Characterisation of the tumour necrosis factor (TNF)-(alpha) response elements in the human ICAM-2 promoter.
ICAM-2 is a cell surface adhesion molecule constitutively expressed on the endothelium, involved in leukocyte recruitment into tissues. We recently showed that pro-inflammatory cytokines tumour necrosis factor (TNF)-(alpha) and interleukin (IL)-1(beta) down-regulate ICAM-2 expression at the transcriptional level. Here we investigate the elements in the ICAM-2 promoter required for the TNF-(alpha)-mediated down-regulation. Site directed mutagenesis of the ICAM-2 promoter implicated three consensus sites for Ets transcription factors in basal activity; two of these sites were also involved in the TNF-(alpha)-induced down-regulation. Electrophoretic mobility shift assays (EMSA) performed in human umbilical vein endothelial cells (HUVEC) showed that all three Ets binding sites (EBS) bind nuclear proteins. TNF-(alpha) treatment (10 ng/ml for 24 hours) decreased binding to the double -135/-127EBS, but not to the -44EBS. The Ets family member Erg was found to be constitutively expressed in HUVEC, and TNF-(alpha) down-regulated Erg protein levels. Furthermore, an Erg cDNA transactivated the ICAM-2 promoter when transiently transfected into both HeLa cells and HUVEC. Protein expression of ICAM-2 and Erg was found to be similarly regulated by TNF-(alpha) in an ex vivo artery model. These data suggest that constitutive endothelial genes ICAM-2 and Erg are on the same pathway of cytokine-dependent regulation of gene expression.
McLaughlin
F
F
Vascular Diseases Unit, GlaxoWellcome Medicines Research Centre, Stevenage, UK.
Ludbrook
V J
VJ
Kola
I
I
Campbell
C J
CJ
Randi
A M
AM
D015703:Antigens, CD / Q000235:genetics*; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002478:Cells, Cultured; D004247:DNA; D015536:Down-Regulation / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020218:Response Elements; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; C065289:ICAM2 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D004247:DNA