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DREAM Mediated Regulation of GCM1 in the Human Placental Trophoblast

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DREAM Mediated Regulation of GCM1 in the Human Placental Trophoblast DREAM Mediated Regulation of GCM1 in the Human Placental Trophoblast Dora Baczyk1*, Mark Kibschull1, Britt Mellstrom2, Khrystyna Levytska1, Marcos Rivas2, Sascha Drewlo1, Stephen J. Lye1,3, Jose R. Naranjo2, John C. P. Kingdom1,3 1 Research Centre for Women’s and Infants’ Health at the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada, 2 Department of Molecular and Cellular Biology, National Centre for Biotechnology, C.S.I.C., Madrid, Spain, 3 Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada Abstract The trophoblast transcription factor glial cell missing-1 (GCM1) regulates differentiation of placental cytotrophoblasts into the syncytiotrophoblast layer in contact with maternal blood. Reduced placental expression of GCM1 and abnormal syncytiotrophoblast structure are features of hypertensive disorder of pregnancy – preeclampsia. In-silico techniques identified the calcium-regulated transcriptional repressor – DREAM (Downstream Regulatory Element Antagonist Modulator) - as a candidate for GCM1 gene expression. Our objective was to determine if DREAM represses GCM1 regulated syncytiotrophoblast formation. EMSA and ChIP assays revealed a direct interaction between DREAM and the GCM1 promoter. siRNA-mediated DREAM silencing in cell culture and placental explant models significantly up-regulated GCM1 expression and reduced cytotrophoblast proliferation. DREAM calcium dependency was verified using ionomycin. Furthermore, the increased DREAM protein expression in preeclamptic placental villi was predominantly nuclear, coinciding with an overall increase in sumolylated DREAM and correlating inversely with GCM1 levels. In conclusion, our data reveal a calcium-regulated pathway whereby GCM1-directed villous trophoblast differentiation is repressed by DREAM. This pathway may be relevant to disease prevention via calcium-supplementation. Citation: Baczyk D, Kibschull M, Mellstrom B, Levytska K, Rivas M, et al. (2013) DREAM Mediated Regulation of GCM1 in the Human Placental Trophoblast. PLoS ONE 8(1): e51837. doi:10.1371/journal.pone.0051837 Editor: David S. Milstone, Brigham and Women’s Hospital, United States of America Received June 25, 2012; Accepted November 7, 2012; Published January 3, 2013 Copyright: ß 2013 Baczyk et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research is supported by Canadian Institute of Health Research (Grant no. 64302) to JK, Department of Obstetrics & Gynecology and Rose Torno Chair at MSH as well as by the Canadian Institutes of Health Research Interface Training Program, Applying Genomics to Human Health & Molly Towell Perinatal Research Foundation Fellowship to SD, and Spanish Science Agency-DGICYT (grants SAF2010-21784; SAF2008-03469) to JN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction of generating a continuous stream of daughter cells to continuously fuse into the overlying SYN. Successful placentation in humans is dependent upon the Development of the mouse labyrinthine trophoblast, analogous promotion of maternal blood flow to the implantation site and the to VCT and SYN in the human placenta, is characterized by cell elaboration of chorionic villi that mediate maternal-fetal exchange cycle arrest and cell-cell fusion promoted by the transcription [1]. Failure of either or both processes during the early stages of factor Glial cell missing-1 (Gcm1) [8]. Mouse embryos lacking placental development can have negative consequences for both Gcm1 die in mid-gestation due to a failure of SYN differentiation the fetus (intrauterine growth restriction [IUGR]) and the mother, [8,9]. In humans, the homolog GCM1 has an expression pattern severe early-onset preeclampsia (sPE), causing death or severe similar to mice [10] and plays a central role in mediating long-term morbidity [2]. differentiation of trophoblast cells along both villous and extra- Human placental development is directed by two distinct villous pathways [11]. GCM1 promotes the expression of the lineages of trophoblast cells. Invasive extravillous cytotrophoblast fusogenic protein syncytin [12,13] and placenta growth factor transform the uteroplacental vasculature [3] while villous cytotro- (PlGF) [14]. phoblast (VCT) give rise to the outer syncytiotrophoblast (SYN) The trophoblast layer of sPE placentas delivered prematurely layer that covers the placental villi [4]. The SYN layer is in direct shows severe depletion of proliferating VCT, accompanied by contact with maternal blood and is responsible for maternal-fetal patchy necrosis or accelerated apoptosis in the outer SYN layer gas, nutrient and waste exchange. Progressive growth of the and SYN knot formation [15]. In parallel, molecular pathology placental villi during gestation, to accommodate exponential studies have demonstrated reduced expression of GCM1 [16] and growth of the fetus, requires an accumulation of SYN [5]. This its downstream fusogenic partner syncytin [17] in sPE placentas is achieved by continuous proliferation of a subset of VCT [6] that while low maternal circulating PlGF levels are predictive of the generate post-mitotic cells capable of syncytial fusion [7]. severity of this disease [18]. Inhibition of GCM1 expression in the Conserving a population of proliferative VCT throughout first trimester floating explant model in-vitro recapitulates these gestation is a key feature of human placentation [6] implying that SYN abnormalities observed in sPE [11]. a population of lineage-restricted progenitor cells remains capable PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e51837 DREAM Mediated Regulation of GCM1 in the Placenta GCM1 is therefore a key regulator of human placental voluntary legal termination of pregnancy. Mount Sinai Hospital development, though the mechanisms governing its regulation (MSH) Research Ethics Board approval (MSH REB #04-0018-U) are poorly understood. To explore potential upstream molecular was obtained for this study and all patients gave written informed mechanisms of GCM1 transcriptional regulation we conducted an consent. Gestational age and viability were established pre- in silico analysis of the GCM1 promoter region that revealed the operatively by ultrasound. All experiments were established within existence of DREAM binding sites. 4–6 hrs from the time of sample collection. DREAM (also known as KChIP-3 and calsenilin) belongs to a Individual clusters of 8–12 week gestation villi (20–30 mg wet family of small calcium sensors that are calcium-dependent wt) were dissected in sterile cold PBS containing calcium and transcriptional repressors [19,20]. DREAM contains 256 amino magnesium, under a microscope. The proximal stem villi were acids and 9 exons (molecular weight of 29 kDa); the human inserted into the underside of sterile polystyrene cubes so as to float placenta only expresses a specific splice variant containing 230 the villous trees in 750 ml of serum-free media (DMEM/F12) with amino acids and 8 exons [21]. Both isoforms encode a protein 1% liquid media supplement ITS+1 (Sigma, St Louis, MO, USA), containing four calcium-binding domains [19]. DREAM is 100 units/ml penicillin, 100 units/ml streptomycin, 2 mM L- expressed in different cellular compartments (nucleus, cytoplasm glutamine, 100 mg/ml gentamycin and 2.5 mg/ml fungizone. and cell membrane) and thus is involved in various distinct cell These explants were maintained in physiologic 8% ambient functions. Nuclear accumulation and thus repressor function of oxygen (40 mmHg)/5% CO2 at 37uC [11,31]. DREAM has been related to sumoylation of the protein [22]. For DREAM siRNA treatment, floating villous explants were DREAM is a multifunctional protein critical for brain function incubated in the presence of 100 nM siRNA or 100 nM non- since i) regulates calcium homeostasis and neuronal viability [23], silencing control (Santa Cruz Biotechnology) for up to 2 days. ii) interacts with presenilins, Alzheimer’s disease- associated First and second trimester placental samples were also collected proteins, to regulate APP processing [24,25] and iii) regulates into 2 ml of RNA Later buffer (Ambion, Streetsville, ON) and neuronal excitability by modulating potassium and calcium stored at 220uC. Third trimester placental samples, from healthy channels [26,27] as well as NMDA receptors [28] [29]. We and severely preeclamptic patients delivering at MSH, Toronto, hypothesized that in the human placenta, DREAM mediates the Canada were collected into RNA later buffer or snap frozen and expression of GCM1 via a direct interaction with its promoter. stored 270uC for future RNA isolation using Qiagen kit. Patient Here we demonstrate that DREAM negatively regulates GCM1 information is summarized in Table 1. Severe pre-eclampsia was gene expression during human placental development in a defined according to accepted criteria developed by the American calcium-regulated manner and we show that sumoylated DREAM College of Obstetricians and Gynecologists (PMID: 16175681). is increased in sPE placentas. These findings may be of relevance to clinical trial observations
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