FLANKING SEQUENCES FOR THE HUMAN INTERCELLULAR-ADHESION MOLECULE-1 NF-KAPPA-B RESPONSE ELEMENT ARE NECESSARY FOR TUMOR-NECROSIS-FACTOR ALPHA-INDUCED GENE-EXPRESSION
Lll. Paxton et al., FLANKING SEQUENCES FOR THE HUMAN INTERCELLULAR-ADHESION MOLECULE-1 NF-KAPPA-B RESPONSE ELEMENT ARE NECESSARY FOR TUMOR-NECROSIS-FACTOR ALPHA-INDUCED GENE-EXPRESSION, The Journal of biological chemistry, 272(25), 1997, pp. 15928-15935
The regulated expression of intercellular adhesion molecule-1 (ICAM-1)
by cytokines such as tumor necrosis factor alpha (TNF-alpha) plays an
important role in inflammation and immune responses, Induction of ICA
M-1 gene transcription by TNF-alpha has previously been shown to be de
pendent upon a region of the ICAM-1 5'-flanking sequences that contain
s a modified kappa B site. We demonstrate here that this modified kapp
a B site alone is insufficient. for induction of transcription by TNF-
alpha, Site-directed mutagenesis of both the kappa B site and specific
flanking nucleotides demonstrates that both the specific 5'- and 3'-f
lanking sequences and the modified kappa B site are necessary for TNF-
alpha induction. Further, site-directed mutagenesis of this modified k
appa B site to a consensus kappa B site allows it to mediate transcrip
tional activation in response to TNF-alpha, even in the absence of spe
cific flanking sequences. Transcription through this minimal ICAM-1 TN
F-alpha-responsive region can be driven by coexpression of p65, and th
e minimal response element interacts with p65 and p50 in supershift mo
bility shift assays. However, when in vitro transcription/translation
products for the Rel proteins ape used in an electrophoretic mobility
shift assay, only p65 is capable of binding the minimal response eleme
nt while both p50 and p65 bind a consensus kappa B oligonucleotide, Ad
ditionally, in the absence of the specific flanking nucleotides, the I
CAM-1 kappa B site is incapable of DNA-protein complex formation in ba
th electrophoretic mobility shift assay and UV cross-linking/SDS -poly
acrylamide gel electrophoresis analysis. These results demonstrate the
requirement for specific flanking sequences surrounding a kappa B bin
ding site for functional transcription factor binding and transactivat
ion and TNF-alpha-mediated induction of ICAM-1.