Nitric oxide regulates interleukin-8 gene expression in activated endothelium by inhibiting NF-kappaB binding to DNA: effects on endothelial function.
Nuclear factor-kappaB (NF-kappaB) binds to nucleotide sequences between -80 and -70 bp upstream of the transcriptional start site in the interleukin-8 (IL-8) promoter and is crucial for transcription of the IL-8 gene. We showed that exogenous nitric oxide in the form of a nitric oxide donor significantly reduced IL-8 mRNA in cytokine-activated ECV304. Similarly, nitric oxide significantly reduced migration of polymorphonuclear neutrophils through cytokine-activated ECV304 monolayers, an IL-8-dependent process. Using a luciferase reporter construct containing the NF-kappaB site of the IL-8 gene, we showed that exposing cytokine-activated ECV304 to exogenous nitric oxide resulted in significant reduction of NF-kappaB binding. Follow-up studies using a luciferase reporter construct possessing a mutated NF-kappaB site confirmed that the luciferase activity observed in the NF-kappaB reporter resulted from NF-kappaB binding. These studies demonstrate that nitric oxide, supplied exogenously into reactions containing activated endothelium, down-regulates pro-inflammatory activity, such as the secretion of chemokines, and functional activity, such as transendothelial migration of neutrophils.
Fowler
A A
AA
Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond 23298-0050, USA. afowler@hsc.vcu.edu
Fisher
B J
BJ
Sweeney
L B
LB
Wallace
T J
TJ
Natarajan
R
R
Ghosh
S S
SS
Ghosh
S
S
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004727:Endothelium / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology; D009569:Nitric Oxide / Q000502:physiology*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger; D009569:Nitric Oxide; D004247:DNA