Transcriptional arrest of the human E-selectin gene.
BACKGROUND
E-selectin transcription requires binding of transcription factors, NF-kappaB, ATF-2, and HMG-I(Y). Here we characterize the mechanism responsible for the transcriptional downregulation of E-selectin expression.


METHODS
Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. HUVEC E-selectin expression was measured by enzyme-linked immunosorbent assay, Northern blotting, and nuclear run-on assays, and NF-kappa B was assessed by electrophoretic gel mobility shift assays (EMSAs).


RESULTS
(1) E-selectin surface expression peaked at 4 h and then diminished over the next 20 h. (2) Transcription of E-selectin began within 1 h of TNF-alpha exposure and ceased by 8 h, despite continuous stimulation of HUVECs with TNF-alpha. (3) EMSAs revealed persistent binding activity of NF-kappa B proteins to two NF-kappa B-binding sites during 24 h of continuous stimulation with TNF-alpha. However, binding activity of proteins that recognize a third NF-kappa B element, -126 to -116 bp from the transcription start site, was lost after 4 h during 24 h of continuous stimulation with TNF-alpha; ATF-2 binding was unchanged over 24 h stimulation with TNF-alpha.


CONCLUSIONS
The termination of E-selectin expression is controlled at the level of transcription, with loss of protein-DNA interactions at only one of three NF-kappa B-binding sites in the E-selectin promoter.
Boyle
E M
EM
Department of Surgery, The University of Washington, Seattle, Washington, 98104-9796, USA.
Sato
T T
TT
Noel
R F
RF
Verrier
E D
ED
Pohlman
T H
TH
D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites; D002478:Cells, Cultured; D015536:Down-Regulation; D019040:E-Selectin / Q000235:genetics* / Q000378:metabolism; D004586:Electrophoresis; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects
D019040:E-Selectin; D016328:NF-kappa B; D010446:Peptide Fragments; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha