Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local Ras activation

Mol Cell Biol. 2010 Jul;30(14):3553-68. doi: 10.1128/MCB.01445-09. Epub 2010 May 10.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Catalase / metabolism
  • Cells, Cultured
  • DNA Primers / genetics
  • Dithiothreitol / pharmacology
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum / ultrastructure
  • Gene Knockdown Techniques
  • Homeostasis
  • Humans
  • Hydrogen Peroxide / metabolism*
  • In Vitro Techniques
  • NADPH Oxidase 4
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Oxidative Stress
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Thapsigargin / pharmacology
  • Tunicamycin / pharmacology
  • Unfolded Protein Response
  • ras Proteins / metabolism*
  • tat Gene Products, Human Immunodeficiency Virus / metabolism


  • DNA Primers
  • Recombinant Fusion Proteins
  • tat Gene Products, Human Immunodeficiency Virus
  • Tunicamycin
  • Thapsigargin
  • Hydrogen Peroxide
  • Catalase
  • NADPH Oxidase 4
  • NADPH Oxidases
  • NOX4 protein, human
  • ras Proteins
  • Dithiothreitol