CLONING OF GUINEA-PIG CYCLOOXYGENASE-2 AND 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE COMPLEMENTARY DEOXYRIBONUCLEIC ACIDS - STEROID-MODULATED GENE-EXPRESSION CORRELATES TO PROSTAGLANDIN-F2-ALPHA SECRETION IN CULTURED ENDOMETRIAL CELLS

Prostaglandin F-2 alpha (PGF(2 alpha)) secretion is lowest at midcycle and highest on day 15 at luteolysis in the cycling guinea pig uterus and is inversely related to serum progesterone levels. An increase in 17-beta estradiol (E(2)) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF(2 alp ha) metabolism at the level of gene expression we established a primar y cell culture model of day 15 cycling guinea pig endometrial cells. W e cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxypros taglandin dehydrogenase (PGDH) that converts PGF(2 alpha) to biologica lly inactive 13,14-dihydro-15-keto PGF(2 alpha) (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homol ogy at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelia l and stromal cell subcultures were primed with medium containing eith er E(2) or medroxyprogesterone acetate (MPA) for 24 h. They were then treated For a further 4 or 24 h either withdrawing the steroid, mainta ining the priming steroid, or supplementing with both steroids, before harvesting conditioned media and RNA. Epithelial cells secreted 30-fo ld more PGF(2 alpha) compared with stromal cells (e.g. 7.8 = 0.7 vs. 0 .26 +/- 0.09 pg/ng DNA . 24 h), and PGF(2 alpha) secretion levels were approximately 15-fold higher than those of PGFM (e.g. 7.8 +/- 0.7 vs. 0.45 +/- 0.16 pg/ng DNA . 24 h, for epithelial cells). COX-1 transcri pts were low and unaffected by treatment in both cell types. COX-2 tra nscripts were more abundant in epithelial than stromal cells. Steroid- modulated, COX-2 dependent changes in PGF(2 alpha) secretion were obse rved. The addition of MPA to E(2) primed cells caused a decrease in PG F(2 alpha) secretion and COX-2 messenger RNA levels after 4 h. Convers ely, the addition of E(2) to MPA primed epithelial cells led to an inc rease in PGF(2 alpha) secretion and COX-2 messenger RNA levels after 4 and 24 h. The withdrawal off, caused a fall in PGF(2 alpha) secretion and COX-2 transcripts after 24 h. In contrast, PGDH transcripts were more abundant in stromal than epithelial cells and were up-regulated b y the addition of MPA to E(2) primed cells. These in vitro observation s are in keeping with the secretory profile seen in vivo in the cyclin g guinea pig uterus suggesting that 1) the fall of E(2) and the coinci ding rise in progesterone seen in the early cycle lead to a reduction in PGF(2 alpha) levels; and 2) the rise of E(2) in the late cycle on a progesterone primed uterus is the stimulus for an increase in uterine PGF(2 alpha), production. Our findings suggest a differential role fo r uterine stroma and epithelium in vivo whereby the former acts to rem ove (via PGDH), and the latter to produce (via COX-2) biologically act ive prostaglandin.