Activation of NF-kappaB induced by H(2)O(2) and TNF-alpha and its effects on ICAM-1 expression in endothelial cells

Am J Physiol Lung Cell Mol Physiol. 2000 Aug;279(2):L302-11. doi: 10.1152/ajplung.2000.279.2.L302.

Abstract

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-kappaB in response to tumor necrosis factor (TNF)-alpha challenge. In the present study, we investigated the effects of H(2)O(2) and TNF-alpha in mediating activation of NF-kappaB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-alpha exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H(2)O(2) added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H(2)O(2) also failed to induce NF-kappaB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-alpha increased the NF-kappaB-driven gene expression approximately 10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H(2)O(2)- and TNF-alpha-induced NF-kappaB complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-alpha exposure induced marked phosphorylation of NF-kappaB p65 in HMEC-1 cells, whereas H(2)O(2) had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-kappaB to nuclear DNA, whereas TNF-alpha generates additional signals that induce phosphorylation of the bound NF-kappaB p65 and confer transcriptional competency to NF-kappaB.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding, Competitive / genetics
  • Cells, Cultured
  • DNA / metabolism
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism*
  • Gene Expression / drug effects
  • Humans
  • Hydrogen Peroxide / pharmacology*
  • Intercellular Adhesion Molecule-1 / biosynthesis*
  • Intercellular Adhesion Molecule-1 / genetics
  • Intracellular Fluid / drug effects
  • Intracellular Fluid / metabolism
  • NF-kappa B / metabolism*
  • Phosphorylation / drug effects
  • Promoter Regions, Genetic / drug effects
  • RNA, Messenger / biosynthesis
  • Reactive Oxygen Species / metabolism
  • Signal Transduction / drug effects
  • Transcription Factor RelA
  • Transcriptional Activation / drug effects
  • Tumor Necrosis Factor-alpha / pharmacology*

Substances

  • NF-kappa B
  • RNA, Messenger
  • Reactive Oxygen Species
  • Transcription Factor RelA
  • Tumor Necrosis Factor-alpha
  • Intercellular Adhesion Molecule-1
  • DNA
  • Hydrogen Peroxide