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Transcriptional arrest of the human E-selectin gene
E M Boyle1, T T Sato, R F Noel
1Department of Surgery, The University of Washington, Seattle, Washington, 98104-9796, USA.
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.
Materials And 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.
Conclusion:
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.