NF-κB activation by oxidative stress and inflammation suppresses contractility in colonic circular smooth muscle cells

Background & Aims: Transcription factor nuclear factor kappaB (NF-kappaB) plays a critical role in transcriptional changes in several diseases, including inflammation. The aim of this study was to investigate whether NF-kappaB is activated by inflammation and oxidative stress in colonic circular smooth muscle cells and whether that leads to suppression of their contractility. Methods: The experiments were performed on freshly dissociated single cells using electrophoretic mobility shift assay, Western immunoblotting, and immunofluorescence imaging. Results: The NF-kappaB DNA binding was similar to6-fold greater in cells from the inflamed colon vs. those from the normal colon. Supershift assay indicated that the antibodies to p65, p50, and c-Rel, but not that to p52, shifted the NF-kappaB band. Western immunoblotting and immunofluorescence imaging also demonstrated the presence of p65, p50, and c-Rel proteins in the cytoplasm and their translocation to the nucleus by H2O2-induced oxidative stress. H2O2 treatment degraded IkappaB(beta), but not IkappaB(alpha), to translocate NF-kappaB to the nucleus. Hydrogen peroxide concentration and time dependently activated NF-kappaB DNA binding and suppressed cell contraction to acetylcholine. NF-kappaB inhibitors significantly inhibited these effects. Inhibition of NF-kappaB prior to and during inflammation in intact dogs also reversed the suppression of contractility. Conclusions: Transcription factor NF-kappaB is activated in colonic circular muscle cells by inflammation and oxidative stress. This activation of NF-kappaB mediates the suppression of cell contractility.