Mifepristone Increases the Cytotoxicity of Uterine Natural Killer Cells by Acting as a Glucocorticoid Antagonist via ERK Activation Yuezhou Chen1, Yan Wang 2 , Yaling Zhuang1, Feng Zhou1, Lili Huang 1 * 1 Women’s Reproductive Health Laboratory of Zhejiang Province, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China, 2 Department of Gastroenterology, Taihe Hospital, Hubei Medical University, Shiyan, Hubei, People’s Republic of China Abstract Background: Mifepristone (RU486), a potent antagonist of progesterone and glucocorticoids, is involved in immune regulation. Our previous studies demonstrated that mifepristone directly augments the cytotoxicity of human uterine natural killer (uNK) cells. However, the mechanism responsible for this increase in cytotoxicity is not known. Here, we explored whether the increased cytotoxicity in uNK cells produced by mifepristone is due to either anti-progesterone or anti-glucocorticoid activity, and also investigated relevant changes in the mitogen-activated protein kinase (MAPK) pathway. Methodology/Principal Findings: Uterine NK cells were isolated from decidual samples and incubated with different concentrations of progesterone, cortisol, or mifepristone. The cytotoxicity and perforin expression of uNK cells were detected by mitochondrial lactate dehydrogenase-based MTS staining and flow cytometry assays, respectively. Phosphorylation of components of the MAPK signaling pathway was detected by Western blot. Cortisol attenuated uNK cell-mediated cytotoxicity in a concentration-dependent manner whereas progesterone had no effect. Mifepristone alone increased the cytotoxicity and perforin expression of uNK cells; these effects were blocked by cortisol. Furthermore, mifepristone increased the phosphorylation of ERK1/2 in a cortisol-reversible manner. Specific ERK1/2 inhibitor PD98059 or U0126 blocked cortisol- and mifepristone-induced responses in uNK cells. Conclusions/Significance: These results suggest that mifepristone acts as a glucocorticoid antagonist to augment uNK cell- mediated cytotoxicity via ERK activation, which may be caused by increased perforin expression. These observations may reveal an important mechanism by which mifepristone upregulates the cytotoxicity of uNK cells. Citation: Chen Y, Wang Y, Zhuang Y, Zhou F, Huang L (2012) Mifepristone Increases the Cytotoxicity of Uterine Natural Killer Cells by Acting as a Glucocorticoid Antagonist via ERK Activation. PLoS ONE 7(5): e36413. doi:10.1371/journal.pone.0036413 Editor: Kang Sun, Fudan University, China Received November 29, 2011; Accepted April 9, 2012; Published May 1, 2012 Copyright: 2012 Chen 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 work was supported by the National Natural Science Foundation of China (No. 81070525). 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: fbhuanglili@163.com Introduction Mifepristone (RU486) is a synthetic 19-norsteroid, and a potent antagonist of progesterone and glucocorticoids. Basic research has demonstrated a variety of potential applications for mifepristone in the fields of gynecology, endocrinology, oncology, and immunol- ogy [1–5]. It has been used primarily as an anti-progesterone drug to produce early pregnancy termination, and as an anti- glucocorticoid drug to ameliorate the clinical manifestations of Cushing’s syndrome [6]. Recently, several studies demonstrated that for the purpose of contraception, low-dose mifepristone retards endometrial development, so-called endometrial contra- ception [7]. Therefore, mifepristone may serve as a novel, estrogen-free, contraceptive pill with little or no change to the menstrual cycle and few adverse side effects. In addition to its antagonistic activities, accumulating evidence suggests that mifepristone may be involved in modulation of the immune response. In vitro, mifepristone significantly inhibits the proliferation of lymphocytes [5,8] and increases the cytotoxicity of natural killer (NK) cells from pregnant subjects [9]. Uterine NK (uNK) cells are the most abundant lymphocytes in endometrial and decidual tissue, characterized as brightly staining for CD56 (CD56+) and dimly staining for CD16 (CD162). Important roles have been proposed for uNK cells in immunotolerance, regulation of trophoblast invasion, and remodeling of the spiral arteries [10]. Altered numbers of uNK cells have been associated with recurrent miscarriage, recurrent implantation failure, fetal growth restric- tion, and preeclampsia [11]. For terminating early pregnancy, Lu et al. [12] demonstrated that mifepristone with misoprostol might increase the expression of CD56+-NK cells in decidua and induce disorder of the decidual micro-environment, which might be one mechanism underlying the abortifacient properties of this drug combination. Our previous studies showed that low-dose mifep- ristone increases the number of CD56+-NK cells, and the percentages of the NK cell subset CD32CD56+CD162 in human endometrial explants [13]. Uterine NK cell-mediated cytotoxicity and perforin expression were also augmented by treatment with PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36413