title: Hypoxic modulation of manganese superoxide dismutase promoter activity and gene expression in lung epithelial cells.
abstract: 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-driven Renilla luciferase (pRL-TK), and were exposed to air/5% CO2 or 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 microM) 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.
authors.affiliation: Birmingham Department of Veterans Affairs Medical Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: T
authors.initials: T
authors.lastname: Ohman
authors.forename: G
authors.initials: G
authors.lastname: Parish
authors.forename: R M
authors.initials: RM
authors.lastname: Jackson
mesh_terms: D000818:Animals; D015687:Cell Hypoxia; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D004847:Epithelial Cells / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008168:Lung / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic; D051381:Rats; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D014157:Transcription Factors; D013482:Superoxide Dismutase