Molecular characterization of bovine prostaglandin G/H synthase-2 and regulation in uterine stromal cells

Prostaglandin G/H synthase (PGHS) is a key rate-limiting enzyme in the pros taglandin biosynthetic pathway, and prostaglandins play a central role in t he control of the reproductive cycle. The objectives of this study were to clone and characterize the primary structure of bovine PGHS-2 and to study its regulation in uterine stromal cells in vitro. The bovine PGHS-2 cDNA wa s cloned by a combination of reverse transcription-polymerase chain reactio n and cDNA library screening. Results showed that the complete bovine PGHS- 2 cDNA is composed of a 5'-untranslated region of 128 bp, an open reading f rame of 1815 bp, and a 3'-untranslated region of 1565 bp containing multipl e repeats (n = 11) of the Shaw-Kamen sequence 5'-ATTTA-3'. The open reading frame encodes a 604-amino acid protein that is 86-97% identical to other m ammalian PGHS-2 homologs. The regulation of PGHS-2 mRNA and protein was stu died in primary cultures of bovine uterine stromal cells stimulated with ph orbol 12-myristate 13-acetate (PMA; 100 nM). Northern and Western blot anal yses reveal a marked induction in PGHS-2 transcript (4.0 kilobases) and pro tein (M-r = 72 000) after 3-12 h of PMA stimulation (P < 0.05). However, th is induction was transient in nature as levels of PGHS-2 mRNA and protein r eturned to basal levels after 24 h of PMA stimulation. In contrast, PMA had no effect on levels of PGHS-1 (P > 0.05). The PMA-dependent induction of P GHS-2 was associated with a significant increase in prostaglandin E-2 secre tion in the culture media (P < 0.05). To study promoter activity of the 5'- flanking DNA region of the bovine PGHS-2 gene, the genomic fragment -1574/ -2 (+1 = transcription start site), as well as a series of 5'-deletion muta nts, were fused upstream of the firefly luciferase gene and transiently tra nsfected into primary cultures of bovine uterine stromal cells. Results sho wed that a first promoter region located between -1574 and -492 and a secon d region between -88 and -39 appear to play important roles in PMA-dependen t regulation of PGHS-2 promoter activity in bovine uterine cells. Thus, thi s study characterizes for the first time the structure of the bovine PGHS-2 transcript and the deduced amino acid sequence of its encoded protein and establishes an in vitro model to study the regulation of PGHS-2 gene expres sion in bovine uterine tissue.