Oxygen-evoked changes in transcriptional activity of the 5′-flanking region of the human amiloride-sensitive sodium channel (αENaC) gene: role of nuclear factor κB

D. L. Baines, M. Janes, D. J. Newman, Oliver G. Best

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

Expression of the alpha-subunit of the amiloride-sensitive sodium channel (alphaENaC) is regulated by a number of factors in the lung, including oxygen partial pressure (PO2). As transcriptional activation is a mechanism for raising cellular mRNA levels, we investigated the effect of physiological changes in PO2 on the activity of the redox-sensitive transcription factor nuclear factor kappaB (NF-kappaB) and transcriptional activity of 5'-flanking regions of the human alphaENaC gene using luciferase reporter-gene vectors transiently transfected into human adult alveolar carcinoma A549 cells. By Western blotting we confirmed the presence of NF-kappaB p65 but not p50 in these cells. Transiently increasing PO2 from 23 to 42 mmHg for 24 h evoked a significant increase in NF-kappaB DNA-binding activity and transactivation of a NF-kappaB-driven luciferase construct (pGLNF-kappaBpro), which was blocked by the NF-kappaB activation inhibitor sulphasalazine (5 mM). Transcriptional activity of alphaENaC-luciferase constructs containing 5'-flanking sequences (including the NF-kappaB consensus) were increased by raising PO2 from 23 to 142 mmHg if they contained transcriptional initiation sites (TIS) for exons 1A and 1B (pGL3E2.2) or the 3' TIS of exon 1B alone (pGL3E0.8). Sulphasalazine had no significant effect on the activity of these constructs, suggesting that the PO2-evoked rise in activity was not a direct consequence of NF-kappaB activation. Conversely, the relative luciferase activity of a construct that lacked the 3' TIS, a 3' intron and splice site but still retained the 5' TIS and NF-kappaB consensus sequence was suppressed significantly by raising PO2. This effect was reversed by sulphasalazine, suggesting that activation of NF-kappaB mediated PO2-evoked suppression of transcription from the exon 1A TIS of alphaENaC.
Original languageEnglish
Pages (from-to)537-545
Number of pages9
JournalThe Biochemical Journal
Volume364
Issue number2
DOIs
Publication statusPublished - Jun 2002
Externally publishedYes

Keywords

  • Base Sequence Blotting, Western Cell Line DNA Primers Electrophoretic Mobility Shift Assay Epithelial Sodium Channels Humans Luciferases/genetics NF-kappa B/*physiology Oxygen/*physiology Promoter Regions, Genetic RNA, Messenger/genetics/metabolism Reverse Transcriptase Polymerase Chain Reaction Sodium Channels/*genetics Transcription, Genetic/*physiology

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