Differential Role of a NF-kappaB Transcriptional Response Element in Endothelial versus Intimal Cell VCAM-1 Expression
Rationale: Human and murine Vcam1 promoters contain 2 adjacent 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 three 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 (LPS) 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/LDL receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished LPS-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 VCAM-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.
- intimal cells
- chromatin imuunoprecipitation
- endothelial cell
- nuclear factor-kappa B
- gene expression
- arterial injury
- Received April 14, 2015.
- Revision received May 20, 2015.
- Accepted May 21, 2015.