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 H₂O₂ 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.

This article is referred to by:

Focal oxidant and Ras signaling on the ER surface activates autophagy

We acknowledge the excellent technical skills of Chengxu Liao.

This work was supported by the NHLBI, grants R01-HL061897 and R01-HL067256, and by institutional support for the Molecular and Cellular Imaging Facility from the University of Texas Southwestern.