Tumor necrosis factor-α- or lipopolysaccharide-induced expression of the murine P-selectin gene in endothelial cells involves novel κB sites and a variant activating transcription factor/cAMP response element

Abstract

Tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) increases expression of the P-selectin gene in murine, but not in human, endothelial cells. These mediators augment expression of a reporter gene driven by the murine, but not the human, P-selectin promoter in transfected endothelial cells. The regions from -593 to -474 and from -229 to -13 in the murine P- selectin promoter are required for TNF-α or LPS to stimulate reporter gene expression. Within these regions, we identified two tandem κB elements, a reverse-oriented κB site and a variant activating transcription factor/cAMP response element (ATF/CRE), that participate in TNF-α-or LPS-induced expression. The tandem κB elements bound to NF-κB heterodimers and p65 homodimers, the reverse-oriented κB site bound to p65 homodimers, and the variant ATF/CRE bound to nuclear proteins that included activating transcription factor-2. Mutations in each individual element eliminated binding to nuclear proteins and decreased by 20-60% the TNF-α- or LPS- induced expression of a reporter gene driven by the murine P-selectin promoter in transfected endothelial cells. Simultaneous mutations of all elements further decreased, but did not abolish, induced expression. Co- overexpression of p50 and p65 enhanced murine P-selectin promoter activity in a κB site-dependent manner. These data indicate that the κB sites and the variant ATF/CRE are required for TNF-α or LPS to optimally induce expression of the murine P-selectin gene. The presence of these elements in the murine, but not the human, P-selectin gene may explain in part why TNF-α or LPS stimulates transcription of P-selectin in a species-specific manner.