Macrophage migration inhibitory factor (MIF)-induced signaling pathways in human endometrial stromal cells of women with and without endometriosis

Abstract

OBJECTIVE: To investigate signal transduction pathways in endometrial stromal cells of women with and without endometriosis in response to MIF, a potent proinflammatory and growth-promoting factor.DESIGN: Primary cultures of endometrial stromal cells isolated from endometrial biopsies of women with and without endometriosis in proliferative and secretory cycle phase exposed to MIF and assessment of extracellular signal-related kinase (Erk)1/2 mitogen-activated protein kinase (MAPK), p38 MAPK and Akt phosphorylation.MATERIALS AND METHODS: Cells were cultured in DMEM-F12 medium with 10% FBS, 1% antibiotics, 5 μg/mL insulin and 5 μg/mL transferrin. Cells grown to confluence were preincubated for 1 h with Erk1/2 MAPK, p38 MAPK and Akt inhibitors (PD98059, SB203580 and LY294002 respectively) and stimulated with different concentrations of MIF (0-50 ng/mL) for different periods of time (0-1 h). Then, the extraction of proteins was performed and phosphorylation of Erk1/2 MAPK, p38 MAPK and Akt was analyzed by Western blot. An unpaired t test and one-way ANOVA followed by the Dunnett's test were performed for this study.RESULTS: Our study showed that MIF stimulated p38 MAPK and Akt phosphorylation in endometrial cells of women with and without endometriosis in the proliferative and the secretory phases of the menstrual cycle. In normal women, the basal level of Erk1/2 MAPK phosphorylation was lower in cells from the secretory phase of the menstrual cycle compared to the proliferative phase, but remained steadily elevated throughout the menstrual cycle in women with endometriosis. Cell exposure to MIF did not result in any detectable increase in Erk1/2 MAPK phosphorylation, whereas in normal women MIF activated Erk1/2 MAPK phosphorylation in the secretory phase. PD98059, SB203580 and LY294002 suppressed the ability of MIF to increase Erk1/2 MAPK, p38 MAPK and Akt phosphorylation, respectively.CONCLUSIONS: p38 and Akt signaling pathways are involved in MIF-mediated activation of endometrial stromal cells of women with and without endometriosis. Activation of Erk1/2 in endometrial cells throughout the menstrual cycle of women with endometriosis may, considering the role of these kinases in cell growth, angiogenesis and tissue remodeling, play an important role in the capability of endometrial cells to implant and develop in ectopic locations. OBJECTIVE: To investigate signal transduction pathways in endometrial stromal cells of women with and without endometriosis in response to MIF, a potent proinflammatory and growth-promoting factor. DESIGN: Primary cultures of endometrial stromal cells isolated from endometrial biopsies of women with and without endometriosis in proliferative and secretory cycle phase exposed to MIF and assessment of extracellular signal-related kinase (Erk)1/2 mitogen-activated protein kinase (MAPK), p38 MAPK and Akt phosphorylation. MATERIALS AND METHODS: Cells were cultured in DMEM-F12 medium with 10% FBS, 1% antibiotics, 5 μg/mL insulin and 5 μg/mL transferrin. Cells grown to confluence were preincubated for 1 h with Erk1/2 MAPK, p38 MAPK and Akt inhibitors (PD98059, SB203580 and LY294002 respectively) and stimulated with different concentrations of MIF (0-50 ng/mL) for different periods of time (0-1 h). Then, the extraction of proteins was performed and phosphorylation of Erk1/2 MAPK, p38 MAPK and Akt was analyzed by Western blot. An unpaired t test and one-way ANOVA followed by the Dunnett's test were performed for this study. RESULTS: Our study showed that MIF stimulated p38 MAPK and Akt phosphorylation in endometrial cells of women with and without endometriosis in the proliferative and the secretory phases of the menstrual cycle. In normal women, the basal level of Erk1/2 MAPK phosphorylation was lower in cells from the secretory phase of the menstrual cycle compared to the proliferative phase, but remained steadily elevated throughout the menstrual cycle in women with endometriosis. Cell exposure to MIF did not result in any detectable increase in Erk1/2 MAPK phosphorylation, whereas in normal women MIF activated Erk1/2 MAPK phosphorylation in the secretory phase. PD98059, SB203580 and LY294002 suppressed the ability of MIF to increase Erk1/2 MAPK, p38 MAPK and Akt phosphorylation, respectively. CONCLUSIONS: p38 and Akt signaling pathways are involved in MIF-mediated activation of endometrial stromal cells of women with and without endometriosis. Activation of Erk1/2 in endometrial cells throughout the menstrual cycle of women with endometriosis may, considering the role of these kinases in cell growth, angiogenesis and tissue remodeling, play an important role in the capability of endometrial cells to implant and develop in ectopic locations.