Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes
Hypoxic regulation of inducible nitric oxide synthase via hypoxia inducible factor-1 in cardiac myocytes
复制标题
DOI：
10.1161/01.res.86.3.319
复制
发表时间：
2000-02-18
影响因子：
20.1
通讯作者：
Johns, RA
中科院分区：
文献类型：
--
作者：
Jung, F;Palmer, LA;Johns, RA
缺氧与心肌组织中一氧化氮合酶（NOS）的调节之间的关系尚不清楚。本实验观察了缺氧诱导因子-1（hypoxia inducible factor-1，HIF-1）对大鼠心肌细胞诱导型一氧化氮合酶（inducible NOS，iNOS）表达的影响。对来自这些动物心脏和来自体外暴露于缺氧12小时的细胞的RNA的北方分析表明HIF-1 RNA表达增加。使用含有iNOS HIF-1 DNA结合位点的寡核苷酸和心肌细胞核提取物的电泳迁移率变化分析显示，在经历缺氧的细胞中诱导特异性DNA结合，使用鼠iNOS启动子瞬时转染心肌细胞导致缺氧下启动子活性比常氧下增加3.43倍，HIF-1位点的突变或缺失消除了缺氧反应。由于细胞因子已显示调节心肌细胞中的iNOS表达，用白细胞介素-1 β刺激培养的新生心肌细胞，在常氧条件下引起iNOS蛋白表达的显著诱导，在缺氧条件下进一步增强。用白细胞介素-1 β刺激的细胞的瞬时转染显示与未刺激的细胞相比，在常氧条件下iNOS启动子活性增加，进一步增加对缺氧的反应，这依赖于HIF-1。这些结果表明，缺氧引起心肌细胞中iNOS表达的增加，并且HIF-1对于iNOS基因表达的缺氧调节是必需的。
The relationship between hypoxia and regulation of nitric oxide synthase (NOS) in myocardial tissue is not well understood. We investigated the role of hypoxia inducible factor-1 (HIF-1) on expression of the inducible NOS (iNOS) in myocardial cells in vivo and in vitro, In situ hybridization in myocardial tissue from rats exposed to hypoxia for 3 weeks demonstrated increased iNOS mRNA expression, Northern analysis of RNA from hearts of those animals and from cells exposed to hypoxia for 12 hours in vitro demonstrated an increase of HIF-1 RNA expression. Electrophoretic mobility shift assays using oligonucleotides containing the iNOS HIF-1 DNA binding site and nuclear extracts from cardiac myocytes showed induction of specific DNA binding in cells subjected to hypoxia, Transient transfection of cardiac myocytes using the murine iNOS promoter resulted in a 3.43-fold increase in promoter activity under hypoxia compared with normoxia, Mutation or deletion of the HIF-1 site eliminated the hypoxic response. As cytokines have been shown to regulate iNOS expression in myocardial cells, cultured neonatal cardiac myocytes were stimulated with interleukin-1 beta causing a dramatic induction of iNOS protein expression under normoxia, with further augmentation under hypoxia, Transient transfection of cells stimulated with interleukin-1 beta showed an increased iNOS promoter activity under normoxic conditions compared with unstimulated cells, with a further increase in response to hypoxia, which was dependent on HIF-1. These results demonstrate that hypoxia causes an increase in iNOS expression in cardiac myocytes and that HIF-1 is essential for the hypoxic regulation of iNOS gene expression.