Osmotic stress (OS)‐Induced Tight Junction (TJ) Disruption in Caco‐2 Cell Monolayers Involves Calcium‐Mediated JNK Activation and JNK‐Mediated MLCK activation

Abstract

We investigated the signaling mechanism involved in OS‐induced TJ disruption in the intestinal epithelium. Caco‐2 cell monolayers were subjected to OS by exposure to 0.4 M mannitol. TJ integrity was evaluated by measuring transepithelial electrical resistance (TER), inulin flux and immunofluorescence localization of TJ proteins, occludin, ZO‐1 and claudin‐4 (Cldn‐4). OS‐induced decrease in TER, increase in inulin flux and redistribution of TJ proteins was attenuated by BAPTA and thapsigargin (TG), the calcium blockers, and by siRNA‐mediated knockdown of JNK1/2. OS rapidly increased phospho‐JNK1/2 (active) levels, which was attenuated by BAPTA and TG. OS also induced a rapid and transient activation of MLCK (increased phospho‐MLC levels), which was attenuated by SP600125 (JNK inhibitor), BAPTA and TG; JNK activation was unaffected by ML‐7. OS‐induced TJ disruption was prevented by ML‐7 (the MLCK inhibitor). OS‐induced TJ disruption was associated with the Tyr‐phosphorylation of occludin, ZO‐1 and Cldn‐4. Inhibition of JNK or MLCK abrogated OS‐induced Tyr‐phosphorylation of TJ proteins. BAPTA and TG also attenuated OS‐induced Tyr‐phosphorylation of TJ proteins. These studies demonstrate that OS‐induced disruption of intestinal epithelial TJ involves elevation of intracellular calcium, calcium‐mediated JNK activation, JNK‐mediated MLCK activation and Tyr‐phosphorylation of TJ proteins.