The human ICAM-2 promoter is endothelial cell-specific in vitro and in vivo and contains critical Sp1 and GATA binding sites.
The expression of intercellular adhesion molecule 2 (ICAM-2) in adult tissues is restricted to vascular endothelial cells and megakaryocytes. We have previously shown that the endothelial-specific in vivo activity of the human ICAM-2 promoter is contained within a small (0.33-kilobase (kbp)) 5'-flanking region of the gene. Here we describe the in vitro characterization of this region. The ICAM-2 promoter is TATA-less, and transcription in endothelial cells initiates at four sites. Reporter gene expression directed by the promoter was 125-fold greater than vector alone in bovine aortic endothelial cells but less than 2-fold vector alone in non-endothelial (COS) cells, confirming that specificity in vivo was paralleled in vitro. The addition of 2.7 kbp of 5'-flanking region to the 0.33-kbp fragment had no effect on promoter activity or specificity. The mutation of an Sp1 motif centered at base pair -194 or an eight-base pair palindrome at -268 each reduced promoter activity by 70%. Mutation of GATA motifs at -145 and -53 reduced promoter activity by 78 and 61%, respectively. Specific binding of bovine aortic endothelial cells nuclear proteins to the Sp1 and GATA sites was demonstrated by gel shift analysis. Promoter activity in COS cells was transactivated 3-4-fold by overexpression of GATA-2. The results presented here suggest that transcription from the ICAM-2 promoter in endothelial cells is regulated by the interplay of several positive-acting factors and provide the basis for further analysis of endothelial-specific gene expression.
Cowan
P J
PJ
Immunology Research Center, St Vincent's Hospital, Fitzroy 3065, Victoria, Australia.
Tsang
D
D
Pedic
C M
CM
Abbott
L R
LR
Shinkel
T A
TA
d'Apice
A J
AJ
Pearse
M J
MJ
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D004727:Endothelium / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D050989:GATA2 Transcription Factor; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; C494712:Gata2 protein, mouse; C065289:ICAM2 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors