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Molecular Characterization of Bovine Prostaglandin G/H Synthase-2 and Regulation in Uterine Stromal Cells¹

^a Centre de recherche en reproduction animale and Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6

ABSTRACT

Prostaglandin G/H synthase (PGHS) is a key rate-limiting enzyme in the prostaglandin biosynthetic pathway, and prostaglandins play a central role in the 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 was cloned by a combination of reverse transcription-polymerase chain reaction 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 frame of 1815 bp, and a 3'-untranslated region of 1565 bp containing multiple 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 mammalian PGHS-2 homologs. The regulation of PGHS-2 mRNA and protein was studied in primary cultures of bovine uterine stromal cells stimulated with phorbol 12-myristate 13-acetate (PMA; 100 nM). Northern and Western blot analyses reveal a marked induction in PGHS-2 transcript (4.0 kilobases) and protein (M_r = 72 000) after 3–12 h of PMA stimulation (P < 0.05). However, this induction was transient in nature as levels of PGHS-2 mRNA and protein returned 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 PGHS-2 was associated with a significant increase in prostaglandin E₂ secretion 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 mutants, were fused upstream of the firefly luciferase gene and transiently transfected into primary cultures of bovine uterine stromal cells. Results showed that a first promoter region located between -1574 and -492 and a second region between -88 and -39 appear to play important roles in PMA-dependent regulation of PGHS-2 promoter activity in bovine uterine cells. Thus, this 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 expression in bovine uterine tissue.

FOOTNOTES

First decision: 20 October 2000.

¹ Supported in part by Canadian Institutes of Health Research (CIHR) Grant MT-13190 (J.S.), Natural Sciences and Engineering Research Council of Canada Grant 8161-98 (A.K.G), and Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR) Grant 99-ER-3016 (J.G.L., D.W.S., and J.S.). J.L. is supported by an FCAR of Québec Doctoral Fellowship, D.B. is supported by a CIHR Doctoral Research Award, and J.S. is supported by a CIHR Investigator Award.

² Correspondence: Jean Sirois, CRRA, Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, Saint-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; siroisje{at}medvet.umontreal.ca

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