Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local Ras activation.
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.
Wu
Ru-Feng
RF
Department of Internal Medicine, Division of Pulmonary and Critical Care, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
Ma
Zhenyi
Z
Liu
Zhe
Z
Terada
Lance S
LS
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
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
J Biol Chem. 2004 Jul 9;279(28):29857-62
15123696
J Cell Biol. 2008 Aug 25;182(4):685-701
18725538
Oncogene. 2008 Oct 27;27(50):6419-33
18955970
Am J Med Sci. 2000 Dec;320(6):379-87
11149550
Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6226-31
19339495
J Biol Chem. 2007 Dec 28;282(52):37412-9
17940286
Mol Cell. 2006 Feb 17;21(4):481-93
16483930
Dev Cell. 2004 Aug;7(2):167-78
15296714
J Cell Biol. 2004 May 10;165(3):347-56
15123740
Cell Death Differ. 2007 Feb;14(2):230-9
16794605
Mol Cell. 2007 Jun 8;26(5):649-62
17560371
Nat Methods. 2006 Apr;3(4):281-6
16554833
J Virol. 2004 Jan;78(2):779-89
14694110
EMBO Rep. 2006 Jan;7(1):46-51
16282985
PLoS One. 2008;3(11):e3731
19009018
J Biol Chem. 1997 Jun 20;272(25):15642-9
9188453
Neuropathol Appl Neurobiol. 2007 Dec;33(6):658-69
17931354
Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11466-71
11027346
J Cell Biol. 2008 Jun 30;181(7):1129-39
18573911
J Biol Chem. 2003 May 9;278(19):16667-74
12609989
Proc Natl Acad Sci U S A. 1995 May 23;92(11):4838-42
7539135
Antioxid Redox Signal. 2005 Mar-Apr;7(3-4):308-17
15706079
J Cell Biol. 2007 Oct 8;179(1):23-31
17908916
J Biol Chem. 1995 Sep 8;270(36):21195-8
7673152
Mol Cell. 2004 Sep 10;15(5):767-76
15350220
J Biol Chem. 2006 Oct 6;281(40):30299-304
16901900
Cell. 2007 Dec 14;131(6):1137-48
18083103
Mol Cell Biol. 2004 Dec;24(24):10703-17
15572675
Mol Cell. 2003 Mar;11(3):619-33
12667446
Nat Cell Biol. 2002 May;4(5):343-50
11988737
Virology. 2001 Jul 20;286(1):62-71
11448159
J Virol. 2007 Dec;81(24):13578-86
17928342
Cell. 2008 Nov 28;135(5):933-47
19026441
Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):299-304
16407158
J Cell Biol. 2006 Aug 28;174(5):615-23
16923830
J Cell Biol. 2007 Dec 3;179(5):895-909
18056408
Cell. 2005 Sep 23;122(6):927-39
16179260
Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7632-6
8395050
Genes Dev. 2004 Dec 15;18(24):3066-77
15601821
J Cell Biol. 2005 Dec 5;171(5):893-904
16330715
Mol Cell. 2007 Jan 26;25(2):193-205
17244528
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18525-30
19011102
PLoS Biol. 2006 Nov;4(12):e423
17132049
Mol Cell Biol. 2006 Dec;26(24):9220-31
17030611
Nat Rev Mol Cell Biol. 2007 Jul;8(7):519-29
17565364
Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9045-50
16754851
Cell Signal. 2006 Jan;18(1):69-82
15927447