title: Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local Ras activation.
abstract: The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H(2)O(2) sensor targeted to different subcellular compartments, we demonstrate specific production of H(2)O(2) by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H(2)O(2) production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.
authors.affiliation: Department of Internal Medicine, Division of Pulmonary and Critical Care, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
authors.forename: Ru-Feng
authors.initials: RF
authors.lastname: Wu
authors.forename: Zhenyi
authors.initials: Z
authors.lastname: Ma
authors.forename: Zhe
authors.initials: Z
authors.lastname: Liu
authors.forename: Lance S
authors.initials: LS
authors.lastname: Terada
mesh_terms: D001483:Base Sequence; D002374:Catalase / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D055785:Gene Knockdown Techniques; D006706:Homeostasis; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D066298:In Vitro Techniques; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D019284:Thapsigargin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D056811:Unfolded Protein Response; D018631:ras Proteins / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D014415:Tunicamycin; D019284:Thapsigargin; D006861:Hydrogen Peroxide; D002374:Catalase; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D018631:ras Proteins; D004229:Dithiothreitol
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