterization of adherent cell population

Cells adhering to FN-coated chamber slides were characterized immunocytochemically by the avidin-biotin immunoperoxidase method, using a mouse monoclonal antibody to human cytokeratin 7 (a specific marker of trophoblast cells) and a rabbit polyclonal antibody to human placental lactogen (hPL; a marker of invasive phenotype EVT) and a polyvalent immunoperoxidase kit (Omnitag, Lipshaw, MI). Briefly, cells cultured on chamber slides were washed (three times with PBS, room temperature), fixed in ethyl alcohol (99.9%, overnight, 4 C), and preincubated with hydrogen peroxide (3% in PBS, 5 min; to quench endogenous peroxidase activity), as previously described (23). The cells were then incubated for 1 h with mouse monoclonal anti-cytokeratin 7 antibody and rabbit polyclonal anti-hPL antibody (both 1:200 dilution; Nichirei Co., Tokyo, Japan; 37 C, overnight, 4 C), subsequently with biotinylated polyvalent antibody, and finally with avidin-horseradish peroxidase. Thereafter, chromogenic reaction was carried out with a freshly prepared 3-amino 9-ethylcarbazole and hydrogen peroxide. The cells were counterstained with Harris hematoxylin, mounted in glycerine phosphate buffer, and examined microscopically. The cells attached to the FN-coated culture dishes were positively stained for cytokeratin 7 and hPL. Specificity of the immunological reactions was assured when the replacement of specific primary antibodies with same dilution of nonimmune murine IgG or rabbit serum (Miles, Elkhart, IN) resulted in a lack of positive immunostaining. To ensure that the cultured cells belong to invasive phenotype of EVT, characterization of adherent cells immunocytochemically using cytokeratin 7 and hPL immunostaining as described were done concomitantly on all the experiments. Cytokeratin 7 was positive in 90–99% of the cases, whereas hPL was positive in 89–96% of the cases.

Immunocytochemical detection, RNA isolation, and Southern blot analysis of T3 receptor

EVTs adhering to FN-coated chamber slides were immunocytochemically stained as described above, using mouse monoclonal antibody against human T3 receptor (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA).

Total RNA was isolated from cultured EVTs, using RNeasy minikit (Qiagen, Inc., Chatsworth, CA). First-strand cDNA was synthesized from 2 µg total RNA using an Omniscript RT kit (Qiagen, Inc., Chatsworth, CA). PCR was performed with 1 µl cDNA as template, 6.25 pM of each primer, 2.5 mM deoxynucleotide triphosphates, 0.125 U Taq DNA polymerase (Roche-Diagnostics Inc., Mannheim, Germany) in 25 µl of reaction buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM of MgCl2 and 0.01% gelatin. The amplification procedure, performed on Gene Amp PCR System 9600-R (Perkin-Elmer Corp., Norwalk, CT), was as follows: initial denaturation (94 C, 5 min), denaturation (94 C, 30 sec), annealing (55 C, 30 sec), and extension (72 C, 30 sec). The reactions were subjected to 35 cycles for c-erb Aß1.

The specificity of the PCR products for c-erb Aß1 was confirmed by Southern blot analysis. The amplified products were electrophoresed on 3% agarose gels, transferred to nylon membrane filters after denaturation with alkaline solution, fixed by UV irradiation, and hybridized with 5'-32P end-labeled oligonucleotide probe. The latter involved prehybridization (65 C, 2 h) and hybridization (65 C,18 h). The membranes were washed twice (20 min, room temperature) with 2x saline sodium citrate (SSC) [1x SSC = 150 mM NaCl and 15 mM trisodium citrate (pH 7.0)], 0.1% sodium dodecyl sulfate and once (5 min, 60 C) with 0.2x SSC and 0.1% sodium dodecyl sulfate. The PCR products were cloned and sequence analysis confirmed the identity of c-erb Aß1 cDNA sequence of the thyroid hormone receptor (24). cDNA was also amplified with primers for ß-actin. Distilled water was used as the negative control. As the positive control, cDNA obtained from human lymphocytes (previously shown to express c-erb Aß1 mRNA), was included in the analysis. At the end of the hybridization procedures, the reaction products were detected on an x-ray film (3 h), and the images were scanned (Scanjet 3C/ADF; Hewlett Packard, Portland, OR), and their intensities were densitometrically quantified (NIH Image version 1.58). The abundance of mRNA was expressed relative to that of ß-actin mRNA in each specimen. A human ß-actin cDNA probe was used. The oligonucleotide sequences used are shown in Table 1Go. The c-erb Aß1 probe had the following sequence: GCATGGAGATCATGTCCCTTCGCGCTGCTG.


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  		TABLE 1. Oligonucleotide primers used for RT-PCR

 
Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL)

Apoptotic cells were identified by direct immunoperoxidase detection of digoxigenin-labeled genomic DNA (Apoptag kit; Oncor, Gaithersburg, MD) as per the manufacturer’s instructions. EVTs cultured on the chamber slides were treated with terminal deoxynucleotidyl transferase enzyme, washed to terminate the reaction, added two drops of antidigoxigenin peroxidase, and detected for peroxidase activity with 3,3-diaminobenzidine substrate. Then 2% H2O2 in PBS was used to quench endogenous peroxidase activity. Distilled water was used to replace terminal deoxynucleotidyl transferase enzyme for the negative control. A section of rat mammary glands obtained 4 d after weaning was included as a positive control. More than 1000 EVT nuclei were counted for each experimental group in determining the mean percentage of apoptosis-positive nuclei of cultured EVTs.

Flow cytometric analysis

Apoptosis in cultured EVTs was also analyzed by flow cytometry, as previously described (25). Briefly, the cells (1 x 106) were trypsinized, pelleted, and fixed in cold ethanol (–20 C). They were washed with PBS, incubated in the dark (30 min, room temperature) with propidium iodide (5 µg/ml) and RNase A (2 µg/ml) in PBS, and analyzed by flow cytometry (488 nm excitation; FACS Scan Flow Cytometer, Becton Dickinson).

Fas-mediated apoptosis

To determine whether T3 regulates Fas-mediated apoptosis in addition to its influence on Fas and Fas ligand expression, cultured EVTs maintained in medium containing 10% FBS were cultured in serum-free medium containing agonistic monoclonal anti-Fas antibody (2 µg/ml; CH-11, Upstate Biotechnology), mouse IgM (as control; 2 µg/ml; Amersham Biosciences, Piscataway, NJ), T3 (10–8 M) plus IgM (2 µg/ml), or agonistic Fas monoclonal antibody (2 µg/ml) plus T3 (10–8 M). Apoptosis was determined by the TUNEL method as described above. Furthermore, apoptotic nuclear morphology was assessed by Hoechst 33258 staining (26). Briefly, cells were fixed in 4% neutral buffered formalin and then resuspended overnight in Hoechst 33258 staining solution (12.5 ng/ml; in dark). Apoptotic cells exhibiting distinct characteristics (cellular shrinkage, condensed chromatin, and fragmented nuclei) were counted. At least 1000 cells were assessed in each treatment group.

Fas and Fas ligand expression, caspase-3, and poly (ADP-ribose) polymerase (PARP) cleavage

Cultured EVTs in chamber slides were immunocytochemically stained as described using rabbit polyclonal antibodies (1:100 dilution) against human Fas and Fas ligand (Santa Cruz Biotechnology) and mouse monoclonal antibodies (1:200 dilution) against the cleavage fragments of human caspase-3 and PARP (Cell Signaling Technology, Beverly, MA). Control procedures undertaken were the same as described above.

Images were randomly captured using a digital camera (Olympus DP50, Tokyo, Japan) mounted onto a microscope and attached to a digital multiscan display (Panasonic, Osaka, Japan). Several images per slide were captured. The intensity of immunostaining was evaluated and scored in one session in a blinded fashion by two observers to reduce variability and avoid experimental bias. Immunostaining of all samples was graded as: not detectable (–), weak but definitely detectable (+), moderate (++), and intense (+++), and the final score credited to each sample was agreed by the two observers.

Statistical analysis

Data were analyzed by one- or two-way ANOVA. Significant differences between treatment groups were determined by Tukey test. Analysis of Fas antigen, Fas ligand, caspase-3, and PARP immunostaining in EVTs cultured in the absence and presence of T3 was carried out using the Mann-Whitney U test. Differences with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunocytochemical characterization of cultured cells attached to FN-coated dishes

Cultured placental cells attached to the FN-coated dishes appeared large, round shaped or polygonal, mononuclear, and some vacuolated with prominent nucleus (data not shown). To confirm the trophoblastic origin of these cells, immunostaining with a monoclonal antibody against cytokeratin 7 was carried out. The attached cells were positive for cytokeratin 7 immunostaining in all the samples, regardless of the duration of the culture period (Fig. 1AGo). The cytoplasms of the attached cells were also positive for hPL immunostaining (Fig. 1BGo). The intensity of immunostaining for hPL was independent of the gestational age of the placental explants and the duration of culture. The cells treated with either nonimmune murine IgG (Fig. 1CGo) or nonimmune rabbit serum (Fig. 1DGo) were immunonegative. Taken together, these findings suggest that the cultured placental cells attached to the FN-coated slides were indeed EVTs.



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  		FIG. 1. Immunocytochemical localization of cytokeratin 7 (A) and hPL (B) in early placental cells adhering to FN-coated chamber slides cultured for 24 h. Immunolocalization of cytokeratin 7 on the cytoplasm and cell membrane were noted in the cultured cells, indicating that those cells were of trophoblast lineage. The expression of hPL on the cytoplasm further implicates that the cultured cells were of the invasive phenotype EVT. The cells treated with either nonimmune murine IgG (C) or nonimmune rabbit serum (D) were immunonegative. Data shown are representative of seven experiments. Bars, 5 µm. Original magnification, x400.

 
Expression of T3 receptor mRNA and protein in cultured EVTs

The expression of T3 receptor mRNA was assessed by RT-PCR and confirmed by Southern blot analysis. As expected, a 212-bp PCR product was detected in cultured early placental EVTs with the use of c-erb Aß1 PCR primers (Fig. 2AGo, top panel). An additional band of 310 bp was also noted. The amplified fragment was identified to be c-erb Aß1 by Southern blot hybridization, and no other band was noted (Fig. 2AGo, bottom panel). No PCR product was detected by Southern blot analysis in the negative control experiments, suggesting that negligible cross-contamination existed.



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  		FIG. 2. A, RT-PCR amplification (top panel) of c-erb Aß1 cDNA and the Southern blot hybridization (bottom panel) of transferred PCR products from early placental EVTs with P32 c-erb Aß1 cDNA probe. The marker used is {phi}X174 HaeIII digest. An expected 212-bp PCR product was detected, and an additional band of size 310 bp was also noted. Distilled water and cDNA from human lymphocytes were used as the negative and positive controls, respectively. The amplified fragment was identified as c-erb Aß1 by Southern blot hybridization, and no other band was noted. Results shown are representative of three independent experiments. B, Immunocytochemical localization of T3 receptor protein in early placental EVTs. T3 receptor protein was evident primarily in the nuclei of cultured EVTs. The cells treated with nonimmune murine IgG were immunonegative (data not shown). Results shown are representative of four independent experiments. Bars, 5 µm. Original magnification, x400.

 
The presence of T3 receptor protein in cultured cells was confirmed immunocytochemically. Immunostaining for T3 receptor protein was evident primarily in the nuclei of cultured cells (Fig. 2BGo). The cells treated with nonimmune murine IgG were immunonegative (data not shown).

Apoptosis

Apoptosis, as assessed by TUNEL, was noted in early placental EVTs cultured in the absence of exogenous thyroid hormone (Fig. 3AGo). TUNEL-positive nuclei were less apparent in EVTs treated with 10–7 M T3 (Fig. 3BGo) and 10–8 M T3 (Fig. 3CGo) but not with 10–9 M T3 (Fig. 3DGo). Rat mammary glands obtained at the fourth day of weaning (positive control) also exhibited the presence of apoptotic cells, as evidenced by the presence of brown stained nuclei (data not shown).



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  		FIG. 3. TUNEL analysis of early placental EVTs cultured for 24 h in the absence (A) or presence (B) of 10–7, 10–8 (C), and 10–9 M T3 (D), as assessed by TUNEL. TUNEL-positive nuclei (arrows) identified by the appearance of brown peroxidatic product were abundantly noted in untreated early placental EVTs. The TUNEL-positive nuclei were less apparent in EVTs treated with 10–7 (B) and 10–8 M T3 (C) but not with 10–9 M T3 (D). Positive control also exhibited the presence of TUNEL-positive cells (data not shown). Results shown are representative of six independent experiments performed in triplicate. Bars, 5 µm. Original magnification, x400.

 
Similarly, quantitative analysis of the apoptosis of cultured early placental EVTs revealed that T3 treatment significantly decreased the apoptosis-positive rate at 10–7 M (P < 0.01) and 10–8 M (P < 0.05) but not at 10–9 M (P > 0.05) when compared with that in untreated EVT cultures (Fig. 4Go, top panel). The antiapoptotic effects of T3 on the cultured EVTs were confirmed by flow cytometry. As shown in Fig. 4Go (bottom panel), the proportion of the EVTs that was apoptotic (<2 N DNA content) was significantly lower in experimental groups treated with either 10–7 M T3 (P < 0.001) or 10–8 M T3 (P < 0.01) when compared with untreated EVTs (controls). Treatment with 10–9 M T3 had no significant effect (P > 0.05).



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  		FIG. 4. Apoptotic response of cultured EVTs as assessed by TUNEL (top panel) and flow cytometry (cells containing < 2 N DNA content; bottom panel). T3 treatment significantly decreased the population of TUNEL-positive cells and cells with < 2 N DNA content at 10–7 and 10–8 M T3 but not at 10–9 M T3, when compared with that with untreated cultures. A total of 1000 cells were counted per experimental group in each analysis. Data shown are means ± SD of six independent experiments performed in triplicate. Compared with untreated control: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
In addition to the maintenance of cell survival under basal state, T3 also suppressed Fas-mediated apoptosis in EVTs in vitro. Although Fas activation by the addition of an agonistic monoclonal anti-Fas antibody (2 µg/ml) to the EVT cultures resulted in a significant increase in apoptosis as determined by TUNEL (Fig. 5Go, top panel; P < 0.05) or Hoechst nuclear stain (Fig. 5Go, bottom panel; P < 0.001), this response was markedly attenuated by the presence of 10–8 M T3 (Fig. 5Go, top and bottom panels; P < 0.01). ANOVA indicated significant effects of anti-Fas antibody (Fig. 5Go, top panel; P < 0.05, bottom panel; P < 0.001) and T3 (Fig. 5Go, top panel; P < 0.01, bottom panel; P < 0.01) as well as a significant interaction between these factors (Fig. 5Go, top and bottom panels; P < 0.001).



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  		FIG. 5. Effects of T3 on Fas-mediated apoptosis in cultured EVTs. EVTs were cultured for 24 h in the presence or absence of 10–8 M T3 and/or agonistic Fas antibody or IgM (as control). The degree of cell death was determined biochemically (TUNEL; top panel) and morphologically (Hoechst nuclear stain; bottom panel). Although Fas activation by an agonistic Fas antibody resulted in a significant increase in apoptosis, as determined by TUNEL and Hoechst nuclear stain, this response was markedly attenuated by the presence of 10–8 M T3. Results shown are means ± SD of six independent experiments performed in triplicate. Compared with control: *, P < 0.05; ***, P < 0.001. Compared with EVTs treated with both anti-Fas and 10–8 M T3: ++, P < 0.01; +++, P < 0.001.

 
T3 down-regulates Fas and Fas ligand expression and suppresses caspase-3 and PARP cleavage

Fas and Fas ligand immunocytochemical staining was prominent in EVTs cultured in the absence of T3 (Fig. 6Go). Their intensities of the immunosignals were less in EVTs treated with either 10–7 M T3 (Fig. 6Go, A-4 and B-4, respectively) or 10–8 M T3 (Fig. 6Go, A-3 and B-3, respectively), compared with those in untreated EVTs (Fig. 6Go, A-1 and B-1), whereas no apparent change in the immunostaining for Fas and Fas ligand was noted in EVTs treated with 10–9 M T3 (Fig. 6Go, A-2 and B-2, respectively). Statistical analysis indicates significant differences in the intensities of immunostaining for Fas and Fas ligand between EVTs cultured in the absence and presence of 10–8 M T3 (both P < 0.05) but not 10–9 M T3 (P > 0.05) or 10–7 M T3 (P > 0.05). The intensity of immunostaining for caspase-3 cleavage fragments was less in EVTs treated with 10–7 M T3 (Fig. 6GoC-4), 10–8 M T3 (Fig. 6GoC-3), and 10–9 M T3 (Fig. 6GoC-2) when compared with that in EVTs cultured in the absence of T3 (Fig. 6GoC-1). Likewise, immunoreactivity of cleaved PARP was markedly suppressed by T3 at these concentrations (Fig. 6Go, D-1 to D-4). Compared with the EVTs cultured in the absence of T3, there was a significantly lower immunoreactivity for the caspase-3 and PARP cleavage fragments in EVTs treated with T3 at 10–7 M (both P < 0.01) and 10–8 M (P < 0.05 and P < 0.01, respectively) but not 10–9 M (both P > 0.05). The replacement of the specific antibody with either nonimmune rabbit serum or nonimmune murine IgG resulted in a lack of positive staining (data not shown). The Fas, Fas ligand, caspase-3, and PARP immunoreactivities in cultured early placental EVTs are summarized in Table 2Go.



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  		FIG. 6. Immunocytochemical localization of Fas (A), Fas ligand (B), caspase-3 cleavage fragments (C), and PARP cleavage fragment (D) in early placental EVTs cultured for 24 h in the absence (panel 1) or presence of 10–9 (panel 2), 10–8 (panel 3), or 10–7 M T3 (panel 4). The immunostaining for Fas was less in EVTs treated with 10–7 (A-4) and 10–8 M T3 (A-3) but not with 10–9 M T3 (A-2), compared with that in untreated EVTs (A-1). The intensity of immunostaining for Fas ligand was similar to that of Fas in untreated and treated cultures (B). The intensity of immunostaining for caspase-3 cleavage fragments was less in EVTs treated with 10–7 (C-4), 10–8 (C-3), and 10–9 M T3 (C-2), compared with that in untreated EVTs (C-1). Likewise, the immunoreactivity of PARP cleavage fragment in EVTs was markedly suppressed by T3 at these concentrations (D). The replacement of primary antibody with nonimmune rabbit serum or nonimmune murine IgG resulted in a lack of positive immunostaining (data not shown). Results shown are representative of five independent experiments. Bars, 5 µm. Original magnification, x400.

 

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  		TABLE 2. Summary of immunolocalization of Fas, Fas ligand, and caspase-3 and PARP cleavage fragments in cultured early placental EVTs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Little is known about the molecular mechanism underlying the regulation of EVT proliferation, apoptosis, and invasiveness. Because the FN receptor integrin-{alpha}5ß1 has been identified in trophoblast and decidua cocultures (21, 27, 28), it seemed likely that this receptor-ligand interaction might be prominent in invasive EVTs. Using FN-coated dishes and FN as a molecular glue of early placental EVTs (29), we successfully established a primary invasive EVT culture system for investigating the possible role of thyroid hormone in the regulation of apoptosis in EVTs. The cells attached to the FN-coated dishes were immunopositive for both cytokeratin 7 and hPL, confirming that the cells are EVTs with the invasive phenotype (30, 31). Parallel investigations done in our laboratory (Ooki, N., H. Matsuo, S. Nakago, H. Murakoshi, J. B. Laoag-Fernandez, and T. Maruo, unpublished data) revealed that the use of ErbB1, a specific marker of both cytotrophoblast and syncytiotrophoblast, and ErbB2, a specific marker for EVT and syncytiotrophoblast (32), demonstrated that ErbB1 was 14.9 ± 3.2% at 24 h culture and became 0.3 ± 0.2% at 48 h culture. After 48 h culture, more than 99% of the cultured cells adherent to FN-coated dishes expressed cytokeratin 7, hPL, and ErbB2 protein but not ErbB1 protein as noted by immunocytochemistry. Moreover, the expression of ErbB2 mRNA was stable but that of ErbB1 remarkably decreased after 24 h culture. This is also supported by the fact that all cells attached to the FN-coated dishes after 24 h culture were mononuclear. In addition, we demonstrated for the first time that T3 receptor mRNA and protein are present in early placental EVTs and T3 maintains survival of EVTs by suppressing apoptosis via down-regulation of Fas/Fas ligand system.

We have previously shown that the villous cytotrophoblast is rich in T3 receptor (19) and that T3-nuclear receptor binding in villous trophoblasts is higher in early placentas than term placentas (19, 20). We have also demonstrated that thyroid hormone (10–8 M) acts as a biological amplifier of early placental villous trophoblast function, including sex steroid and peptide production (22). Furthermore, the placenta is also a rich source of iodothyronine deiodinases (33), implicating the role of these enzymes in the regulation of T3 in the placenta (34, 35, 36). Three types of deiodinase exist, and the type III or the deactivating enzyme had been reported to be present mainly in the placenta (37). Moreover, changes in deiodinase expression have been demonstrated in normal placentas after treatment with T3 concentrations in vitro (34).

In this connection, the present study revealed that treatment with 10–8 M T3 significantly reduced apoptosis in cultured early placental EVTs. Consistent with this observation is our finding that treatment with 10–8 M T3 markedly reduced Fas and Fas ligand expression in cultured early placental EVTs, suggesting the suppressed apoptosis observed in the presence of thyroid hormone may be mediated through the down-regulation of these cell death factors in early placental EVTs. The Fas/Fas ligand system is an important mechanism for the induction of apoptosis in various cell lineages (38) and its proapoptotic activity is dependent on the level of Fas expression on target cells and the relative amounts of cytoplasmic, membrane-bound and secreted Fas ligand (39), although the significance of the simultaneous expression of Fas and Fas ligand in the same cells has not been clearly defined (40).

The down-regulation of Fas and Fas ligand expression by T3 is likely to be mediated via its nuclear receptors in early placental EVTs. Because 10–8 M T3 is within the physiological range of circulating levels of T3, the observed responses may reflect a vital role of thyroid hormone in the regulation of apoptosis in early placental EVTs and are supportive of our previous reports (17, 22, 41) that an appropriate thyroid hormone concentration is important for the maintenance of early pregnancy. Although the basal apoptotic rate of cultured early placental EVTs was relatively high, it might be a consequence of autoinduction of Fas/Fas ligand, as has been demonstrated in a variety of normal and tumor cells (42, 43, 44, 45, 46). Although both Fas and Fas ligand are believed to be expressed in first-trimester proliferative EVTs and the invasive EVTs expressed only Fas ligand (47), recent study by Murakoshi et al. (15) has shown that the expression of both the death receptor and its ligand is minimal in proliferative EVTs but predominant in the invasive counterparts. Whereas the reason(s) for these differences is not known, it is possible that, in the course of differentiation of proliferative EVT to the invasive phenotype, the expression of the Fas/Fas ligand system becomes variable in early pregnancy. In addition, studies on Fas-mediated apoptosis in trophoblast cells revealed that treatment with agonistic Fas antibody enhanced the reduction of trophoblast cell viability induced by treatment with serum from preeclamptic women (48). Agonistic Fas antibody also augmented the apoptosis in human granulosa-luteal cells (49) and rat granulosa cells (50). In the present study, we demonstrated that not only was agonist Fas antibody capable of inducing apoptosis in invasive EVTs, but also culture of these cells with T3 suppressed the Fas antibody-induced apoptotic response. These findings are consistent with our hypothesis that the T3-induced reduction of Fas expression in invasive EVTs indeed might lead to decreased Fas-mediated apoptosis in these cells.

Fas-Fas ligand binding results in the activation of cas-pase-3, a cysteinyl aspartate specific protease known to play a central role in the execution of the apoptotic program (51, 52, 53, 54) and to be responsible for PARP cleavage during cell death (55, 56, 57). Our current knowledge on the expression and activity of caspases in placental villi and trophoblasts is limited (58, 59, 60, 61). Comparative studies on caspases in human early and term trophoblasts have shown that caspase activity is highest in cytotrophoblasts and decreases with trophoblastic differentiation (59, 61). PARP, an enzyme involved in DNA repair and a substrate of caspase-3, is present in less abundance in the syncytium than in the cytotrophoblast, and the cleavage of PARP by caspase-3 is a significant event in the apoptotic cascade (62, 63). Although caspase-3 and PARP appear to be important in the regulation for EVT cell fate, to the authors’ knowledge, there has been no report on the expression of caspase-3 and PARP in first-trimester EVTs.

The role of thyroid hormone in the regulation of its target cell fate appears to be cell type and species specific. Studies on Xenopus laevis tadpole (64, 65) have shown that thyroid hormone-induced muscle death was mediated by the thyroid hormone receptor and activation of a caspase-/caspase-3-like protease-dependent death pathway. T4 treatment has been shown to increase rat pancreatic ß-cell apoptosis, as evidenced by increased TUNEL- and caspase-3-positive cells (66). Caspase-3 activity was also noted to increase in goiter (67), whereas low thyroid hormone levels in astronauts after space flight is associated with an increased thyrocyte PARP activity and apoptosis (68). Thyroid hormone has a regulatory role on PARP activity (69). In patients with untreated Graves’ disease, increased soluble Fas ligand elicits apoptosis in mononuclear cells and thyroid hormone regulates soluble Fas content (70, 71). Tamura et al. (72) demonstrated that the increased number of apoptotic cells during goiter formation is associated with increased Fas expression in the thyrocytes.

The cellular mechanism(s) by which T3 regulates Fas and Fas ligand expression in early placental EVTs is not known. It has been demonstrated in various systems that the expression of Fas and Fas ligand is regulated via the signaling of p53 (73, 74, 75, 76) and Akt/Forkhead (77, 78, 79, 80), respectively. Whether T3 suppresses Fas and Fas ligand expression via down-regulation of p53 and up-regulation of Akt-mediated Forkhead phosphorylation, respectively, remains to be determined.

In conclusion, we have demonstrated the presence of T3 receptor mRNA and protein in early placental EVTs. We have also shown that T3, in the physiologic concentration of 10–8 M, down-regulates apoptosis of early placental EVTs through the inhibition of Fas and Fas ligand expression and caspase-3 and PARP cleavage. The decrease in Fas expression is accompanied by suppressed Fas-mediated apoptosis. Accordingly, T3 may promote the invasion of EVTs to the decidua by inhibiting apoptosis of EVTs in early pregnancy. Although 10–8 M concentration of T3 is above that of physiological serum levels, the tissue concentration may not necessarily be the same. Further investigations are required to determine the precise molecular mechanisms by which T3 down-regulates these death factors and inhibits apoptosis in early placental EVTs.


   Footnotes
 
This work was supported in part by Grants in Aid for Scientific Research 13877274 from the Japanese Ministry of Education, Science, and Culture, the Ogyaa-Donation Foundation of Japan Association of Obstetricians and Gynecologists, and Postdoctoral Fellowship (to J.B.L.-F.) and Visiting Professorship (to B.K.T.) of the Japan Society for the Promotion of Science.

Abbreviations: EVT, Extravillous trophoblast; FBS, fetal bovine serum; FN, fibronectin; hPL, human placental lactogen; PARP, poly (ADP-ribose) polymerase; SSC, saline sodium citrate; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling.

Received December 24, 2003.

Accepted May 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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