STAT3 Enhances Intracellular Fas-Mediated Apoptotic Signals in HHUA Human Endometrial Epithelial Cells

MOLECULAR MEDICINE REPORTS 4: 307-312, 2011 STAT3 enhances intracellular Fas-mediated apoptotic signals in HHUA human endometrial epithelial cells TETSUJI TANAKA1, TAO BAI1, HIROTOSHI UTSUNOMIYA2, TAKAHIRO FUKUMOTO3 and KAZUNORI YUKAWA4 Departments of 1Obstetrics and Gynecology, and 2Experimental Pathology, Wakayama Medical University, Wakayama 641-0012; 3Research Center for Infection-Associated Cancer, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815; 4Department of Physiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan Received September 15, 2010; Accepted December 24, 2010 DOI: 10.3892/mmr.2011.424 Abstract. Several endometrial signal transducer and activator epithelium, and the number of endometrial apoptotic cells of transcription 3 (STAT3)-activating cytokines are reported is reported to increase during the secretory phase of the to be essential for blastocyst implantation, with inhibition menstrual cycle (1-3), the blastocyst implantation period. of STAT3 activation in the endometrium also reported to Fas-mediated apoptosis is thought to affect blastocyst implan- prevent implantation. To investigate STAT3 signals in endo- tation, since the Fas antigen, a cell surface apoptotic death metrial epithelial cells, the activation and inactivation effects receptor, is expressed in the pre-implantation human endo- of STAT3 signals were examined in the human endometrial metrial epithelium (4-6), and endometrial epithelial apoptosis epithelial cell line HHUA, which is thought to retain many of occurs during blastocyst implantation (7,8). Moreover, specific the intracellular signaling pathways found in normal human stimulation of Fas antigen induces the apoptosis of normal endometrial epithelial cells. Five STAT3-activating cytok- human endometrial epithelial cells (6,9,10). ines, IL-11, IL-10, LIF, oncostatin M and leptin, enhanced Recent studies in cytokine-null or receptor-null mice the Fas-mediated apoptosis of the HHUA cells without any have revealed that several interleukin (IL)-6 family cytok- increase in cell surface Fas antigen expression. STAT3 siRNA ines may play essential roles in blastocyst implantation and/ transfection suppressed STAT3 expression in HHUA cells and or endometrial stromal decidualization. Leukemia inhibitory significantly inhibited Fas-mediated cell death. These results factor (LIF)-null female mice exhibited impaired blastocyst indicate that intracellular apoptotic signals in HHUA cells are implantation that can be partially restored by intraperitoneal constitutively activated and regulated by STAT3-mediated administration of LIF (11). Additionally, transfer of recovered signals. This apoptosis-promoting effect of STAT3 in HHUA embryos from LIF-null mice to wild-type pseudopregnant cells is completely different from many previous reports female mice resulted in implantation and pregnancy. Female demonstrating anti-apoptotic effects by STAT3 activation. mice with a null mutation in the IL-11 receptor α chain are The STAT3 signals in HHUA cells may be specific to the also reported to be infertile due to defective decidualization human endometrial epithelial cell lineage in the regulation of (12). Oncostatin M (OSM) binds competitively to the LIF blastocyst implantation. receptor to regulate LIF functions on targeted cells, although the physiological functions of OSM in the human endometrium Introduction are not entirely understood (13). Both IL-11 and LIF bind to each specific receptor and activate signal transducer and acti- Unlike most normal adult tissues, cyclic growth and tissue vator of transcription 3 (STAT3), while specific inhibition of remodeling occurs within the uterine endometrium throughout STAT3 activation in the endometrium prevents implantation the reproductive years. Tissue remodeling is regulated by a (14). These results indicate that STAT3-activating cytokines balance between cell growth and selective cell death (apop- may play important roles in human blastocyst implanta- tosis). Apoptotic cells are found in normal human endometrial tion. The first step of blastocyst implantation is endometrial epithelial cell apoptosis following blastocyst attachment to the epithelium. Therefore, we examined the effects of STAT3- activating cytokines on the Fas-mediated apoptosis of human endometrial epithelial cells. Correspondence to: Dr Tetsuji Tanaka, Department of Obstetrics In the present experiments, five STAT3-activating and Gynecology, Wakayama Medical University, 811-1 Kimi-idera, Wakayama 641-0012, Japan cytokines, LIF, IL-11, OSM, leptin and IL-10, were used. E-mail: [email protected] The leptin/leptin receptor system has been reported to be a regulator of the blastocyst implantation process (15). IL-10 is Key words: signal transducer and activator of transcription 3, an anti-inflammatory cytokine expressed in the endometrium apoptosis, endometrium, implantation, Fas and placenta, although IL-10-null mice do not have impaired embryo implantation (16). In the present study, the HHUA 308 TANAKA et al: STAT3 ACTIVATES APOptOTIC SIGNALS IN HUMAN ENDOMETRIAL EPIthELIAL CELLS human endometrial epithelial cell line was selected because day 4, genomic DNA was extracted from all cells, including the cells express high levels of Fas antigen as well as functional dead cells, using a SepaGene DNA extraction kit (Sankyo- estrogen receptors and progesterone receptors, similar to the Junyaku Co. Ltd., Tokyo, Japan) and treated with 100 µg/ml normal human endometrial epithelium during the implanta- RNase A (Sigma, St. Louis, MO, USA) in TE buffer (10 mM tion period (17), and form glandular luminal structures in Tris, pH 8.0, 2 mM EDTA) for 90 min at 37˚C to remove any collagen gel cultures, similar to the structures of normal glan- contaminating RNA. Approximately 20 µg of the genomic dular epithelial cells (18). Karyotyping analysis performed DNA was electrophoresed on a 1.2% (w/v) agarose gel at 50 V on 20 HHUA cells revealed normal 46XX karyotypes (19). for approximately 2 h, stained with 5 µg/ml ethidium bromide HHUA cells are known to express functional Fas antigens on and visualized by UV illumination. The final concentrations their cell surface that mediate specific apoptotic signals (9). of recombinant human cytokines in the culture media were Based on these characteristics, HHUA cells are considered 100 ng/ml for IL-11, IL-10, LIF and OSM, and 200 ng/ml for to retain many of the intracellular signaling pathways found leptin. in normal human endometrial epithelial cells. Hence, in the present study, HHUA cells were used to examine the effects Semi-quantitative flow cytometry.HH UA cells were detached of five STAT3-activating cytokines on basic cellular functions, and re-cultured in dishes as described above. Untreated such as proliferation, viability and apoptosis. HHUA cells and HHUA cells treated with each cytokine for 2 days were detached from the dishes using 3 mM EDTA in Materials and methods phosphate-buffered saline (PBS) and stained. Cells (3x105) were incubated with an excess of mouse anti-human Fas Cell line and culture. The HHUA cell line (17) was obtained (CD95) monoclonal antibody (clone UB2; MBL) for 20 min at from the Riken Cell Bank (Tsukuba, Japan). All cells in 4˚C, then washed twice with washing buffer (PBS containing these experiments were cultured in OPTI-MEM (Invitrogen, 2% fetal calf serum and 0.1% NaN3) and incubated with a Carlsbad, CA, USA) supplemented with 5% fetal bovine serum secondary antibody [FITC-conjugated goat anti-mouse IgG (Equitech Bio Inc., Ingram, TX, USA), penicillin (100 U/ (H+L); Dako-Japan, Kyoto, Japan] for 20 min at 4˚C. After two ml), streptomycin (100 U/ml) and fungizone (0.25 µg/ml; washes, the cells were suspended in 200 µl of washing buffer Invitrogen) in the presence of 5% CO2 and 95% air at 37˚C. and analyzed with a FACSCalibur™ (Becton Dickinson, Mountain View, CA, USA). The final concentrations of recom- Cell proliferation assay. The cell proliferation effects of binant human cytokines in culture media were 100 ng/ml for human recombinant cytokines or mouse anti-human Fas IL-11, IL-10, LIF and OSM, and 200 ng/ml for leptin. monoclonal IgM (clone CH-11; MBL, Nagoya, Japan) on HHUA cells were measured. HHUA cells were seeded onto Transfection of STAT3 siRNAs. The STAT3 siRNA and the 96-well plates at 5x103 cells/well (n=6) and incubated with negative control siRNA were obtained from a SureSilencing™ various concentrations of recombinant human cytokines or Human STAT3 siRNA kit (SuperArray Biosci Corp., anti-Fas IgM for 48 h. At the end of the treatments, viable cell Frederick, MD, USA). Lipofectamine 2000 (Invitrogen) was numbers were determined using a cell counting kit (Dojin used as the transfection reagent according to the manufac- Chemical Laboratory Co. Ltd., Tokyo, Japan) according to the turer's instructions. For the experiments, cells were seeded manufacturer's instructions. The absorbance at 450 nm was onto 6-well plates (2.5x105 cells/well) or 10-cm dishes (2x106 measured using a microplate reader. A viability of 100% was cells/dish), cultured for 24 h and then transfected with the defined as the absorbance obtained for cells without any treat- STAT3 siRNAs or control siRNA. Subsequently, the cells ment. All five STAT3-activating cytokines used in the cultures were cultured for 72-120 h for protein assays before being were purchased from PeproTech EC Ltd. (London, UK). harvested as indicated. Cell viability assay. Cell viability was examined using the cell Western blotting.

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