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Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α‐mediated stress signaling
(2016)
Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine–threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species‐generating NADPH oxidase‐4 (Nox4) is induced downstream of ATF4, binds to a PP1‐targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4‐regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia–reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4–GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.
Tolerizing CTL by sustained hepatic PD-L1 expression provides a new therapy spproach in mouse sepsis
(2019)
Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option.
Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection.
Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers.
Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.
CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity
(2016)
The NADPH oxidases are important transmembrane proteins producing reactive oxygen species (ROS). Within the Nox family, different modes of activation can be discriminated. Nox1-3 are dependent on different cytosolic subunits, Nox4 seems to be constitutively active and Nox5 is directly activated by calcium. With the exception of Nox5, all Nox family members are thought to depend on the small transmembrane protein p22phox. With the discovery of the CRISPR/Cas9-system, a tool to alter genomic DNA sequences has become available. So far, this method has not been widely used in the redox community. On such basis, we decided to study the requirement of p22phox in the Nox complex using CRISPR/Cas9-mediated knockout. Knockout of the gene of p22phox, CYBA, led to an ablation of activity of Nox4 and Nox1 but not of Nox5. Production of hydrogen peroxide or superoxide after knockout could be rescued with either human or rat p22phox, but not with the DUOX-maturation factors DUOXA1/A2. Furthermore, different mutations of p22phox were studied regarding the influence on Nox4-dependent H2O2 production. P22phox Q130* and Y121H affected maturation and activity of Nox4. Hence, Nox5-dependent O2•- production is independent of p22phox, but native p22phox is needed for maturation of Nox4 and production of H2O2.
Aims: Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling.
Methods and results: We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4−/−) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4−/− mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P < 0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P < 0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 μm2, P < 0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4−/− mice (+29% vs. −21%, P < 0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4−/− mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A.
Conclusion: Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.
Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.
Objective: The NADPH oxidase Nox4 is an important source of H2O2. Nox4-derived H2O2 limits vascular inflammation and promotes smooth muscle differentiation. On this basis, the role of Nox4 for restenosis development was determined in the mouse carotid artery injury model. Methods and results: Genetic deletion of Nox4 by a tamoxifen-activated Cre-Lox-system did not impact on neointima formation in the carotid artery wire injury model. To understand this unexpected finding, time-resolved single-cell RNA-sequencing (scRNAseq) from injured carotid arteries of control mice and massive-analysis-of-cDNA-ends (MACE)-RNAseq from the neointima harvested by laser capture microdissection of control and Nox4 knockout mice was performed. This revealed that resting smooth muscle cells (SMCs) and fibroblasts exhibit high Nox4 expression, but that the proliferating de-differentiated SMCs, which give rise to the neointima, have low Nox4 expression. In line with this, the first weeks after injury, gene expression was unchanged between the carotid artery neointimas of control and Nox4 knockout mice. Conclusion: Upon vascular injury, Nox4 expression is transiently lost in the cells which comprise the neointima. NADPH oxidase 4 therefore does not interfere with restenosis development after wire-induced vascular injury.
Activation of TRPC6 channels is essential for lung ischaemia–reperfusion induced oedema in mice
(2012)
Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.
Regular exercise has widespread health benefits. Fundamental to these beneficial effects is the ability of the heart to intermittently and substantially increase its performance without incurring damage, but the underlying homeostatic mechanisms are unclear. We identify the ROS-generating NADPH oxidase-4 (Nox4) as an essential regulator of exercise performance in mice. Myocardial Nox4 levels increase during acute exercise and trigger activation of the transcription factor Nrf2, with the induction of multiple endogenous antioxidants. Cardiomyocyte-specific Nox4-deficient (csNox4KO) mice display a loss of exercise-induced Nrf2 activation, cardiac oxidative stress and reduced exercise performance. Cardiomyocyte-specific Nrf2-deficient (csNrf2KO) mice exhibit similar compromised exercise capacity, with mitochondrial and cardiac dysfunction. Supplementation with an Nrf2 activator or a mitochondria-targeted antioxidant effectively restores cardiac performance and exercise capacity in csNox4KO and csNrf2KO mice respectively. The Nox4/Nrf2 axis therefore drives a hormetic response that is required for optimal cardiac mitochondrial and contractile function during physiological exercise.
