cAMP and tumor necrosis factor competitively regulate transcriptional activation through and nuclear factor binding to the cAMP-responsive element/activating transcription factor element of the endothelial leukocyte adhesion molecule-1 (E-selectin) promoter.
The cAMP-responsive element/activating transcription factor (CRE/ATF) element (also known as NF-ELAM1) of the endothelial leukocyte adhesion molecule-1 (ELAM-1) promoter is necessary for full cytokine responsiveness. It differs from a consensus cAMP-responsive element (CRE) by 1 nucleotide (G-->A conversion) and does not mediate transcriptional activation in response to cAMP. We reported previously that cAMP actually decreases ELAM-1 synthesis induced by tumor necrosis factor (TNF). We now show that cAMP decreases the ELAM-1 promoter response to TNF in transient transfection assays in bovine aortic endothelial cells and that cAMP-mediated inhibition maps to the CRE/ATF element. Electrophoretic mobility shift assays using the ELAM-1 CRE/ATF DNA sequence reveal three complexes. Antibody supershift assays suggest the slowest migrating form (complex 1) contains ATF2, the middle form (complex 2) contains ATF2 and c-Jun, and the fastest migrating form (complex 3) contains a CRE-binding protein. TNF increases c-Jun-containing complex 2 while diminishing complex 1, whereas cAMP decreases complex 2 and increases complex 1. Complex 3 is unchanged by either treatment, and the CRE-binding protein is not phosphorylated. Our data suggest that a change in the composition of the proteins binding to the CRE/ATF promoter element contributes to the competing effects of TNF and cAMP on ELAM-1 gene expression.
De Luca
L G
LG
Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812.
Johnson
D R
DR
Whitley
M Z
MZ
Collins
T
T
Pober
J S
JS
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D001483:Base Sequence; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D019040:E-Selectin; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D004268:DNA-Binding Proteins; D019040:E-Selectin; D014409:Tumor Necrosis Factor-alpha; D000242:Cyclic AMP