Ca2+‐Induced Mitochondrial Oxidative Stress Mediates Disruption of Intestinal Epithelial Tight Junction: Activation of a Similar Signaling Cascade in Colon by Chronic Restraint Stress
Authors: Meena Avtar S. , Gangwar Ruchika , Shukla Pradeep K , Manda Bhargavi , Pallikkuth Sandeep , Rao Radhakrishna (RK) 
Published: November 2023

BackgroundIntestinal epithelial tight junctions (TJ) plays a crucial role in establishing the mucosal barrier function, by which it prevents diffusion of toxins, allergens and pathogens into intestinal tissue and systemic circulation. Our previous studies showed that osmotic stress (OS), dextran sulfate sodium (DSS) and cyclic stretch disrupt intestinal epithelial TJ and induce barrier dysfunction by a common mechanism involving Ca2+ channels, Ca2+ influx and Ask1/MAPKK/JNK/c‐Src signaling cascade. Oxidative stress is well known to disrupt intestinal epithelial TJ. IN the present study we investigated the role of mitochondrial oxidative stress in OS and DSS‐induced TJ disruption in Caco‐2 cell monolayers in vitro and activation of a similar signaling cascade in mouse colon by chronic restraint stress (CRS) in vivo.MethodsCaco‐2 cell monolayers were treated with cyclosporin A (CsA; mitochondrial membrane permeability blocker) 30 min prior to OS (0.65M medium adjusted with mannitol on apical surface) or DSS (3% w/v). Intracellular Ca2+ ([Ca2+]i was measured by loading cells with Fura‐2, and production of reactive oxygen species (ROS) by loading cells with MitoSox and DCF‐DA. Barrier function was evaluated by measuring transepithelial electrical resistance (TER) and FITC‐inulin flux, and TJ integrity by immunofluorescence confocal microscopy for occludin and ZO‐1. For CRS, mice were restrained for 2 h daily for up to 8 days (CRS) or injected with corticosterone (25 mg/kg/day, s.c.) for 7 days. [Ca2+]i in colonic mucosa ex vivo was measured by fluorescence radiometric analysis using Indo‐1 as a probe. Colonic sections were stained for ZO‐1, F‐actin, phospho‐JNK, phospho‐cSrc and protein thiols.ResultsPretreatment of Caco‐2 cell monolayers with CsA almost completely blocked OS and DSS‐induced decrease in TER, increase in inulin permeability and redistribution of occludin and ZO‐1 from the junctions. Both OS and DSS rapidly increased [Ca2+]i, ROS production and JNK/c‐Src activation in Caco‐2 cells, which was effectively blocked by CsA pretreatment, indicating the potential role of mitochondrial membrane permeability and oxidative stress in OS and DSS‐induced TJ disruption. Mito‐TEMPO, mitochondria‐targeted superoxide scavenger, blocked the effects of OS and DSS on TJ integrity and barrier function. CRS elevated [Ca2+]i starting day 4, which was associated with redistribution of occludin and ZO‐1 from the epithelial junctions staring day 4. CRS for 5 days showed a dramatic elevation of phospho‐JNK and phospho‐cSrc indicating the activation of JNK and c‐Src. Corticosterone administration also elevated phospho‐JNK and phospho‐cSrc, and induced redistribution of ZO‐1 from the epithelial junctions. Corticosterone depleted reduced‐protein thiols and elevated oxidized‐protein thiols, suggesting that corticosterone‐mediated signaling induces oxidative stress.ConclusionThese data show that Ca2+‐dependent mitochondrial oxidative stress mediates OS and DSS‐induced JNK/c‐Src activation and TJ disruption in the intestinal epithelium. Data also show that a similar mechanism is activated during CRS and corticosterone‐induced TJ disruption in the colonic epithelium. Supported by NIH grant DK55532.