Transcriptional Arrest of the Human E-Selectin Gene

• Published in: The Journal of surgical research 1999-04, Vol.82 (2), p.194-200
• Main Authors: Boyle, Edward M., Sato, Thomas T., Noel, Robert F., Verrier, Edward D., Pohlman, Timothy H.
• Format: Article
• Language: English
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; ATF-2; Base Sequence - genetics; Binding Sites; Biological and medical sciences; Cells, Cultured; Down-Regulation; E-selectin; E-Selectin - genetics; E-Selectin - metabolism; Electrophoresis; Emergency and intensive care: infection, septic shock; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; endothelium-vascular; Gene Expression - physiology; gene-expression-regulation; human; Humans; Intensive care medicine; Medical sciences; NF-kappa B - genetics; NF-kappa B - metabolism; NF-κB; Peptide Fragments - genetics; Peptide Fragments - metabolism; RNA, Messenger - metabolism; TNF-α; Transcription, Genetic - physiology; Tumor Necrosis Factor-alpha - pharmacology; Umbilical Veins - cytology; Umbilical Veins - drug effects
• ISSN: 0022-4804; 1095-8673
• DOI: 10.1006/jsre.1998.5536

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.