Negative transcriptional regulation of the VCAM-1 gene by fluid shear stress in murine endothelial cells

To explore the mechanism of shear stress-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1) expression in murine endothelial cells (ECs), we examined the effect of shear stress on VCAM-1 gene transcription and assessed the cis-acting elements involved in this phenomenon. VCAM-1 mRNA expression was downregulated at the transcriptional level as defined by nuclear run-on assay and transient transfection of VCAM-1 promoter-luciferase gene constructs. The luciferase assay on the VCAM-1 deletion mutants revealed that the cis-acting element is contained between -694 and -329 bp upstream from the transcription initiation site. Gel shift assay using overlapping oligonucleotide probes of this region showed that oligonucleotides containing a double AP-1 consensus sequence (TGACTCA) formed distinct complexes with nuclear proteins extracted from shear-stressed cells. Mutation of either one or both of two AP-1 consensus sequences completely abolished the ability of the promoter to respond to shear stress. These results suggest that fluid shear stress downregulates the transcription of the VCAM-1 gene via an upstream cis-element, a double AP-1 consensus sequence, in murine lymph node venule ECs.