H2O2 and tumor necrosis factor-α induce differential binding of the redox-responsive transcription factors AP-1 and NF-κB to the interleukin-8 promoter in endothelial and epithelial cells

We previously demonstrated that tumor necrosis factor-alpha (TNF alpha) and H2O2 differentially regulate interleukin-8 (IL-8) and intercellular adhesion molecule (ICAM-1) gene expression in endothelial and epithelial cells. H2O2 induced IL-8 expression in the A549 and BEAS-2B epithelial cell lines, but not in the human microvessel endothelial cell line, HMEC-1 or human umbilical vein endothelial cells. In contrast, H2O2 induced ICAM-1 only in endothelial cells, Unlike H2O2, the proinflammatory cytokine TNF alpha induced IL-8 and ICAM-1 in both cell types. In this study, me examine the role of the redox-responsive transcription factors AP-1 and nuclear factor-kappa B (NF-kappa B) in the differential expression of IL-8, DNA binding studies using nuclear protein extracts from HMEC-1 and A549 cells stimulated with H2O2 or TNFa demonstrated differential activation and promoter binding of AP-1 and NF-kappa B. H2O2 activated AP-1 but not NF-kappa B in A549, whereas TNF alpha activated AP-1 as well as NF-kappa B. In HMEC-1, TNF alpha activated NF-kappa B but not AP-1, while H2O2 did not activate either transcription factor. The differential activation of the factors was also reflected in their differential binding to the IL-8 promoter. Moreover, the H2O2 concentration dependent increase in epithelial IL-8 mRNA expression directly corresponded to the H2O2 concentration dependent binding of AP-1 to the IL-8 promoter. Supershift analysis revealed H2O2 as well as TNF alpha induced AP-1 complexes containing c-Fos and JunD. TNF alpha induced NF-kappa B complexes containing Rel A (p65), Immunohistochemical staining of HMEC-1 and A549 cells revealed TNFa stimulated nuclear localization of Rel A, whereas no translocation of Rel A was detected in either cell type stimulated by H2O2. These data indicate that the cell type-specific induction of IL-8 gene expression by H2O2 and TNF alpha in HMEC-1 and A549 cells can be explained by the differential binding of AP-1 and NF-kappa B to the IL-8 promoter.