Potential role of nuclear factor kappaB and reactive oxygen species in cAMP and cytokine regulation of surfactant protein-A gene expression in lung type II cells.
The human surfactant protein-A2 (hSP-A2) gene is developmentally regulated, expressed in type II pneumonocytes, and induced by cAMP. cAMP induction of hSP-A2 expression is O2 dependent and mediated by increased phosphorylation, DNA binding, and transcriptional activation of thyroid transcription factor-1 (TTF-1). The TTF-1-binding element (TBE) at -175 bp contains a reverse-oriented nuclear factor-kappaB (NF-kappaB) binding site. IL-1 increased SP-A expression in lung type II cells and had additive stimulatory effects with cAMP. Nuclear extracts from cAMP- or IL-1-treated type II cells manifested increased binding to NF-kappaB consensus and TBE probes; cAMP and IL-1 had additive effects. Competitive and antibody supershift EMSA revealed that NF-kappaB and TTF-1 interact with TBE. IL-1 treatment of type II cells caused rapid (1 h) increases in nuclear levels of NF-kappaB (p50 and p65) and in binding to NF-kappaB and TBE probes; nuclear levels of TTF-1 were unaffected. Bt2cAMP increased binding to NF-kappaB and TBE probes more slowly; no changes in nuclear levels of p50, p65, or TTF-1 were evident, suggesting that IL-1 and cAMP act by different mechanisms. A role for endogenous NF-kappaB in cAMP and IL-1 regulation of SP-A was suggested by findings that dominant-negative forms of inhibitor of kappaB reduced binding of type II cell nuclear proteins to TBE and inhibited SP-A expression. In cotransfection assays, NF-kappaB and TTF-1 cooperatively interacted at TBE to stimulate SP-A promoter activity; this was further enhanced by IL-1. In coimmunoprecipitation assays using type II cell nuclear extracts, TTF-1 was found to interact with p65 in vivo. Finally, antioxidant inhibitors of NF-kappaB reduced type II cell nuclear protein binding to TBE and blocked stimulatory effects of cAMP on SP-A expression. This provides intriguing evidence that permissive effects of O2/reactive oxygen species on cAMP regulation of SP-A expression may be mediated by cooperative interactions of TTF-1 and NF-kappaB at the TBE.
Islam
Kazi Nazrul
KN
Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038, USA.
Mendelson
Carole R
CR
D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology*; D008168:Lung / Q000187:drug effects* / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D037662:Pulmonary Surfactant-Associated Protein A / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D013859:Thiocarbamates / Q000494:pharmacology; D000074482:Thyroid Nuclear Factor 1; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism
C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000614412:NKX2-1 protein, human; D009687:Nuclear Proteins; D037662:Pulmonary Surfactant-Associated Protein A; D017382:Reactive Oxygen Species; D013859:Thiocarbamates; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; C066229:prolinedithiocarbamate; D000072000:NF-KappaB Inhibitor alpha; D011392:Proline; D000242:Cyclic AMP