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J Biol Chem, Vol. 273, Issue 16, 10068-10077, April 17, 1998

Tumor Necrosis Factor-- or Lipopolysaccharide-induced Expression of the Murine P-selectin Gene in Endothelial Cells Involves Novel B Sites and a Variant Activating Transcription Factor/cAMP Response Element

From the Departments of Medicine and Biochemistry & Molecular Biology, W. K. Warren Medical Research Institute, University of Oklahoma Health Sciences Center, and Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

Tumor necrosis factor- (TNF-) 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- or LPS to stimulate reporter gene expression. Within these regions, we identified two tandem B elements, a reverse-oriented B site and a variant activating transcription factor/cAMP response element (ATF/CRE), that participate in TNF-- or LPS-induced expression. The tandem B elements bound to NF-B heterodimers and p65 homodimers, the reverse-oriented B 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-- 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 B site-dependent manner. These data indicate that the B sites and the variant ATF/CRE are required for TNF- 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- or LPS stimulates transcription of P-selectin in a species-specific manner.

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