title: H2O2 activates G protein, α 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 Gα12 activation by H(2)O(2) leading to tight junction disruption and demonstrate a critical role for Gα12 activation during bilateral renal ischemia/reperfusion injury. Madin-Darby canine kidney (MDCK) cells with inducible Gα12 (Gα12-MDCK) and silenced Gα12 (shGα12-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 Gα12, but reassembly was accelerated by Gα12 depletion. In contrast, silencing of Gα12 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 Gα12/Src signaling pathway. Gα12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QLα12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, Gα12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates Gα12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.
authors.affiliation: Renal Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
authors.forename: Wanfeng
authors.initials: W
authors.lastname: Yu
authors.forename: Sarah
authors.initials: S
authors.lastname: Beaudry
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Negoro
authors.forename: Ilene
authors.initials: I
authors.lastname: Boucher
authors.forename: Mei
authors.initials: M
authors.lastname: Tran
authors.forename: Tianqing
authors.initials: T
authors.lastname: Kong
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Denker
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007365:Intercellular Junctions / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010766:Phosphorylation; D015427:Reperfusion Injury / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D044365:GTP-Binding Protein alpha Subunits, G12-G13