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H2O2 activates G protein, ? 12 to disrupt the junctional complex and enhance ischemia reperfusion injury.


ABSTRACT: The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify G?12 activation by H(2)O(2) leading to tight junction disruption and demonstrate a critical role for G?12 activation during bilateral renal ischemia/reperfusion injury. Madin-Darby canine kidney (MDCK) cells with inducible G?12 (G?12-MDCK) and silenced G?12 (shG?12-MDCK) were subjected to ATP depletion/repletion and H(2)O(2)/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by G?12, but reassembly was accelerated by G?12 depletion. In contrast, silencing of G?12 completely protected cells from H(2)O(2)-stimulated barrier disruption, a response that rapidly occurred in control cells. H(2)O(2) activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H(2)O(2)-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H(2)O(2) led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through G?12/Src signaling pathway. G?12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QL?12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, G?12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates G?12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.

PROVIDER: S-EPMC3340086 | BioStudies | 2012-01-01T00:00:00Z

REPOSITORIES: biostudies

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