Gene Expression in Endocrine Tissues
(W474) LONG PROMOTER AND TRANSCRIPTIONAL REGULATION OF PROSTAGLANDIN F AND E SYNTHASES BY IL1B IN HUMAN ENDOMETRIAL CELLS.
Chapdelaine, Pierre1, Fortier, Michel1, 2, 1 Laval University Hospital Center, Quebec, Qc, Canada2 Laval University, Quebec, Qc, Canada
ABSTRACT- Prostaglandins (PGs) are recognized as key regulators of female reproductive function and associated with recognition and establishment of pregnancy or gynecological pathologies such as endometrial carcinomas, menorrhagia, dysmenorrhoea and endometriosis. Our laboratory has identified and characterised new enzymes involved in PG biosynthesis in the bovine and found the corresponding isoforms in the human endometrium. Among those, microsomal prostaglandin synthase 1 (mPGES1) and a prostaglandin F synthase (AKR1B1) appear as the primary enzymes involved respectively in terminal production of prostaglandin E2 (PGE2) and F2 (PGF2) in epithelial and stromal cells of the human endometrium. We have shown previously that the expression of these enzymes and the production of PGE2 and PGF2are increased in endometrial cells following stimulation with Interleukin-1 (IL1). To study the regulation of the terminal synthase genes, we have developed a nested PCR strategy to obtain a 4.2 kb long promoter for mPGES1 and a 4.5 kb long promoter for AKR1B1. The long promoter PCR fragments (amplified with PFU turbo DNA polymerase) were cloned directly by fusion in pGL3 basic vector containing the luciferase gene. Human endometrial cell lines (HIESC2 for stromal cells and HIEEC22 for epithelial cells) were transfected separately with the two reporter constructs in presence of lipofectamine 2000 and treated for 24 hours with IL1 (1ng/ml). The promoters activity increased the basal expression of mPGES1 and AKR1B1 50 and 200 folds respectively by comparison with the pGL3 basic vector. IL-1 (1ng/ml) increased the promoter activity of mPGES1 up to 3 folds and AKR1B1 up to 10 folds. No difference was observed between stromal and epithelial cells. This study opens the field to investigate further the strategic associations between the different biosynthetic enzymes of the PG cascade necessary to produce selectively PGE2 and PGF2 in human endometrial cells.
KEY WORDS: prostaglandin biosynthesis, gene regulation, AKR1B1, mPGES1