Cav1.3 Channels and Intracellular Calcium Mediate Osmotic Stress-induced N-terminal c-Jun Kinase Activation and Disruption of Tight Junctions in Caco-2 Cell Monolayers

We investigated the role of a Ca2+ channel and intracellular calcium concentration ([Ca2+](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 Ca2+ depletion. Depletion of extracellular Ca2+ at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca2+ depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca2+ depletion partially reduced osmotic stress-induced rise in [Ca2+](i), whereas thapsigargin and extracellular Ca2+ depletion together resulted in almost complete loss of rise in [Ca2+](i). L-type Ca2+ channel blockers (isradipine and diltiazem) or knockdown of the Ca(v)1.3 channel abrogated [Ca2+](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca2+ depletion, thapsigargin, or Ca(v)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca2+ 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(v)1.3 and rise in [Ca2+](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca2+](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 Ca2+ also contributes to osmotic stress-induced tight junction disruption.