FEMALE REPRODUCTIVE TRACT - B
Wednesday, August 4, 2004
10:30 AM–12:30 PM
(810) TNF UP-REGULATED THE EXPRESSION OF MIF IN HUMAN ENDOMETRIAL STROMAL CELLS IN VITRO: PROBABLY THROUGH NF-B.
Cao, Wenguang1, 2, Therriault, MJ2, Akoum, Ali2, Maheux, R2, 1 ONPRC, Oregon Health and Science University, Beaverton, OR2 Centre de Recherche, Hospital Saint-Francois d'Assise, Faculte de Medecine, QC, QP, Canada
ABSTRACT- Endometriosis is a disease characterized by the growth of endometrial or endometrium-like tissue outside of the uterus. Our previous studies showed that macrophage migration inhibitory factor (MIF) was highly expressed and actively involved in endometriotic pathogenesis. It is reported that TNF may also be involved in the pathogenesis of endometriosis. Expression of TNF may also affect the expression of MIF in endometrial stromal cells. Our goal was to determine if a relationship exists between TNF and MIF in endometrial stromal cells. Methods: Endometrial stromal cells were cultured in DMEM/F-12 medium supplied with 10% FBS, 0.1% insulin and 0.055% transferrin. When the cells reached confluence, the medium was replaced with no serum, insulin, transferrin or phenol red added, and the cultures were treated with TNF(0.1ng/ml to 100ng/ml) with or without curcumin (10-8 M), for 6 to 48 hours. Similar cultures were prepared on chamber slides for fluorescent immunocytochemistry with FITC. Medium and cells were collected. RNA was extracted from the cells and analyzed by RT-PCR. Nuclear and cytosolic protein was extracted from the cells for Western blotting and electrophoretic mobility shift assay (EMSA). Medium was analyzed for MIF by ELISA. Results: Both MIF protein detected by ELISA (P < 0.05) and transcript detected by RT-PCR was increased significantly after TNF treatment. MIF protein was depressed by co-treatment with curcumin. FITC also showed an increase in MIF. Western blot revealed that p-ikB was increased by TNF. EMSA displayed a specific binding of NF-kB P50 and a super shift band with anti-NF-kB P50 antibodies. The specific band either disappeared with a competitive sequence or was markedly eliminated by curcumin. We conclude that TNF up-regulated the expression of MIF in human endometrial stromal cells in vitro through activation of a nuclear factor. This study was sponsored by Canadian Institute of Health Science.
KEY WORDS: Endometrial , TNF, Endometriosis, MIF