Transcriptional Arrest of the Human E-Selectin Gene

Abstract

Background.E-selectin transcription requires binding of transcription factors, NF-κB, 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-α for 24 h. HUVEC E-selectin expression was measured by enzyme-linked immunosorbent assay, Northern blotting, and nuclear run-on assays, and NF-κB 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-α exposure and ceased by 8 h, despite continuous stimulation of HUVECs with TNF-α. (3) EMSAs revealed persistent binding activity of NF-κB proteins to two NF-κB-binding sites during 24 h of continuous stimulation with TNF-α. However, binding activity of proteins that recognize a third NF-κB element, −126 to −116 bp from the transcription start site, was lost after 4 h during 24 h of continuous stimulation with TNF-α; ATF-2 binding was unchanged over 24 h stimulation with TNF-α.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-κB-binding sites in the E-selectin promoter.