Mifepristone Augments uNK Cell-Cytotoxicity Figure 5. Effects of mifepristone on the ERK signaling pathway in uNK cells. Western blot of p-ERK and total ERK in uNK cells treated with mifepristone 6 cortisol. To detect p-ERK and total ERK, total cell lysates were harvested after a 30 min treatment with mifepristone (1.0 mM) 6 cortisol (1.0 mM). Uterine NK cells were isolated and cultured in the medium without IL-2 for 12 h as a negative control of MAPK/ERK activation. In the drug treatment groups, uNK cells were cultured in the medium supplemented with IL-2. Uterine NK cells were cultured in the medium with IL-2 and IL-15 for 15 min as a positive control. A typical blot (A) and densitometric scans of triplicate blots (B) are shown. Data were analyzed using ANOVA and expressed as means 6 SEM. *, P,0.05. doi:10.1371/journal.pone.0036413.g005 Mifepristone augments uNK cell-mediated cytotoxicity and perforin expression by activation of the ERK pathway Because mifepristone appeared to increase ERK activation, we further investigated the possible role of the ERK pathway in mifepristone-induced cytotoxicity and changes in perforin expres- sion in uNK cells. Uterine NK cells were pretreated for 30 min with either of two specific ERK1/2 inhibitors, PD98059 or U0126. They were then treated with mifepristone with or without cortisol for 24 h. The effect of mifepristone 6 cortisol on uNK cell-mediated cytotoxicity was determined by the MTS assay. After pretreatment with PD98059 or U0126, mifepristone had no effect on uNK cell-mediated cytotoxicity, with or without cortisol (P.0.05, Fig. 6). No changes in perforin expression, due to cortisol or mifepristone, were observed when uNK cells were pretreated with PD98059 or U0126 (Fig. 7). Discussion Accumulating evidence supports a requirement for optimal postovulatory increases of uNK cell numbers for successful embryo implantation and placentation [31,32]. Altered numbers of uNK cells and their function may be responsible for reproductive disorders [11]. Our previous research confirmed that low-dose mifepristone increases the number of CD56+-NK cells and the percentages of the NK cell subset CD3 2 CD56+ CD162 in human endometrial explants [13]. Mifepristone can also directly augment uNK cell-mediated cytotoxicity through the increased expression of perforin (unpublished result). However, the specific mechanisms responsible for the effects due to mifepristone in uNK cells are unclear. Changes in the production of steroids play a major role in modulation of local immunosuppression at the maternal-fetal interface. The number of uNK cells within the endometrial stroma varies during the menstrual cycle and throughout pregnancy. It is tempting to speculate that progesterone may be involved in the ever-changing status of uNK cells. In the present study, however, we found that progesterone at different concentrations does not affect uNK cell-mediated cytotoxicity. Despite expression of the classical PR in peripheral blood NK cells, strong evidence indicates that neither isoform of the PR is expressed in uNK cells [17]. The proliferation, target cell lysis and cytokine secretion of high-purity uNK cells was not influenced by progesterone in vitro [18]. The current results are consistent with previous studies and support the concept that progesterone has no direct effect on the biological activity of uNK cells. We found that mifepristone augments the cytotoxicity of uNK cells in culture, and this effect was blocked by cortisol but not progesterone. This suggested that mifepristone could regulate uNK cell-mediated cytotoxicity by antagonizing the GR rather than the PR. Accumulating evidence indicates that mifepristone has a variety of putative effects on cancer cells and immune cells [1–5], with the mechanism of action involving antagonism of hormone receptors [33,34]. Binding of mifepristone to the receptor induces the loss of associated heat shock proteins and PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36413