H2O2 and tumor necrosis factor-alpha induce differential binding of the redox-responsive transcription factors AP-1 and NF-kappaB to the interleukin-8 promoter in endothelial and epithelial cells.
We previously demonstrated that tumor necrosis factor-alpha (TNFalpha) 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 TNFalpha induced IL-8 and ICAM-1 in both cell types. In this study, we examine the role of the redox-responsive transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB) in the differential expression of IL-8. DNA binding studies using nuclear protein extracts from HMEC-1 and A549 cells stimulated with H2O2 or TNFalpha demonstrated differential activation and promoter binding of AP-1 and NF-kappaB. H2O2 activated AP-1 but not NF-kappaB in A549, whereas TNFalpha activated AP-1 as well as NF-kappaB. In HMEC-1, TNFalpha activated NF-kappaB 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 TNFalpha induced AP-1 complexes containing c-Fos and JunD. TNFalpha induced NF-kappaB complexes containing Rel A (p65). Immunohistochemical staining of HMEC-1 and A549 cells revealed TNFalpha 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 TNFalpha in HMEC-1 and A549 cells can be explained by the differential binding of AP-1 and NF-kappaB to the IL-8 promoter.
Lakshminarayanan
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Department of Immunology/Microbiology, Rush-Presbyterian, St. Luke's Medical Center, Chicago, Illinois 60612, USA.
Drab-Weiss
E A
EA
Roebuck
K A
KA
D001483:Base Sequence; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D016209:Interleukin-8 / Q000235:genetics*; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D017931:DNA Primers; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; D013438:Sulfhydryl Compounds; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D006861:Hydrogen Peroxide