Potential Role of Nuclear Factor B and Reactive Oxygen Species in cAMP and Cytokine Regulation of Surfactant Protein-A Gene Expression in Lung Type II Cells

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Abstract
The human surfactant protein-A2 (hSP-A2) gene is developmentally regulated, expressed in type II pneumonocytes, and induced by cAMP. cAMP in- duction of hSP-A2 expression is O2 dependent and mediated by increased phosphorylation, DNA bind- ing, and transcriptional activation of thyroid tran- scription factor-1 (TTF-1). The TTF-1-binding ele- ment (TBE) at 175 bp contains a reverse-oriented nuclear factor-B (NF-B) binding site. IL-1 in- creased SP-A expression in lung type II cells and had additive stimulatory effects with cAMP. Nu- clear extracts from cAMP- or IL-1-treated type II cells manifested increased binding to NF-B con- sensus and TBE probes; cAMP and IL-1 had addi- tive effects. Competitive and antibody supershift EMSA revealed that NF-B and TTF-1 interact with TBE. IL-1 treatment of type II cells caused rapid (1 h) increases in nuclear levels of NF-B (p50 and p65) and in binding to NF-B and TBE probes; nu- clear levels of TTF-1 were unaffected. Bt2cAMP increased binding to NF-B 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 endoge- nous NF-B in cAMP and IL-1 regulation of SP-A was suggested by findings that dominant-negative forms of inhibitor of B reduced binding of type II cell nuclear proteins to TBE and inhibited SP-A expression. In cotransfection assays, NF-B 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 inhib- itors of NF-B 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 ox- ygen species on cAMP regulation of SP-A expres- sion may be mediated by cooperative interactions of TTF-1 and NF-B at the TBE. (Molecular Endo- crinology 16: 1428-1440, 2002)

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