NF-κB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-κB site in the ICAM-1 gene

Xue J, Thippegowda PB, Hu G, Bachmaier K, Christman JW, Malik AB, Tiruppathi C. NF-kappa B regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-kappa B site in the ICAM-1 gene. Physiol Genomics 38: 42-53, 2009. First published April 7, 2009; doi:10.1152/physiolgenomics.00012.2009.-Activation of NF-kappa 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-kappa 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-kappa B binding sites in intron-1 (+ 70, NF-kappa B site 1; +611, NF-kappa B 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-kappa B 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-kappa B site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1,385 to +234) construct similar to 25- fold and mutation of intronic NF-kappa 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-alpha- induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-kappa B site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.