Differential Role Of An Nf-Kappa B Transcriptional Response Element In Endothelial Versus Intimal Cell Vcam-1 Expression

Rationale: Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-B (NF-B)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown.Objective: Determine the role of the 5 NF-B-binding element in expression of the endogenous Vcam1 gene.Methods and Results: Homologous recombination in embryonic stem cells was used to inactivate the 5 NF-B element in the Vcam1 promoter and alter 3 nucleotides in the 5 untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-B p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions.Conclusions: This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-B transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.