Liu J, Antaya M, Goff A K, Boerboom D, Silversides D W, Lussier J G, Sirois 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.
Biol Reprod. 2001 Mar;64(3):983-91. doi: 10.1095/biolreprod64.3.983.
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.
前列腺素G/H合酶（PGHS）是前列腺素生物合成途径中的关键限速酶，而前列腺素在生殖周期的调控中起着核心作用。本研究的目的是克隆并鉴定牛PGHS-2的一级结构，并在体外研究其在子宫基质细胞中的调控机制。通过逆转录-聚合酶链反应和cDNA文库筛选相结合的方法克隆了牛PGHS-2 cDNA。结果表明，完整的牛PGHS-2 cDNA由128 bp的5'-非翻译区、1815 bp的开放阅读框和1565 bp的3'-非翻译区组成，该3'-非翻译区含有多个Shaw-Kamen序列5'-ATTTA-3'的重复序列（n = 11）。开放阅读框编码一个604个氨基酸的蛋白质，与其他哺乳动物的PGHS-2同源物具有86-97%的同一性。在用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA；100 nM）刺激牛子宫基质细胞的原代培养物中，研究了PGHS-2 mRNA和蛋白质的调控。Northern和Western印迹分析显示，PMA刺激3-12小时后，PGHS-2转录本（4.0千碱基）和蛋白质（M(r)=72 000）有明显诱导（P<0.05）。然而，这种诱导本质上是短暂的，因为PMA刺激24小时后，PGHS-2 mRNA和蛋白质水平恢复到基础水平。相比之下，PMA对PGHS-1水平没有影响（P>0.05）。PGHS-2的PMA依赖性诱导与培养基中前列腺素E2分泌的显著增加有关（P<0.05）。为了研究牛PGHS-2基因5'-侧翼DNA区域的启动子活性，将基因组片段-1574/-2（+1=转录起始位点）以及一系列5'-缺失突变体与萤火虫荧光素酶基因上游融合，并瞬时转染到牛子宫基质细胞的原代培养物中。结果表明，位于-1574和-492之间的第一个启动子区域以及位于-88和-39之间的第二个区域似乎在牛子宫细胞中PGHS-2启动子活性的PMA依赖性调控中起重要作用。因此，本研究首次鉴定了牛PGHS-2转录本的结构及其编码蛋白质的推导氨基酸序列，并建立了一个体外模型来研究牛子宫组织中PGHS-2基因表达的调控。
