NF-kappaB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-kappaB site in the ICAM-1 gene.
Activation of NF-kappaB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-kappaB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-kappaB binding sites in intron-1 (+70, NF-kappaB site 1; +611, NF-kappaB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-kappaB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-kappaB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1,385 to +234) construct approximately 25-fold and mutation of intronic NF-kappaB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-alpha-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-kappaB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
Jiaping
J
Xue
Prabhakar B
PB
Thippegowda
Guochang
G
Hu
Kurt
K
Bachmaier
John W
JW
Christman
Asrar B
AB
Malik
Chinnaswamy
C
Tiruppathi
D001483:Base Sequence; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics*; D007438:Introns; D016328:NF-kappa B / Q000502:physiology*; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D013917:Thrombin / Q000502:physiology*
D017931:DNA Primers; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D034741:RNA, Small Interfering; D018799:Intercellular Adhesion Molecule-1; D013917:Thrombin