Proteomic Characterization of the Cellular Response to Nitrosative Stress Mediated by S-Nitrosoglutathione Reductase Inhibition

The S-nitrosoglutathione-metabolizing enzyme, GSNO reductase
(GSNOR), has emerged as an important regulator of protein S-nitrosylation.
GSNOR ablation is protective in models of asthma and heart failure,
raising the idea that GSNOR inhibitors might hold therapeutic value.
Here, we investigated the effects of a small molecule inhibitor of
GSNOR (GSNORi) in mouse RAW 264.7 macrophages. We found that GSNORi
increased protein S-nitrosylation in cytokine-stimulated cells, and
we utilized stable isotope labeling of amino acids in cell culture
(SILAC) to quantify the cellular response to this “nitrosative
stress”. The expression of several cytokine-inducible immunomodulators,
including osteopontin, cyclooxygenase-2, and nitric oxide synthase
isoform 2 (NOS2), were decreased by GSNORi. In addition, selective
targets of the redox-regulated transcription factor, nuclear factor
(erythroid-derived 2)-like 2 (Nrf2)including heme oxygenase
1 (HO-1) and glutamate cysteine ligase modulatory subunitwere
induced by GSNORi in a NOS2- and Nrf2-dependent manner. In cytokine-stimulated
cells, Nrf2 protected from GSNORi-induced glutathione depletion and
cytotoxicity and HO-1 activity was required for down-regulation of
NOS2. Interestingly, GSNORi also affected a marked increase in NOS2
protein stability. Collectively, these data provide the most complete
description of the global effects of GSNOR inhibition and demonstrate
several important mechanisms for inducible response to GSNORi-mediated
nitrosative stress.