Expression of the α-subunit of the amiloride-sensitive sodium channel (αENaC) 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 κB (NF-κB) and transcriptional activity of 5′-flanking regions of the human αENaC gene using luciferase reporter-gene vectors transiently transfected into human adult alveolar carcinoma A549 cells. By Western blotting we confirmed the presence of NF-κB p65 but not p50 in these cells. Transiently increasing Po2 from 23 to 42mmHg for 24h evoked a significant increase in NF-κB DNA-binding activity and transactivation of a NF-κB-driven luciferase construct (pGLNF-κBpro), which was blocked by the NF-κB activation inhibitor sulphasalazine (5mM). Transcriptional activity of αENaC-luciferase constructs containing 5′-flanking sequences (including the NF-κB consensus) were increased by raising Po2 from 23 to 142mm Hg 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-κB 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-κB consensus sequence was suppressed significantly by raising Po2. This effect was reversed by sulphasalazine, suggesting that activation of NF-κB mediated Po2-evoked suppression of transcription from the exon 1A TIS of αENaC.
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June 2002
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Research Article|
June 01 2002
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
Deborah L. BAINES;
Deborah L. BAINES
1
∗St. George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 0RE, U.K.
1To whom correspondence should be addressed (e-mail d.baines@sghms.ac.uk).
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Mandy JANES;
Mandy JANES
†S.W. Thames Institute for Renal Research, St. Helier Hospital, Carshalton, Surrey SM5 1AA, U.K.
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David J. NEWMAN;
David J. NEWMAN
†S.W. Thames Institute for Renal Research, St. Helier Hospital, Carshalton, Surrey SM5 1AA, U.K.
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Oliver G. BEST
Oliver G. BEST
‡Tayside Institute of Child Health, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, U.K.
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Publisher: Portland Press Ltd
Received:
November 12 2001
Revision Received:
January 31 2002
Accepted:
March 27 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 364 (2): 537–545.
Article history
Received:
November 12 2001
Revision Received:
January 31 2002
Accepted:
March 27 2002
Citation
Deborah L. BAINES, Mandy JANES, David J. NEWMAN, Oliver G. BEST; 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. Biochem J 1 June 2002; 364 (2): 537–545. doi: https://doi.org/10.1042/bj20011651
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