Induction of cyclooxygenase-2 expression and prostaglandin E2 secretion by macrophage migration inhibitory factor in ectopic endometrial cells: role in the pathophysiology of endometriosis

Abstract

OBJECTIVE: To assess whether macrophage migration inhibitory factor (MIF), a potent proinflammatory and angiogenic factor found at elevated levels in the peritoneal fluid and active endometriotic lesions of women with endometriosis, may influence the expression of cyclooxygenases (COX) 1 and 2 and the secretion of prostaglandin E2 (PGE2) in ectopic endometrial cells. DESIGN: Primary cultures of ectopic endometrial cells exposed to different concentrations of MIF and assessment of COX-1 and COX-2 expression and PGE2 secretion. MATERIALS AND METHODS: Ectopic endometrial cells were isolated and cultured from endometriotic lesions (n=9) according to routinely used procedures. At confluence, cells were starved 16h in DMEM F12 and then stimulated with various concentrations of MIF (0-50 ng/mL) for several periods of time (0-24 h). In some cultures, cells were co-incubated with a specific inhibitor of MIF, ISO-1 or with NS-398, a specific inhibitor of COX-2 activity. Expression of COX-1 and COX-2 was analysed by quantitative real-time PCR at mRNA level and by Western Blot and immunocytofluorescence at protein level. PGE2 concentration in supernatants was measured using a PGE2 Enzyme Immunoassay (EIA) kit. Statistical analysis was performed using ANOVA followed by the Dunnet's or the Bonferonni's multiple comparison test. RESULTS: MIF stimulates the synthesis of COX-2 in endometriotic cells in vitro, whereas COX-1 is not affected. Cell exposure to MIF led to a dose- and time-dependent upregulation of COX-2 mRNA expression, as shown by quantitative real-time PCR (p<0.01), and ISO-1 significantly inhibited such a MIF-induced effect. Results from Western blot and immunocytofluorescence further demonstrated an increase in COX-2 protein synthesis in response to MIF. Analysis of PGE2 secretion revealed a significant increase in cells exposed to MIF (p<0.01), whereas addition of NS-398 resulted in a significant inhibition of the MIF-induced PGE2 secretion (p<0.01). CONCLUSIONS: MIF up-regulates COX-2 expression and PGE2 secretion in ectopic endometrial cells. Considering the wide spectrum of PGE2 inflammatory, growth-promoting and angiogenic properties and implication in various female reproductive functions, these data unveil a new mechanism by which MIF may favour the development of endometriotic lesions and the manifestation of the disease's clinical symptoms.