Negative transcriptional regulation of human colonic smooth muscle Ca _v1.2 channels by p50 and p65 subunits of nuclear factor-κB

Background & Aims: The expression of Ca_v1.2 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-κB 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 α_1C subunit of Ca_v1.2 channels decreased time dependently in response to tumor necrosis factor α. 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 α_1C. Three putative κB binding motifs were identified on the 5′ flanking region of exon 1b of the human L-type calcium channel α_1C gene. Progressive 5′ deletions of the promoter and point mutations of the κB binding motifs indicated that the two 5′ binding sites, but not the third 3′ binding site, were essential for the suppression of α_1C. Transient transfection of human colonic circular muscle strips with antisense oligonucleotides to p50 and p65 decreased expression of the 2 nuclear factor-κB units and reversed the suppression of α_1C, as well as that of the contractile response to acetylcholine, by 24 hours of treatment with tumor necrosis factor α. Conclusions: The activation of p50 and p65 by tumor necrosis factor α suppresses the expression of the α_1C subunit of Ca_v1.2 channels in human colonic circular smooth muscle cells and their contractile response to acetylcholine. Nuclear factor-κB must bind concurrently to the two 5′ κB motifs on the promoter of α_1C to produce this effect.