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.