H2O2 and tumor necrosis factor-alpha induce differential binding of the redox-responsive transcription factors AP-1 and NF-kappa B 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 adhesi on molecule (ICAM-1) gene expression in endothelial and epithelial cells. H 2O2 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 umb ilical vein endothelial cells. In contrast, H2O2 induced ICAM-1 only in end othelial 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 us ing nuclear protein extracts from HMEC-1 and A549 cells stimulated with H2O 2 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 TN F alpha activated AP-1 as well as NF-kappa B. In HMEC-1, TNF alpha activate d 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 t heir differential binding to the IL-8 promoter. Moreover, the H2O2 concentr ation dependent increase in epithelial IL-8 mRNA expression directly corres ponded to the H2O2 concentration dependent binding of AP-1 to the IL-8 prom oter. Supershift analysis revealed H2O2 as well as TNF alpha induced AP-1 c omplexes containing c-Fos and JunD. TNF alpha induced NF-kappa B complexes containing Rel A (p65), Immunohistochemical staining of HMEC-1 and A549 cel ls revealed TNFa stimulated nuclear localization of Rel A, whereas no trans location of Rel A was detected in either cell type stimulated by H2O2. Thes e data indicate that the cell type-specific induction of IL-8 gene expressi on by H2O2 and TNF alpha in HMEC-1 and A549 cells can be explained by the d ifferential binding of AP-1 and NF-kappa B to the IL-8 promoter.