bims-noxint 2019-04-21 papers

Issue of 2019–04–21
five papers selected by
Laia Caja Puigsubira, Uppsala University

Int J Mol Sci. 2019 Apr 02. pii: E1644. [Epub ahead of print]20(7):

Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells.

Ryugo Sakurada, Keiichi Odagiri, Akio Hakamata, Chiaki Kamiya, Jiazhang Wei, Hiroshi Watanabe.

BACKGROUND: Previous studies demonstrated that calcium/calmodulin (Ca2+/CaM) activates nicotinamide adenine dinucleotide phosphate oxidases (NOX). In endothelial cells, the elevation of intracellular Ca2+ level consists of two components: Ca2+ mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca2+ entry. However, little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. Here, we investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+/CaM-dependent pathway.
METHODS: We measured ROS production using a fluorescent indicator in endothelial cells and performed phosphorylation assays.
RESULTS: Bradykinin (BK) increased NOX-derived cytosolic ROS. When cells were exposed to BK with either a nominal Ca2+-free or 1 mM of extracellular Ca2+ concentration modified Tyrode's solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca2+ by BAPTA/AM or the depletion of ER Ca2+ contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor did not affect BK-induced ROS production. Furthermore, BK stimulation did not increase phosphorylation of NOX2, NOX4, and NOX5.
CONCLUSIONS: BK-induced NOX-derived ROS production was mediated via a Ca2+/CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents.

Keywords: NADPH oxidase; calcium; calmodulin; endoplasmic reticulum; endothelial cell; reactive oxygen species

DOI: https://doi.org/10.3390/ijms20071644

Blood Adv. 2019 Apr 23. 3(8): 1272-1284

Nox2 NADPH oxidase is dispensable for platelet activation or arterial thrombosis in mice.

Vijay K Sonkar, Rahul Kumar, Melissa Jensen, Brett A Wagner, Anjali A Sharathkumar, Francis J Miller, MaryBeth Fasano, Steven R Lentz, Garry R Buettner, Sanjana Dayal.

Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

DOI: https://doi.org/10.1182/bloodadvances.2018025569

Cell Rep. 2019 Apr 16. pii: S2211-1247(19)30382-1. [Epub ahead of print]27(3): 699-707.e4

Keap1-Nrf2 System Plays an Important Role in Invariant Natural Killer T Cell Development and Homeostasis.

Kalyani Pyaram, Ajay Kumar, Yeung-Hyen Kim, Sanjeev Noel, Sekhar P Reddy, Hamid Rabb, Cheong-Hee Chang.

Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) proteins work in concert to regulate the levels of reactive oxygen species (ROS). The Keap1-Nrf2 antioxidant system also participates in T cell differentiation and inflammation, but its role in innate T cell development and functions remains unclear. We report that T cell-specific deletion of Keap1 results in defective development and reduced numbers of invariant natural killer T (NKT) cells in the thymus and the peripheral organs in a cell-intrinsic manner. The frequency of NKT2 and NKT17 cells increases while NKT1 decreases in these mice. Keap1-deficient NKT cells show increased rates of proliferation and apoptosis, as well as increased glucose uptake and mitochondrial function, but reduced ROS, CD122, and Bcl2 expression. In NKT cells deficient in Nrf2 and Keap1, all these phenotypic and metabolic defects are corrected. Thus, the Keap1-Nrf2 system contributes to NKT cell development and homeostasis by regulating cell metabolism.

Keywords: NKT cells; antioxidant system; cell metabolism; innate T cells; reactive oxygen species

DOI: https://doi.org/10.1016/j.celrep.2019.03.052

Proc Natl Acad Sci U S A. 2019 Apr 18. pii: 201821323. [Epub ahead of print]

Cystine/glutamate antiporter xCT (SLC7A11) facilitates oncogenic RAS transformation by preserving intracellular redox balance.

Jonathan K M Lim, Alberto Delaidelli, Sean W Minaker, Hai-Feng Zhang, Milena Colovic, Hua Yang, Gian Luca Negri, Silvia von Karstedt, William W Lockwood, Paul Schaffer, Gabriel Leprivier, Poul H Sorensen.

The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach, we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo by mitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.

Keywords: RAS; antioxidants; oncogene; xCT

DOI: https://doi.org/10.1073/pnas.1821323116

Biochem Biophys Res Commun. 2019 Apr 16. pii: S0006-291X(19)30673-4. [Epub ahead of print]

MicroRNA-146b-5p protects oligodendrocyte precursor cells from oxygen/glucose deprivation-induced injury through regulating Keap1/Nrf2 signaling via targeting bromodomain-containing protein 4.

Xiaoquan Li, Wei Zhang, Mi Xiao, Fanghui Wang, Ping Zhou, Jian Yang, Xinlin Chen.

The injury of oligodendrocyte precursor cells (OPCs) contributes to the pathology of hypoxic-ischemic encephalopathy in newborns. MicroRNAs (miRNAs) have emerge as critical regulators of hypoxic-ischemic encephalopathy; however, the role of miRNAs in regulating OPC injury remains largely unknown. MiRNA-146b-5p (miR-146b-5p) has been reported to exert a cytoprotective function under various pathological conditions. In this study, we aimed to investigate the potential function of miR-146b-45p in regulating oxygen/glucose deprivation (OGD)-induced injury of OPCs and explore the underlying mechanism. Herein, we found that miR-146b-5p expression was reduced in OPCs exposed to OGD. Functional experiments showed that miR-146b-5p overexpression promoted cell growth and viability, and reduced the apoptosis and oxidative stress in OGD-injured OPCs, while miR-146b-5p inhibition showed an opposite effect. Interestingly, bromodomain-containing protein 4 (Brd4) was identified as a target gene of miR-146b-5p. Brd4 expression was negatively modulated by miR-146b-5p in OPCs. Moreover, the inhibition of Brd4 showed a protective effect in OGD-injured OPCs. Notably, miR-146b-5p overexpression or Brd4 inhibition down-regulated kelch-like ECH-associated protein 1 (Keap1) expression, but promoted nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear expression and enhanced the transcriptional activity of the antioxidant response element (ARE). However, the overexpression of Brd4 significantly abrogated miR-146b-5p mediated protection effect in OGD exposed OPCs. Taken together, these results demonstrate that the overexpression of miR-146b-5p attenuates OGD-induced injury in OPCs through targeting Brd4 and regulating Keap1/Nrf2/ARE antioxidant signaling, suggesting a potential role of miR-146b-5p/Brd4 in the pathophysiology of neonatal hypoxic-ischemic brain injury.

Keywords: Brd4; Keap1; Nrf2; Oligodendrocyte precursor cells; miR-146b-5p

DOI: https://doi.org/10.1016/j.bbrc.2019.04.045