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The Progesterone-Responsive Gene 14-3-3T Enhances the Transcriptional Activity of Progesterone Receptor in Uterine Cells

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The Progesterone-Responsive Gene 14-3-3T Enhances the Transcriptional Activity of Progesterone Receptor in Uterine Cells 193 The progesterone-responsive gene 14-3-3t enhances the transcriptional activity of progesterone receptor in uterine cells Masanori Ito1,2,3, Tomohiko Urano1,2, Hisahiko Hiroi3, Mikio Momoeda3,4, Mayuko Saito3, Yumi Hosokawa3, Ryo Tsutsumi3, Fumiko Zenri3, Minako Koizumi3, Hanako Nakae3, Kuniko Horie-Inoue5, Tomoyuki Fujii3, Tetsu Yano3, Shiro Kozuma3, Satoshi Inoue1,2,5 and Yuji Taketani3 Departments of 1Geriatric Medicine, 2Anti-Aging Medicine and 3Obstetrics and Gynaecology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan 4Department of Integrated Women’s Health, St Luke’s International Hospital, 9-1 Akashi-cho, Chuo-ku, Tokyo 104-8560, Japan 5Research Center for Genomic Medicine, Saitama Medical School, 1397-1, Yamane, Hidaka-shi, Saitama 350-1241, Japan (Correspondence should be addressed to S Inoue at Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo; Email: [email protected]) Abstract Members of the 14-3-3 family are intracellular dimeric phosphoserine-binding proteins that can associate with and modulate the activities of many proteins. In our efforts to isolate the genes regulated by progesterone (P4) using suppressive subtractive hybridization, we previously found that 14-3-3t is one of the genes upregulated by P4. In this study, we demonstrated by quantitative RT-PCR (qRT-PCR), western blot analyses, and immunohistochemistry that 14-3-3t mRNA and protein levels were increased in the rat uterus after P4 treatment. Furthermore, qRT-PCR indicated that P4 increased 14-3-3t mRNA levels in human endometrial epithelial cells and endometrial stromal cells (ESCs). Western blot and qRT-PCR analyses revealed that in vitro decidualization using cAMP and medroxyprogesterone 17-acetate increased levels of 14-3-3t mRNA and protein in ESCs. We have shown by qRT-PCR and western blot analyses that P4 increased the mRNA and protein levels of 14-3-3t in Ishikawa cells that stably express P4 receptor-B (PR-B). Immunocytochemistry revealed that 14-3-3t colocalizes with PR and translocates from the cytoplasm to the nucleus in response to P4. Moreover, by luciferase reporter assay, we demonstrated that 14-3-3t enhances the transcriptional activity of PR-B. Taken together, we propose that 14-3-3t is a P4-responsive gene in uterine cells that modulates P4 signaling. Journal of Molecular Endocrinology (2012) 49, 193–202 Introduction mRNA subgroups that are transcribed from a single gene under the control of separate A and B promoters The endometrium undergoes cyclic changes controlled (Kastner et al. 1990, Giangrande & McDonnell 1999, by the ovarian steroid hormones estrogen and pro- Richer et al. 2002, Leonhardt et al. 2003). PRs and their gesterone (P4). During the proliferative phase, estro- cofactors are expressed in both the rat uterus and the gen secreted from the granulosa cells in the ovary human endometrium and are involved in the stimulates endometrial proliferation. After ovulation, regulation of their responsive genes (Vienomen et al. estrogen and P4 are secreted by the ovary to facilitate 2004). Some P4-responsive genes have been reported, endometrial decidualization. Estrogen and P4 actions such as calcitonin (Zhu et al. 1998), corticotrophin- are mediated by their specific receptors, namely, releasing hormone (Makrigiannakis et al.1995), estrogen receptor and P4 receptor (PR) respectively. insulin-like growth factor-binding protein-1 (IGFBP1; P4 also plays an important role in implantation, and PR Gao et al. 1999), heparin-binding epidermal growth is a member of the nuclear receptor superfamily that factor-like growth factor (Zhang et al. 1994), thrombo- regulates the transcription of an array of responsive spondin-1 (Iruela-Arispe et al. 1996), and FK506-binding genes (Mangelsdorf et al. 1995). PR is detected as two protein 51 (FKBP51; Hubler et al. 2003) – these genes distinct proteins, namely, PR-A and PR-B, which possess are related to uterine differentiation in normal different molecular weights. In humans, the molecular female reproduction. weight of the PR-A protein is 94 kDa while that of PR-B Several investigators have identified the genes is 120 kDa. These proteins are translated from distinct regulated by P4 in the uterus using cDNA microarray Journal of Molecular Endocrinology (2012) 49, 193–202 DOI: 10.1530/JME-12-0112 0952–5041/12/049–193 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 01:14:38AM via free access 194 M ITO and others . Progesterone-responsive 14-3-3t in uterine cells analyses (Cheon et al. 2002, Jeong et al. 2005). Recently, were administered only corn oil. The E2-treated and gene-targeting technology created PR-A knockout P4-treated rats were killed, and each organ was collected (PR-A KO) mice that display reproductive anomalies, at the appropriate time after E2 and P4 injections including the inability to ovulate, endometrial hyper- respectively. After excising the uterus, it was rapidly plasia and inflammation, and defects in embryo frozen and stored at K80 8C until use. implantation (Lydon et al. 1995, Conneely et al. 2001). Female infertility in PR-A KO mice is associated with defective embryo implantation and lack of decidualiza- RNA isolation and cDNA preparation tion of uterine stromal cells in response to P4 The total RNA was isolated from the rat tissues using the (Mulac-Jericevic et al. 2000). ToTALLY RNA kit (Ambion, Austin, TX, USA), To isolate the genes regulated by P4 in the rat uterus, following the manufacturer’s instruction. The RNA we previously performed suppressive subtractive hybrid- concentration was determined by NanoDrop (Thermo ization (Ohno et al. 2008), and 14-3-3t was identified as Scientific, Wilmington, DE, USA). cDNA was synthe- one of the genes that were upregulated in the rat uterus sized from 500 ng of total RNA by RT using PrimeScript after P4 treatment. The 14-3-3 proteins form a family of Reverse Transcriptase (TaKaRa, Tokyo, Japan), highly conserved molecules that are expressed in a wide following the manufacturer’s instruction. range of organisms and tissues (Obsilova´ et al. 2008). There are nine isotypes (a, b, g, d, 3, h, s, t, and z)in vertebrates, and additional isotypes exist in yeasts, Materials plants, amphibians, and invertebrates (Aitken 2006). All these proteins have a molecular mass of w30 kDa DMEM/F-12, OPTI-MEM I reduced serum medium ! and exist as homodimers or heterodimers. Further- 1 , and fetal bovine serum (FBS) were purchased more, 14-3-3 proteins regulate various cellular activities from Invitrogen Life Technologies, Inc. Charcoal– by binding to phosphorylated signaling molecules, dextran-treated FBS was purchased from Hyclone including Raf-1, Cbl, Bad, Cdc25C, and IGF1 receptor. (Logan, UT, USA). E2,P4,8-bromoadenosinecAMP In addition, 14-3-3 proteins have been shown to sodium salt (8-Br-cAMP), and medroxyprogesterone enhance the transcriptional activity of glucocorticoid 17-acetate (MPA) were from Sigma–Aldrich Corp. The receptor (GR) and androgen receptor (AR) (Wakui luciferase assay kit was obtained from Promega Corp. The et al. 1997, Haendler et al. 2001, Zilliacus et al. 2001, Kim primary antibodies used for western blot analyses in this et al. 2005, Quayle & Sadar 2007, Titus et al. 2009). study were polyclonal anti-14-3-3t (Santa Cruz Bio- Moreover, Liang et al. (2006) demonstrated that technology), monoclonal anti-b-actin (Sigma), anti-HA aldosterone induced increased levels of 14-3-3b mRNA (Santa Cruz Biotechnology), and monoclonal anti-FLAG and protein in mouse cortical collecting duct epi- (Sigma). The secondary antibodies used in this study thelium. In this study, we examined the expression and were anti-rabbit and anti-mouse from Santa Cruz regulation of 14-3-3t in the rat uterus, cultured human Biotechnology, and the fluorescent secondary antibodies endometrial cells, and human endometrium. Further- were anti-mouse labeled with Alexa Fluor 488 and more, we analyzed the effect of 14-3-3t on PR-B anti-rabbit labeled with Alexa Fluor 594 from Invitrogen. transcriptional activity and the mechanism underlying the change induced by 14-3-3t in the transcriptional Cell culture activity of PR-B. Human endometrial adenocarcinoma Ishikawa cells (purchased from ATCC, Rockville, MD, USA) were routinely cultured at 37 8Cin5%CO2 atmosphere Materials and methods in phenol red-containing DMEM/F-12, 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Animals and treatment The HEK293 cells stably expressing PR-B were routinely In this study, we used 7-week-old female Sprague cultured under the same conditions as the human Dawley rats (Japan SLC, Hamamatsu, Japan). All endometrial adenocarcinoma Ishikawa cells. animals were maintained in accordance with the institutional guidelines for care and use of laboratory Isolation and culture of endometrial stromal cells animals. Our research was approved by the Research and treatments Ethics committee of the University of Tokyo. The rats were administered the hormone or vehicle 2 weeks after The endometrial tissues were obtained from women being ovariectomized. The ovariectomized rats were undergoing hysterectomies for benign diseases unre- s.c. injected with 20 mg17b-estradiol (E2) or 2 mg P4 lated to endometrial pathology. All patients had regular (Sigma) dissolved in corn oil. The control rats menstrual cycles, and none had received hormonal Journal of Molecular Endocrinology (2012) 49, 193–202 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 01:14:38AM via free access Progesterone-responsive 14-3-3t in uterine cells . M ITO and others 195 treatment for at least three cycles before the surgery. and 1 min at 60 8C) using the SYBR Green PCR master The specimens were annotated according to the criteria mix (Applied Biosystems) and the ABI 7000 Fast Real- as described (Noyes et al.
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