Abstract
We investigated the mechanisms by which H2O2 increases intercellular adhesion molecule 1 (ICAM-1; CD54) expression in endothelial cells. The H2O2-induced increase in ICAM-1 mRNA was inhibited by actinomycin D, by the antioxidant N-acetylcysteine, and by 3-amino-benzamide (which blocks oxidant-induced AP-1 activity), but not by pyrrolidine dithiocarbamate (which blocks oxidant-induced NF-kappa B activity). Nuclear run-on and transient transfections of ICAM-1 promoter constructs indicated that H2O2 stimulated ICAM-1 gene transcription by activation of a distinct region of the ICAM-1 promoter. The H2O2-responsive element was localized to sequences between -981 and -769 (relative to start codon). Located within this region are two 16-base pair repeats, each containing binding sites for the transcription factors AP-1 and Ets. A similar composite AP-1/Ets element isolated from the macrophage scavenger receptor gene conferred H2O2 responsiveness to a minimal promoter. Mutation of the 16-base pair repeats within the ICAM-1 promoter prevented H2O2-induced DNA binding activity, and their deletion abrogated the H2O2-induced transcriptional activity. In contrast, TNF alpha induced ICAM-1 transcription via activation of promoter sequences between -393 and -176, a region with C/EBP and NF-kappa B binding sites. The results indicate that H2O2 activates ICAM-1 transcription through AP-1/Ets elements within the ICAM-1 promoter, which are distinct from NF-kappa B-mediated ICAM-1 expression induced by TNF alpha.
Highlights
In human umbilical vein endothelial cells (HUVEC), we found that oxidant-induced ICAM-1 expression was associated with increased ICAM-1 mRNA levels occurring 1 h after H 20 2 exposure (Lo et al, 1993)
The AP-1/Ets elements responded to TNFa, the TNFa-induced ICAM-1 expression was mediated by promoter sequences between 393 and 176 bp upstream of the gene, containing binding sites for C/EBP and NF-KB
H202 Ind uces de N ovo mRNA Synth esis-We have previou sl y reported that exposure of human umbilical ve in e nd ot helial cells (HUVEC ) to 100 J.l.M H zO z for 1 h r esulted in maximal a ccu m u la t ion of s t e a dy-s t a te ICMl-1 message, which could be d e t e ct e d a s early a s 30 min after oxidant ex pos u re (Lo et al ., 19 9 3 )
Summary
Materials-Diethylpyrocarbonate, DMEM, heparin, HEPES, 3.0% H 20 2, MOPS, PMSF, spermidine, spermine, pyrrolidine dithiocarbamate (PDTC), and N-acetylcysteine The cells were refed with fresh medium containing 10% fetal calf serum 4 h before lipofection (Malone et al, 1989) with LipofectAMINE as described by Life Technologies, Inc. Transfection of 100-mm plates at 80% confluency typically contained 8 iLg of reporter plasmid (ICAM-1 LUC) and 2 iLg of f3-gal expression plasmid DNA. The cell pellet was washed once with ice-cold PBS and once with RSB buffer Nuclea r protei n pellets were res us pended in 200 ul of nu clea r dia lysis bu ffer (20 mxi HEPES, pH 7.9,20% glycerol, 100 mxt KCl, 0.2 mxi EDTA, 2.0 mxt DTT, a nd 0.1 rnxt PMSF ) a nd dialyzed twice for 90 min agai nst 200 ml ofNDB in th e cold.
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