Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH The nuclear receptor REV-ERBa represses the transcription of growth/differentiation factor 10 and 15 genes in rat endometrium stromal cells Lijia Zhao1, Keishiro Isayama1, Huatao Chen1,*, Nobuhiko Yamauchi1, Yasufumi Shigeyoshi2, Seiichi Hashimoto3 & Masa-aki Hattori1 1 Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan 2 Department of Anatomy and Neurobiology, Kinki University School of Medicine, Osaka, Japan 3 Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Keywords Abstract Circadian clock, decidualization, growth/ differentiation factors, REV-ERBa. Cellular oscillators in the uterus play critical roles in the gestation processes of mammals through entraining of the clock proteins to numerous downstream Correspondence genes, including growth/differentiation factor (Gdf)10 and Gdf15. The expres- Masa-aki Hattori, Department of Animal and sion of Gdf10 and Gdf15 is significantly increased in the uterus during decidu- Marine Bioresource Sciences, Graduate alization, but the mechanism underlying the regulation of Gdf gene expression School of Agriculture, Kyushu University, in the uterus is poorly understood. Here, we focused on the function of the Hakozaki, Higashi-ku, Fukuoka 812-8581, cellular oscillators in the expression of Gdf family by using uterine endome- Japan. Tel: +81-92-642-2938 trial stromal cells (UESCs) isolated from pregnant Per2-dLuc transgenic rats. Fax: +81-92-642-2938 A significant decline of Per2-dLuc bioluminescence activity was induced in E-mail: [email protected] in vitro decidualized UESCs, and concomitantly the expression of canonical clock genes was downregulated. Conversely, the expression of Gdf10 and ⁄ Present address Gdf15 of the Gdf was upregulated. In UESCs transfected with Bmal1-specific College of Veterinary Medicine, Northwest A siRNA, in which Rev-erba expression was downregulated, Gdf10 and Gdf15 & F University, Yangling, Shaanxi, 712100, were upregulated. However, Gdf5, Gdf7, and Gdf11 were not significantly China affected by Bmal1 silencing. The expression of Gdf10 and Gdf15 was enhanced Funding Information after treatment with a REV-ERBa antagonist in the presence or absence of This work was supported in part by a Grant- progesterone. Chromatin immunoprecipitation-PCR analysis revealed the inhi- in-Aid for Scientific Research (B) from the bitory effect of REV-ERBa on the expression of Gdf10 and Gdf15 in UESCs by Japan Society for the Promotion of Sciences recognizing their gene promoters. Collectively, our findings indicate that the (JSPS No. 22380152, 24658246) (to M-A attenuation of REV-ERBa leads to an upregulation of Gdf10 and Gdf15 in Hattori). Lijia Zhao (Grant No. decidual cells, in which cellular oscillators are impaired. Our results provide 201308050010) and Huatao Chen (Grant No. 2010630069) are sponsored by the novel evidence regarding the functions of cellular oscillators regulating the China Scholarship Council. Keishiro Isayama expression of downstream genes during the differentiation of UESCs. was supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (Grant No. 6117). Received: 24 November 2015; Accepted: 30 November 2015 doi: 10.14814/phy2.12663 Physiol Rep, 4 (2), 2016, e12663, doi: 10.14814/phy2.12663 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2016 | Vol. 4 | Iss. 2 | e12663 the American Physiological Society and The Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Inhibitory Role of REV-ERBa in Uterus Gdf10 and Gdf15 L. Zhao et al. Introduction sion of the transcription of Bmal1 through direct binding to the RORE located in the Bmal1 promoter (Albrecht Growth/differentiation factors (GDFs) are members of the and Eichele 2003; Brown et al. 2005). In addition to regu- transforming growth factor-b (TGF-b) superfamily, and lating each other to sustain oscillations, REV-ERBa also they are involved in a variety of cellular functions and controls the expression of numerous downstream genes biological processes such as cell proliferation, differentia- through binding to ROREs at their promoters. tion, and remodeling (Lee 1991; McPherron et al. 1997; BMAL1, a critical component of clock proteins, is Whitman 1998). The expression of GDFs is involved in indispensable in maintaining the integrity of the circadian embryonic development and the development of female feedback loop and the homeostasis of numerous behav- reproductive tissues. The GDFs such as GDF1, GDF3, iors and physiological processes (Kondratov et al. 2006; GDF10, and GDF15 are spatiotemporally expressed in the Alvarez et al. 2008; Grechez-Cassiau et al. 2008; Ratajczak embryo and uterus. For example, GDF10 is highly et al. 2009). Several studies provided evidence demon- expressed in the uterus during the menstrual cycle and strating that the physiologic significance of BMAL1 is pregnancy (Zhao et al. 1999) and plays a role in head for- related to mammalian reproductive functions (Ratajczak mation (Hino et al. 2004). GDF15, which is also known et al. 2009; Boden et al. 2010; Liu et al. 2014). REV-ERBa as macrophage inhibitory cytokine-1 (MIC-1), is highly usually functions as a transcriptional repressor for the expressed in human placenta and is thought to have a lack of activation function (AF-2) domain present at the predictive aspect for pregnancy outcomes (Lawton et al. C-terminal of the ligand-binding domain (Yin and Lazar 1997; Fairlie et al. 1999; Tong et al. 2004). GDF15 is 2005; Phelan et al. 2010; Crumbley and Burris 2011). principally expressed in villous cytotrophoblast cells, REV-ERBa recruits the endogenous nuclear receptor core- extravillous trophoblasts, decidual stromal cells, and pla- pressor (N-CoR)/histone deacetylase3 complex to repress centa (Lawton et al. 1997; Fairlie et al. 1999; Tong et al. its target gene transcription, thereby regulating a diverse 2004). A study using extravillous trophoblast cells demon- array of cellular processes (Yin and Lazar 2005; Yin et al. strated an inhibitory effect of GDF15 on cell viability by 2006). REV-ERBa was originally regarded as an orphan apoptosis and growth inhibition (Morrish et al. 2001). nuclear receptor (Miyajima et al. 1989), and thereafter GDF15 also functions as a potent regulator of matrix heme was identified as its natural ligand (Yin et al. 2007; metalloproteinases, which controls the degradation of the Meng et al. 2008; Grant et al. 2010; Kojetin et al. 2011). decidual matrix and thus affects the invasion of tro- GSK4112 is synthesized as a chemical agonist of REV- phoblast cells (Marjono et al. 2003). However, the mecha- ERBa, and it represses REV-ERBa target genes (Grant nism underlying the regulation of Gdf gene expression in et al. 2010; Chen et al. 2012; Gibbs et al. 2012; Chini the uterus remains poorly understood. et al. 2013). A chemical REV-ERBa antagonist, SR8278, There are many E-box and ROR/REV-ERB response was reported (Kojetin et al. 2011), and it increases the elements (ROREs), which are the circadian clock-con- transcription of REV-ERBa target genes (Kojetin et al. trolled cis-regulatory elements, in the promoter regions of 2011; Isayama et al. 2015; Tasaki et al. 2015). the Gdf genes such as Gdf10 and Gdf15 (NCBI Reference Rev-erba has a key role in several physiological actions Sequence: NC_005115.4). Numerous peripheral circadian such as adipocyte differentiation, glucose metabolism, and clocks are partially self-operative and independent in their thermogenesis (Chawla and Lazar 1993; Cho et al. 2012; responses to external and internal stimuli other than the Gerhart-Hines et al. 2013). The circadian system consist- stimuli originating from the suprachiasmatic nucleus, ing of clock genes is also disrupted in differentiating cells known as the central circadian clock (Hara et al. 2001; of rat ovaries and uteri (Alvarez and Sehgal 2005; He Vollmers et al. 2009; Tahara et al. 2012; Wu et al. 2012). et al. 2007a). Several studies have demonstrated that cir- The molecular mechanism of the mammalian circadian cadian clock genes are rhythmically expressed in the clock involves a primary conservative interlocked tran- uterus (Dolatshad et al. 2006; He et al. 2007a; Hirata scriptional-translational feedback loop (Ko and Takahashi et al. 2009; Akiyama et al. 2010). In rodents and humans, 2006). This loop is comprised of a core group of clock the uterus endometrial stromal cells (UESCs) undergo genes and their protein products, which are mostly the proliferation and differentiation into decidual cells in transcription factors. The transcriptional activators response to ovarian steroids and blastocyst implantation BMAL1 and CLOCK form a heterodimer, which drives at the early stage of pregnancy (Clarke and Sutherland the expression of the Per1-3 and Cry1-2 genes by recog- 1990; Zhang et al. 1994; Dey et al. 2004). Decidualization nizing E-box cis-elements in their promoters (Gekakis is critical to the establishment of fetal-maternal commu- et al. 1998; Hogenesch et al. 1998; Ueda et al. 2005). The nication and the progression of implantation and this CLOCK-BMAL1 heterodimer also induces expression of process ultimately results in the formation of the placenta. the nuclear receptor, REV-ERBa, resulting in the repres-