Negative transcriptional regulation of human colonic smooth muscle cav1.2 channels by p50 and p65 subunits of nuclear Factor-κB

Background & Aims: The expression of Ca(v)1.2 channels in colonic circular smooth muscle cells and the contractility of these cells are suppressed in inflammation. Our aim was to investigate whether the activation of p50 and p65 nuclear factor-kappa B subunits mediates these effects. Methods: Primary cultures of human colonic circular smooth muscle cells and muscle strips were used. Results: The messenger RNA and protein expression of the pore-forming alpha(1c) subunit of Ca(v)1.2 channels decreased time dependently in response to tumor necrosis factor et. This effect was blocked by prior transient transfection of the cells with antisense oligonucleotides to p50 or p65. The overexpression of p50 and p65 inhibited the constitutive expression of alpha(1c). Three putative kappa B binding motifs were identified on the 5' flanking region of exon 1b of the human L-type calcium channel alpha(1c) gene. Progressive 5' deletions of the promoter and point mutations of the kappa B binding motifs indicated that the two 5' binding sites, but not the third 3' binding site, were essential for the suppression of alpha(1c). Transient transfection of human colonic circular muscle strips with antisense oligonucleoticles to p50 and p65 decreased expression of the 2 nuclear factor-kappa B units and reversed the suppression of alpha(1c), as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor alpha. Conclusions: The activation of p50 and p65 by tumor necrosis factor alpha suppresses the expression of the alpha(1c) subunit of Ca(v)1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-kappa B must bind concurrently to the two 5' kappa B motifs on the promoter of alpha(1c) to produce this effect.