Lu De-qin, Li Hui-ge, Ye Shi-qiao, Ye Hong, Jin Si, Wang Di-xun
Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Yi Xue Za Zhi. 2004 Jan 17;84(2):146-51.
To explore the regulation of eNOS gene expression in pulmonary arterial endothelial cells (PAECs) by protein kinase C (PKC) and its isoforms during hypoxia.
Primary cultured porcine PAECs were exposed to 5%O(2) for 2, 6, 12, 24, 48 hours. The eNOS mRNA level was measured by RT-PCR. Western blot technology was used to detect the contents of eNOS protein and the 8 PKC isoforms. After addition of selective PKC inhibitors, bisindolylmaleimideI (BIMI, 1 micro mol/L) or Gö6983 (1 micro mol/L), PAECs were exposed to 5%O(2) for 24 hours, then the expression of eNOS mRNA was detected by RT-PCR. Promoter activity of eNOS gene was determined by luciferase reporter gene assay. PAECs were transfected transiently with 1.6 kb fragment of the human eNOS promoter driving a luciferaes reporter gene, then exposed to 5%O(2). 24 h later, the activity of luciferase and beta-galactosidase was examined and the relative luciferase activity, representing the eNOS promotor activity, was calculated. After addition of actinomycine D (5 micro g/ml) and exposure to 5%O(2) or normoxia for 6, 12, 24 hours, and eNOS mRNA in PAECs was measured by RT-PCR.
After exposed to hypoxia for 24 hours, the expression of eNOS mRNA and protein level increased by 171% +/- 18% (P < 0.05) and 166% +/- 21% (P < 0.01) respectively. These up-regulation effects were prevented by BIM I and Gö6983. Further experiments showed that among 8 isoforms of PKC detected in this study, only nPKCepsilon protein expression was changed in PAECs after exposure to hypoxia for 24 h. After exposure to hypoxia nPKCepsilon was translocated from cytosol to cell membrane, showing the activation of nPKCepsilon during hypoxia. Reporter gene assay showed that hypoxia enhanced eNOS promoter activity up to 2.3 +/- 0.7 fold. In addition, hypoxia did not change the stability of eNOS mRNA.
Hypoxia may up-regulate eNOS expression in PAECs by transcriptional mechanism through nPKCepsilon signaling pathway. Higher levels of mRNA observed during hypoxia are due to increased transcription, not to increased stability of mRNA.
探讨缺氧时蛋白激酶C（PKC）及其亚型对肺动脉内皮细胞（PAECs）中eNOS基因表达的调控作用。
将原代培养的猪PAECs置于5%O₂环境中2、6、12、24、48小时。采用RT-PCR检测eNOS mRNA水平。运用蛋白质印迹技术检测eNOS蛋白及8种PKC亚型的含量。加入选择性PKC抑制剂双吲哚马来酰亚胺I（BIMI，1 μmol/L）或Gö6983（1 μmol/L）后，将PAECs置于5%O₂环境中24小时，然后用RT-PCR检测eNOS mRNA的表达。通过荧光素酶报告基因检测法测定eNOS基因的启动子活性。将携带荧光素酶报告基因的人eNOS启动子1.6 kb片段瞬时转染PAECs，然后置于5%O₂环境中。24小时后，检测荧光素酶和β-半乳糖苷酶的活性，并计算代表eNOS启动子活性的相对荧光素酶活性。加入放线菌素D（5 μg/ml）后，将PAECs置于5%O₂或常氧环境中6、12、24小时，用RT-PCR检测PAECs中eNOS mRNA的含量。
缺氧24小时后，eNOS mRNA表达和蛋白水平分别升高171%±18%（P<0.05）和166%±21%（P<0.01）。BIM I和Gö6983可抑制这些上调作用。进一步实验表明，在本研究检测的8种PKC亚型中，缺氧24小时后PAECs中仅nPKCepsilon蛋白表达发生变化。缺氧后nPKCepsilon从胞质转位至细胞膜，表明缺氧时nPKCepsilon被激活。报告基因检测显示，缺氧使eNOS启动子活性增强至2.3±0.7倍。此外，缺氧未改变eNOS mRNA的稳定性。
缺氧可能通过nPKCepsilon信号通路以转录机制上调PAECs中eNOS的表达。缺氧时观察到的较高mRNA水平是由于转录增加，而非mRNA稳定性增加。
