TY - JOUR
T1 - Nox4-derived H2O2 mediates endoplasmic reticulum signaling through local ras activation
AU - Wu, Ru Feng
AU - Ma, Zhenyi
AU - Liu, Zhe
AU - Terada, Lance S.
PY - 2010/7
Y1 - 2010/7
N2 - 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 2O2 sensor targeted to different subcellular compartments, we demonstrate specific production of H2O2 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 H2O2 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.
AB - 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 2O2 sensor targeted to different subcellular compartments, we demonstrate specific production of H2O2 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 H2O2 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.
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U2 - 10.1128/MCB.01445-09
DO - 10.1128/MCB.01445-09
M3 - Article
C2 - 20457808
AN - SCOPUS:77954382142
SN - 0270-7306
VL - 30
SP - 3553
EP - 3568
JO - Molecular and cellular biology
JF - Molecular and cellular biology
IS - 14
ER -