Calcium-mediated oxidative stress: a common mechanism in tight junction disruption by different types of cellular stress

Abstract

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction (TJ) disruption was investigated in Caco-2 cell monolayers <i>in vitro</i> and restraint stress-induced barrier dysfunction in mouse colon <i>in vivo</i> Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca²⁺ by 1,2-bis-(<i>o</i>-aminophenoxy)ethane-<i>N</i>,<i>N</i>,<i>N</i>',<i>N</i>'-tetraacetic acid. Knockdown of Ca_V1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated TJ disruption and barrier dysfunction. <i>N</i>-Acetyl l-cysteine (NAC) and l-<i>N</i>^G-Nitroarginine methyl ester (l-NAME) blocked stress-induced TJ disruption and barrier dysfunction. NAC and l-NAME also blocked stress-induced activation of c-Jun <i>N</i>-terminal kinase (JNK) and c-Src. ROS was colocalized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, TJ disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and TJ disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca²⁺, activation of JNK and c-Src, and disruption of TJ in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, TJ disruption and protein thiol oxidation in colonic mucosa. The present study demonstrates that oxidative stress is a common signal in the mechanism of TJ disruption in the intestinal epithelium by different types of cellular stress <i>in vitro</i> and bio behavioral stress <i>in vivo</i>.