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Differential Role of an NF-κB Transcriptional Response Element in Endothelial Versus Intimal Cell VCAM-1 Expression

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Rationale:

Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)–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-κB–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-κB 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-κB 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-κB 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.
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Keywords: NF-κB; atherosclerosis; chromatin immunoprecipitation; endothelial cells; gene expression; tunica intima; vascular cell adhesion molecule-1

Document Type: Research Article

Publication date: July 3, 2015

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