REGULATION OF CYCLOOXYGENASE GENES IN DEVELOPING LUNG

肺发育中环加氧酶基因的调控

• 批准号: 2231527
• 负责人: PHILIP W SHAUL
• 金额: $ 20.14万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2231527
• 关键词: SDS polyacrylamide gel electrophoresis; embryo /fetus cell /tissue; gene expression; growth /development; hormone regulation /control mechanism; immunocytochemistry; in situ hybridization; lung; messenger RNA; molecular cloning; newborn animals; northern blottings; oxygen tension; oxygenases; physiologic stressor; polymerase chain reaction; prostacyclins; prostaglandin E; protein biosynthesis; protein degradation; radioimmunoassay; sheep; vascular endothelium; vascular smooth muscle; western blottings

Prostacyclin (PGI2) and prostaglandin E2 (PGE2) are critical mediators of vasomotor tone, airway reactivity, and surfactant synthesis in the developing lung. They are produced primarily in vascular cells by cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2. We have demonstrated O2 modulation of PGI2 and PGE2 synthesis in ovine fetal and newborn intrapulmonary arteries (PA), with hypoxia causing decreased synthesis in fetal PA and increased synthesis in newborn PA. We have also demonstrated a 32-fold maturational rise in PGI2 synthesis. Both the O2- and age-associated alterations in synthesis are related to changes in COX protein expression. The OVERALL OBJECTIVE of this proposal is to determine the molecular mechanisms by which COX gene expression in pulmonary vascular cells is modified by changes in O2 and by maturation, focusing the latter studies on modulation by hormonal factors and shear stress. Aim No. 1 is to determine the effects of O2 on COX expression. Fetal and newborn ovine PA endothelial cells (PAEC) and vascular smooth muscle (VSM) cells will be subjected to varying O2 and changes in PGI2 and PGE2 synthesis, and COX-1 and COX-2 protein and mRNA levels will be assessed. COX protein synthesis and degradation, and COX gene transcription and mRNA stability will also be evaluated. We hypothesize that hypoxia downregulates COX expression in fetal cells and upregulates expression in newborn cells. Aim No. 2 is to determine the effects of estrogen and glucocorticoids on COX expression in fetal PAEC and VSM, using a similar approach. Estrogen enhances and glucocorticoids attenuate PGI2 synthesis in other cell types, and estrogen levels rise in the fetus and cortisol levels fall postnatally as PA COX protein increases. As such, we hypothesize that COX expression is upregulated by estrogen and downregulated by glucocorticoids. Aim No. 3 is to determine if COX expression is upregulated by prolonged shear stress, which increases PG2 synthesis in nonpulmonary endothelium. Fetal PAEC will be subjected to 24 h of shear, and changes in COX function, protein and mRNA expression will be assessed. Aim No. 4 is to test the hypothesis that there is cell- specific developmental regulation of COX expression. Immunohistochemical analyses and in situ hybridization for COX-1 and COX-2 will be performed in fetal, newborn and adult ovine lung. By revealing the molecular processes modulating PGI2 and PGE2 synthesis in the developing lung, these studies will increase our understanding of the mechanisms underlying successful pulmonary vascular, airway and alveolar function in the neonatal period.

前列环蛋白（PGI2）和前列腺素E2（PGE2）是关键介体 血管舒张张张，气道反应性和表面活性剂合成 发育肺部。它们主要由 环氧合酶（COX）存在于两个同工型COX-1和COX-2中。我们 已经证明了卵子胎儿中PGI2和PGE2合成的O2调制 和新生儿肺内动脉（PA），缺氧降低 胎儿PA的合成和新生儿PA中的合成增加。我们也有 在PGI2合成中显示出32倍的成熟升高。两个O2- 合成中与年龄相关的变化与Cox的变化有关 蛋白表达。该提议的总体目的是 确定Cox基因表达在的分子机制 肺血管细胞是通过O2的变化和成熟的变化来改变的 将后者的研究集中在荷尔蒙因子和剪切的调制上 压力。目标1是确定O2对COX表达的影响。 胎儿和新生儿椭圆形PA内皮细胞（PAEC）和血管光滑 肌肉（VSM）细胞将受到不同的O2和PGI2的变化。 PGE2合成，Cox-1和Cox-2蛋白和mRNA水平将是 评估。 Cox蛋白质合成和降解和Cox基因 也将评估转录和mRNA稳定性。我们假设 缺氧下调胎儿细胞中的Cox表达并上调 在新生细胞中的表达。目标2是确定 胎儿PAEC和VSM中COX表达的雌激素和糖皮质激素， 使用类似的方法。雌激素增强和糖皮质激素减弱 其他细胞类型中的PGI2合成，胎儿的雌激素水平上升 随着PA COX蛋白的增加，皮质醇水平在产后下降。作为 这样，我们假设Cox表达被雌激素和 被糖皮质激素下调。目标3是确定Cox是否 表达被延长的剪切应力上调，这增加了PG2 非肺内皮的合成。胎儿PAEC将受到 剪切24小时，COX功能，蛋白质和mRNA表达的变化 将被评估。目标第4号是测试有细胞的假设 COX表达的特定发育调节。免疫组织化学 将进行COX-1和COX-2的分析和原位杂交 在胎儿，新生儿和成年卵子肺中。通过揭示分子 在发育中的肺中调节PGI2和PGE2合成的过程， 这些研究将增加我们对机制的理解 潜在的成功肺血管，气道和肺泡功能 在新生儿时期。