TNF-alpha induced NFκB signaling and p65 (RelA) overexpression repress Cldn5 promoter in mouse brain endothelial cells

Inflammatory cytokine TNFα enhances permeability of brain capillaries constituting blood brain barrier (BBB). In the monoculture endothelial models of BBB TNFα alters tight junction (TJ) structure and protein content. Claudin-5 (Cldn5) is a key TJ protein whose expression in the brain endothelial cells is critical to the function of BBB. TNFα reduces Cldn5 promoter activity and mRNA expression in mouse brain derived endothelial cells but the regulatory elements and signaling mechanism involved are not defined. Here we report that TNFα acts through NFκB signaling and requires a conserved promoter region for the down-regulation of Cldn5 expression. Overexpression of the NFκB subunit p65 (RelA) alone repressed Cldn5 promoter activity in mouse brain endothelial cells. We observed partial loss of Cldn5 protein expression after prolonged TNFα treatment in primary endothelial culture isolated from C56BL/6 mice brain. Taken together, our results confirm and extend previous observations of TNFα induced down-regulation of Cldn5 expression in mouse brain endothelial cells.

► TNFα downregulates Cldn5 promoter activity.
► Promoter fragment −900/−750, relative to transcription start site of murine Cldn5 gene is functionally important for TNFα dependent downregulation.
► Molecular abrogation of TNFα induced NFκB signaling overrides the suppressive effect of TNFα on Cldn5 promoter.
► NFκB–p65 (RelA) binds to kB-sites in Cldn5 promoter and cotransfection of NFκB–p65 (RelA) expression plasmid with Cldn5 promoter–luciferase construct mimics TNFα induced Cldn5 promoter suppression.
► Suppressive effect of TNFα on Cldn5 expression is observable in primary cerebral endothelial cells.