Mifepristone Augments uNK Cell-Cytotoxicity Figure 4. Effects of mifepristone on the phosphorylation level of MAPK in uNK cells. A, a representative immunoblot is shown after the treatment of uNK cells with mifepristone (1.0 mM) for 0, 15, 30, 60, and 120 min. 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 group, 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. Immunodetection of MAPK members used specific antibodies for phosphorylated and total proteins of ERK1/2, P38 and JNK. B, densitometric scans of triplicate blots are shown. Experiments were independently repeated 3 times in each group. Data were analyzed using ANOVA and expressed as means 6 SEM. *, P,0.05. doi:10.1371/journal.pone.0036413.g004 Upregulation of perforin expression by mifepristone in uNK cells is reversed by cortisol We found that, 65 and 200 nmol/L mifepristone had no significant influence on human uNK-cell perforin expression in vitro. Compared with control group, human uNK-cell perforin expression (49.1362.92% vs. 36.2360.85%, P,0.05) (Fig. 2C) significantly increased in 1000 nmol/L (1.0 mM) mifepristone group. We then explored the effects of cortisol on changes in perforin expression induced by mifepristone in uNK cells. Cortisol (1.0 mM) significantly inhibited the mifepristone-induced increase in perforin expression (36.264.9% vs. 28.562.3%, P,0.05) and mifepristone significantly increased perforin expression (36.264.9% vs. 49.162.9%, P,0.05). When uNK cells were treated with mifepristone (1.0 mM) in the presence of cortisol, the upregulation of perforin expression by mifepristone in uNK cells was suppressed (49.162.9% vs. 33.163.5%, P,0.05; Fig. 3C). Mifepristone increases MAPK/ERK activation in uNK cells To confirm whether or not the MAPK pathway is involved in immune regulation by mifepristone, the expression and activation of ERK, p38 and JNK in uNK cells were determined by Western blot. Uterine NK cells were treated with 1.0 mM mifepristone for different times. The results indicated that mifepristone induced a time-dependent activation of the ERK pathway. Activation of ERK in uNK cells occurred at 15 min after mifepristone stimulation, peaked at 30 min, and decreased at 60 and 120 min (Fig. 4). In contrast, the phosphorylation levels of p38 and JNK in uNK cells were not altered at the different time points after mifepristone stimulation. Furthermore, 1.0 mM mifepristone significantly increased ERK activation at 30 min after the stimulation (P,0.05) and 1.0 mM cortisol significantly inhibited ERK activation (P,0.05). When uNK cells were simultaneously treated with cortisol and mifepristone, activation of the ERK pathway by mifepristone was suppressed (P,0.05, Fig. 5). PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e36413