Ca v1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 cell monolayers

Abstract

We investigated the role of a Ca 2+ channel and intracellular calcium concentration ([Ca 2+] i) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2- aminophenoxyl)ethane-N, N, N′, N′-tetraacetic acid (BAPTA)-mediated intracellular Ca 2+ depletion. Depletion of extracellular Ca 2+ at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca 2+ depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca 2+ depletion partially reduced osmotic stress-induced rise in [Ca 2+] i, whereas thapsigargin and extracellular Ca 2+ depletion together resulted in almost complete loss of rise in [Ca 2+] i. L-type Ca 2+ channel blockers (isradipine and diltiazem) or knockdown of the Ca V1.3 channel abrogated [Ca 2+] i rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca 2+ depletion, thapsigargin, or Ca V1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca 2+ depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca V1.3 and rise in [Ca 2+] i play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca 2+] i mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca 2+ also contributes to osmotic stress-induced tight junction disruption. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.