Hypoxic modulation of manganese superoxide dismutase promoter activity and gene expression in lung epithelial cells.
Hypoxic modulation of manganese superoxide dismutase promoter activity and gene expression in lung epithelial cells.
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缺氧调节肺上皮细胞中锰超氧化物歧化酶启动子活性和基因表达。
DOI：
10.1165/ajrcmb.21.1.3521
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Jackson,RM
中科院分区：
文献类型：
--
作者：
Ohman,T;Parish,G;Jackson,RM
我们研究了低氧(2.5%O2)对瞬时转染肺上皮细胞(A549、L2和E1a-T2)和成纤维细胞(R9Ab)中大鼠锰超氧化物歧化酶(MnSOD)基因启动子-荧光素酶报告结构的影响。我们克隆了2,505，1,064,507,405和289的MnSOD启动子-荧光素酶报告结构(数字是指转录起始点5‘方向的碱基对长度)：2,505，1,064,507,405和289，克隆到无启动子的萤火虫荧光素酶载体pGL2-Basic中。将含或不含胸苷激酶驱动的肾氨酸氨基转移酶(PRL-TK)的MnSOD启动子-报告基因载体导入肺细胞，分别暴露于空气/5%CO2或低氧(2.5%O2/5%CO2/Balance N_2)24 h后，经双向方差分析发现，A549细胞株(人肺癌)中荧光素酶的表达显著增加。低氧时最大的表达(>；3倍增加)与2,505-bp的MnSOD启动子(归一化为细胞蛋白)有关。叠氮(10μM)不增加报告基因的表达。289个碱基的启动子足以在空气中表达该报告，并在低氧条件下增加其表达。通过构建荧光素酶报告结构，在A549细胞中评估了大鼠MnSOD5‘区域的启动子活性，在低氧条件下增加了启动子活性。这种增加是A549细胞所特有的，在其他细胞中没有出现。
We investigated the effects of hypoxia (< 2.5% O2) on rat manganese superoxide dismutase (MnSOD) gene promoter–luciferase reporter constructs in transiently transfected lung epithelial cells (A549, L2, and E1A-T2) and fibroblasts (R9Ab). We cloned MnSOD promoter–luciferase reporter constructs (numbers refer to length in base pairs [bp] in the 5′ direction from the transcription initiation site): 2,505, 1,064, 507, 405, and 289 into pGL2-Basic, a promoterless, firefly luciferase vector. Lung cells were transfected with MnSOD promoter–reporter constructs with or without thymidine kinase-drivenRenillaluciferase (pRL-TK), and were exposed to air/5% CO2or hypoxia (2.5% O2/5% CO2/balance N2) for 24 h. Hypoxia caused a significant (by two-way analysis of variance) consistent increase in luciferase in the A549 cell (human lung carcinoma) line. Greatest expression (> 3-fold increase) in hypoxia was associated with the 2,505-bp MnSOD promoter (normalized to cellular protein). Azide (10 μM) did not increase expression of the MnSOD reporter constructs. The 289-bp promoter was sufficient to express the reporter in air and to increase its expression in hypoxia. Promoter activity of the rat MnSOD 5′ region, assessed by luciferase reporter constructs in A549 cells, increased in hypoxia. The increase was exclusive to A549 cells and did not occur in other cells.