An essential role for EROS in redox-dependent endothelial signal transduction.

The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

Conserved cysteines prevent C-mannosylation of mucin Cys domains.

Mucins are major components of the mucus. Besides the highly O-glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide-forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C-mannosylation, mucin CysDs have been suggested to be targets for C-mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucin CysDs in Chinese hamster ovary (CHO) cells and analyzed the C-mannosylation status. Mass spectrometric analysis revealed that the putative C-mannose site is not or only barely C-mannosylated. However, mutation of the adjacent cysteine residues enabled C-mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C-mannosylated, but introducing a cysteine at the -2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.

Differential endothelial hydrogen peroxide signaling via Nox isoforms: Critical roles for Rac1 and modulation by statins.

Statins have manifold protective effects on the cardiovascular system. In addition to lowering LDL cholesterol levels, statins also have antioxidant effects on cardiovascular tissues involving intracellular redox pathways that are incompletely understood. Inhibition of HMG-CoA reductase by statins not only modulates cholesterol synthesis, but also blocks the synthesis of lipids necessary for the post-translational modification of signaling proteins, including the GTPase Rac1. Here we studied the mechanisms whereby Rac1 and statins modulate the intracellular oxidant hydrogen peroxide (H2O2) via NADPH oxidase (Nox) isoforms. In live-cell imaging experiments using the H2O2 biosensor HyPer7, we observed robust H2O2 generation in human umbilical vein endothelial cells (HUVEC) following activation of cell surface receptors for histamine or vascular endothelial growth factor (VEGF). Both VEGF- and histamine-stimulated H2O2 responses were abrogated by siRNA-mediated knockdown of Rac1. VEGF responses required the Nox isoforms Nox2 and Nox4, while histamine-stimulated H2O2 signals are independent of Nox4 but still required Nox2. Endothelial H2O2 responses to both histamine and VEGF were completely inhibited by simvastatin. In resting endothelial cells, Rac1 is targeted to the cell membrane and cytoplasm, but simvastatin treatment promotes translocation of Rac1 to the cell nucleus. The effects of simvastatin both on receptor-dependent H2O2 production and Rac1 translocation are rescued by treatment of cells with mevalonic acid, which is the enzymatic product of the HMG-CoA reductase that is inhibited by statins. Taken together, these studies establish that receptor-modulated H2O2 responses to histamine and VEGF involve distinct Nox isoforms, both of which are completely dependent on Rac1 prenylation.

The importance of aquaporin-8 for cytokine-mediated toxicity in rat insulin-producing cells.

Aquaporin-8 (AQP8) is a peroxiporin, a transmembrane water and hydrogen peroxide (H2O2) transport protein expressed in the mitochondrial and plasma membranes of pancreatic ß-cells. AQP8 protein expression is low under physiological conditions, but it increases after cytokine exposure both, in vitro and in vivo, possibly related to a NF-κB consensus sequence in the promoter. AQP8 knockdown (KD) insulin-producing RINm5F cells are particularly susceptible to cytokine-mediated oxidative stress. Cytokine (a mixture of IL-1ß, TNF-α, and IFN-γ) treated AQP8 KD cells exhibited pronounced sensitivity to reactive oxygen and nitrogen species (ROS and RNS), resulting in a significant loss of ß-cell viability due to enhanced toxicity of the increased concentrations of H2O2 and hydroxyl radicals (●OH) in mitochondria of AQP8 KD cells. This viability loss went along with increased caspase activities, reduced nitrite concentration (representative of nitric oxide (NO●) accumulation) and increased lipid peroxidation. The explanation for the increased toxicity of the proinflammatory cytokines in AQP8 KD cells resides in the fact that efflux of the H2O2 generated during oxidative stress in the ß-cell mitochondria is hampered through the loss of the peroxiporin channels in the mitochondrial membranes of the AQP8 KD cells. The increased proinflammatory cytokine toxicity due to loss of AQP8 expression in the KD ß-cell mitochondria is thus the result of increased rates of apoptosis. This decreased cell viability is caused by increased levels of oxidative stress along with a ferroptosis-mediated cell death component due to decreased NO● generation.

AQP8 is a crucial H2O2 transporter in insulin-producing RINm5F cells.

Peroxiporins are distinct aquaporins (AQP) which, beside water, also facilitate the bidirectional transport of hydrogen peroxide (H2O2) across cellular membranes. H2O2 serves as the major reactive oxygen species that mediates essential cell signaling events. In pancreatic ß-cells, H2O2 has been associated with the regulation of cell growth but in excess it leads to failure of insulin secretion, making it important for diabetes mellitus (DM) pathogenesis. In the present study, the role of aquaporin-8 (AQP8) as a peroxiporin was investigated in RINm5F cells. The role of AQP8 was studied in an insulin-producing cell model, on the basis of stable AQP8 overexpression (AQP8↑) and CRISPR/Cas9-mediated AQP8 knockdown (KD). A complete AQP8 knock-out was found to result in cell death, however we demonstrate that mild lentiviral re-expression through a Tet-On-regulated genetically modified AQP8 leads to cell survival, enabling functional characterization. Proliferation and insulin content were found to be increased in AQP8↑ cells underlining the importance of AQP8 in the regulation of H2O2 homeostasis in pancreatic ß-cells. Colocalization analyses of V5-tagged AQP8 proteins based on confocal microscopic imaging revealed its membrane targeting to both the mitochondria and the plasma membrane, but not to the ER, the Golgi apparatus, insulin vesicles, or peroxisomes. By using the fluorescence H2O2 specific biosensor HyPer together with endogenous generation of H2O2 using d-amino acid oxidase, live cell imaging revealed enhanced H2O2 flux to the same subcellular regions in AQP8 overexpressing cells pointing to its importance in the development of type-1 DM. Moreover, the novel ultrasensitive H2O2 sensor HyPer7.2 clearly unveiled AQP8 as a H2O2 transporter in RINm5F cells. In summary, these studies establish that AQP8 is an important H2O2 pore in insulin-producing RINm5F cells involved in the transport of H2O2 through the mitochondria and cell membrane and may help to explain the H2O2 transport and toxicity in pancreatic ß-cells.