Submitted on January 16, 2009
Revised on March 31, 2009
Accepted on April 1, 2009
NF-
B regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-B site in the ICAM-1 gene
Jiaping Xue1, Prabhakar B Thippegowda1, Guochang Hu2, Kurt Bachmaier1, John W. Christman1, Asrar B. Malik3, and Chinnaswamy Tiruppathi1*
1 University of Illinois
2 University of Illinois at Chicago
3 University of Illinois, College of Medicine
* To whom correspondence should be addressed. E-mail: tiruc{at}uic.edu.
Activation of NF-
B 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-B binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-B binding sites in intron-1 (+ 70, NF-B site 1; +611, NF-B site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation (CHIP) results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-B site 1 of the ICAM-1 gene. Electrophoretic mobility shift and super shift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-B site 1 in thrombin stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1385 to +234) construct ~25-fold and mutation of intronic NF-B 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-
-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-B site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.
