Cloning of Guinea Pig Cyclooxygenase-2 and 15-Hydroxyprostaglandin Dehydrogenase Complementary Deoxyribonucleic Acids: Steroid-Modulated Gene Expression Correlates to Prostaglandin F2  Secretion in Cultured Endometrial Cells

Abstract

Prostaglandin F 2␣ (PGF 2␣ ) 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-␤ estradiol (E 2 ) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF 2␣ metabolism at the level of gene expression we established a primary cell culture model of day 15 cycling guinea pig endometrial cells. We cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxyprostaglandin dehydrogenase (PGDH) that converts PGF 2␣ to biologically inactive 13,14-dihydro-15-keto PGF 2␣ (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homology at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelial and stromal cell subcultures were primed with medium containing either E 2 or medroxyprogesterone acetate (MPA) for 24 h. They were then treated for a further 4 or 24 h either withdrawing the steroid, maintaining the priming steroid, or supplementing with both steroids, before harvesting conditioned media and RNA. Epithelial cells secreted 30-fold more PGF 2␣ compared with stromal cells (e.g. 7.8 Ϯ 0.7 vs. 0.26 Ϯ 0.09 pg/ng DNA•24 h), and PGF 2␣ secretion levels were approximately 15-fold higher than those of PGFM (e.g. 7.8 Ϯ 0.7 vs. 0.45 Ϯ 0.16 pg/ng DNA⅐24 h, for epithelial cells). COX-1 transcripts were low and unaffected by treatment in both cell types. COX-2 transcripts were more abundant in epithelial than stromal cells. Steroid-modulated, COX-2 dependent changes in PGF 2␣ secretion were observed. The addition of MPA to E 2 primed cells caused a decrease in PGF 2␣ secretion and COX-2 messenger RNA levels after 4 h. Conversely, the addition of E 2 to MPA primed epithelial cells led to an increase in PGF 2␣ secretion and COX-2 messenger RNA levels after 4 and 24 h. The withdrawal of E 2 caused a fall in PGF 2␣ secretion and COX-2 transcripts after 24 h. In contrast, PGDH transcripts were more abundant in stromal than epithelial cells and were up-regulated by the addition of MPA to E 2 primed cells. These in vitro observations are in keeping with the secretory profile seen in vivo in the cycling guinea pig uterus suggesting that 1) the fall of E 2 and the coinciding rise in progesterone seen in the early cycle lead to a reduction in PGF 2␣ 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␣ production. Our findings suggest a differential role for uterine stroma and epithelium in vivo whereby the former acts to remove (via PGDH), and the latter to produce (via COX-2) biologically active prostaglandin. (Endocrinology 138: [237][238][239][240][241][242][243][244][245][246][247] 1997)

Induction of Cyclo-oxygenase-2 Expression in Naturally Occurring Gastric Ulcers

“…These domains include a putative transmembrane region, heme-coordinating histidines 295 and 374, the cyclooxygenase active-site tyrosine 371, the aspirin acetylation-site serine 516, and four putative N-linked glycosylation sites (DeWitt et al 1990;Shimokawa and Smith 1991;Lecomte et al 1994;Wennogle et al 1995). The presence of multiple repeats of the ShawKamen's sequence (5Ј-ATTTA-3Ј) in the 3Ј-untranslated region in the porcine COX-2 transcript is also a feature reported for COX-2 in several other species (Kujubu et al 1991;Hla and Neilson 1992;Feng et al 1993;Bracken et al 1997;Zhang et al 1996;Guan et al 1997;Boerboom and Sirois 1998;Song et al 1998;Liu et al 2001). This motif has previously been shown to be present in several immediate early genes and to confer instability to mRNAs (Caput et al 1986;Shaw and Kamen 1986).…”

“…Comparative analysis showed that the amino acid sequence of porcine COX-2 is very similar to that of other mammalian homologues, being 89%, 88%, 87%, 90%, 91%, 91%, 90%, 88%, 87% to human, rat, mouse, horse, cow, ovine, rabbit, guinea pig, and mink COX-2, respectively (Kujubu et al 1991;Hla and Neilson 1992;Feng et al 1993;Zhang et al 1996;Bracken et al 1997;Guan et al 1997;Boerboom and Sirois 1998;Song et al 1998;Liu et al 2001). The porcine enzyme includes all known structural and functional domains involved in COX-2 activity.…”

Specific trans-acting proteins interact with auxiliary RNA polyadenylation elements in the COX-2 3′-UTR

“…It is now evident that the stability of the mRNA plays one role in the regulation of COX-2 (Ristimaki et al 1996;Gou et al 1998;Cok and Morrison 2001;Dixon et al 2000Dixon et al , 2001Dixon et al , 2003. In addition, alternative COX-2 transcripts differing only in their 39-UTRs have been found in a number of cell and tissue types (Ristimaki et al 1996;Bracken et al 1997;Lukiw and Bazan 1997;Newton et al 1997Newton et al , 1998Dixon et al 2001;Schmidt et al 2003). Therefore, RNA stability and alternative 39-end processing are two important regulatory mechanisms that can contribute to the expression control of COX-2.…”

Cloning of equine prostaglandin dehydrogenase and its gonadotropin-dependent regulation in theca and mural granulosa cells of equine preovulatory follicles during the ovulatory process

“…PGDH cDNA has been cloned from several species and organs, including human placenta, mouse lungs, rat intestine, and bovine and porcine uterus (Ensor et al 1990, Matsuo et al 1996, Bracken et al 1997, Zhang et al 1997, Parent et al 2006. Human PGDH encodes a protein of 266 amino acids with an apparent molecular weight of 29 kDa (Ensor et al 1990).…”