Detection of a positive feedback loop between macrophage migration inhibitory factor (MIF) and estrogen in endometriosis

Abstract

OBJECTIVE: To determine the interaction between MIF and estradiol (E2) in human ectopic endometrial cells. DESIGN: Primary cultures of ectopic endometrial cells exposed to E2 or to MIF and assessment of MIF and aromatase regulation. MATERIALS AND METHODS: Endometriotic stromal cells were isolated from human endometriotic lesions. Cells were then stimulated with E2 (10-9 M and 10-8 M) or MIF (10 and 50 ng/mL) for different periods of time (0-48 h). MIF secretion and mRNA levels were measured by ELISA and real time RT-PCR respectively. The transcriptional activity of MIF gene was analyzed by transfection with different MIF promoter-luciferase reporter constructs. The presence of estrogen receptors ERαand ERβ was assessed by immunocytofluorescence and the role of these receptors was evaluated by transfection with expression vectors. Aromatase protein was analyzed by immunocytofluorescence and western blot, and mRNA levels were assessed by real-time RT-PCR. An unpaired t test and one-way ANOVA followed by the Dunnett test were performed. RESULTS: Our study showed that E2 significantly induced MIF protein and mRNA expression in endometriotic stromal cells in a dose- and time-dependent manner. Treatment with Fulvestrant (a pure estrogen receptor antagonist) suppressed the ability of E2 to induce MIF protein secretion and mRNA synthesis. The MIF promoter activity was significantly stimulated with E2 compared to control. Transfection of endometriotic stromal cells with estrogen receptors ERαand ERβ resulted in a statistically significant increase of the E2-induced activation of the MIF protein and mRNA levels compared to culture medium without stimulation. However, when we compared the responses to cells transfected with control vector, the only statistically significant difference has been observed with ERβ. Moreover, MIF significantly stimulated aromatase protein and mRNA expression. CONCLUSIONS: The present study revealed the existence of a positive feedback loop operating locally in ectopic endometrial implants by which estrogen acts directly on ectopic endometrial cells to upregulate the expression of MIF, which, in turn, displays the capability of inducing the expression of aromatase, the key and rate-limiting enzyme for estrogen synthesis. Such immuno-endocrine interplay may have a considerable impact on endometriosis development.