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 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.
Liu
J
J
Centre de recherche en reproduction animale and Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada J2S 7C6.
Antaya
M
M
Goff
A K
AK
Boerboom
D
D
Silversides
D W
DW
Lussier
J G
JG
Sirois
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D051546:Cyclooxygenase 2; D018076:DNA, Complementary / Q000235:genetics; D015232:Dinoprostone / Q000096:biosynthesis; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011863:Radioimmunoassay / Q000662:veterinary; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017386:Sequence Homology, Amino Acid; D017154:Stromal Cells / Q000201:enzymology / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection / Q000662:veterinary; D014599:Uterus / Q000166:cytology / Q000201:enzymology* / Q000502:physiology
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate