bims-noxint 2019-10-20 papers

bims-noxint

Biomed News

on NADPH oxidases in tumorigenesis

Issue of 2019–10–20
nine papers selected by
Laia Caja Puigsubira, Uppsala University

NOX4 is the main NADPH oxidase involved in the early stages of hematopoietic differentiation from human induced pluripotent stem cells.
Spironolactone suppresses aldosterone-induced Kv1.5 expression by attenuating mineralocorticoid receptor-Nox1/2/4-mediated ROS generation in neonatal rat atrial myocytes.
Neurotensin receptors regulate transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner.
The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production.
miR-590-3p Inhibits Pyroptosis in Diabetic Retinopathy by Targeting NLRP1 and Inactivating the NOX4 Signaling Pathway.
Potential Neuroprotective Treatment of Stroke: Targeting Excitotoxicity, Oxidative Stress, and Inflammation.
Salix tetrasperma Roxb. Extract Alleviates Neuropathic Pain in Rats via Modulation of the NF-κB/TNF-α/NOX/iNOS Pathway.
Inhibition of NADPH Oxidase-Derived Reactive Oxygen Species Decreases Expression of Inflammatory Cytokines in A549 Cells.
Toll-like receptor 4 protects against irradiation-induced hematopoietic injury by promoting granulopoiesis and alleviating marrow adipogenesis.

Free Radic Biol Med. 2019 Oct 15. pii: S0891-5849(19)31571-0. [Epub ahead of print]

NOX4 is the main NADPH oxidase involved in the early stages of hematopoietic differentiation from human induced pluripotent stem cells.

Julie Brault, Bénédicte Vigne, Mathieu Meunier, Sylvain Beaumel, Michelle Mollin, Sophie Park, Marie José Stasia.

  Reactive oxygen species (ROS) produced in hematopoietic stem cells (HSCs) are involved in the balance between quiescence, self-renewal, proliferation and differentiation processes. However the role of NOX enzymes on the early stages of hematopoietic differentiation is poorly investigated. For that, we used induced pluripotent stem cells (iPSCs) derived from X-linked Chronic Granulomatous Disease (X0CGD) patients with deficiency in NOX2, and AR220CGD patients with deficiency in p22phox subunit which decreases NOX1, NOX2, NOX3 and NOX4 activity. CD34+ hematopoietic progenitors were obtained after 7, 10 and 13 days of iPS/OP9 co-culture differentiation system. Neither NOX expression nor activity was found in Wild-type (WT), X0CGD and AR220CGD iPSCs. Although NOX2 and NOX4 mRNA were found in WT, X0CGD and AR220CGD iPSC-derived CD34+ cells at day 10 and 13 of differentiation, NOX4 protein was the only NOX enzyme expressed in these cells. A NADPH oxidase activity was measured in WT and X0CGD iPSC-derived CD34+ cells but not in AR220CGD iPSC-derived CD34+ cells because of the absence of p22phox, which is essential for the NOX4 activity. The absence of NOX4 activity and the poor NOX-independent ROS production in AR220CGD iPSC-derived CD34+ cells favored the CD34+ cells production but lowered their hematopoietic potential compared to WT and X0CGD iPSC-derived CD34+ cells. In addition we found a large production of primitive AR220CGD iPSC-derived progenitors at day 7 compared to the WT and X0CGD cell types. In conclusion NOX4 is the major NOX enzyme involved in the early stages of hematopoietic differentiation from iPSCs and its activity can modulate the production, the hematopoietic potential and the phenotype of iPSC-derived CD34+.

Keywords:  Chronic granulomatous disease; Hematopoietic differentiation; Induced pluripotent stem cells; NADPH oxidase; NOX4; Reactive oxygen species

DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.10.005
Biochem Biophys Res Commun. 2019 Oct 09. pii: S0006-291X(19)31927-8. [Epub ahead of print]

Spironolactone suppresses aldosterone-induced Kv1.5 expression by attenuating mineralocorticoid receptor-Nox1/2/4-mediated ROS generation in neonatal rat atrial myocytes.

Guihua Lu, Jie Li, Yuansheng Zhai, Qinglang Li, Dongmei Xie, Juhong Zhang, Ying Xiao, Xiuren Gao.

  Our previous investigation indicated that angiotensin II (Ang II) enhances the expression of Kv1.5, a promising target for the treatment of atrial fibrillation (AF), by activating reactive oxygen species (ROS)-dependent phosphorylation of Smad 2/3 (forming P-Smad 2/3) and ERK 1/2 (forming P-ERK 1/2). A recent study indicated that aldosterone (Aldo) upregulates atrial Kv1.5 protein in a rat AF model, but the mechanism remains unknown. The present study aimed to clarify the mechanism underlying Aldo-induced Kv1.5 expression and to test whether spironolactone may modulate atrial Kv1.5. Our Western blot analysis indicated that the Aldo/mineralocorticoid receptor (MR) interacts with Ang II/AT1R in upregulating Kv1.5 expression in cultured neonatal atrial myocytes (NRAMs). Blockade of MR with spironolactone and of AT1R with losartan significantly suppressed Kv1.5 expression induction by combined Aldo and Ang II treatment. Aldo increased the protein expression of Nox1, Nox2 and Nox4, but this effect was abolished by spironolactone pretreatment. The Aldo-induced upregulation of Kv1.5 was also reversed by the Src protein tyrosine kinase family inhibitor PP2, the Nox2 inhibitor gp91ds-tat and the Nox1/Nox4 inhibitor GKT137831 but not by the Rac GTPase inhibitor NSC23766. Flow cytometry showed that the Aldo-induced ROS production was inhibited by spironolactone, PP2, gp91ds-tat and GKT137831. Spironolactone suppressed the Aldo-induced protein expression phosphorylated Src (P-Src), P-Smad 2/3 and P-ERK 1/2. In conclusion, we have demonstrated that spironolactone suppresses Aldo-induced Kv1.5 expression by attenuating MR-Nox1/2/4-mediated ROS generation in NRAMs.

Keywords:  Aldosterone; Atrial fibrillation; Kv1.5; Mineralocorticoid receptor; NADPH oxidase; Reactive oxygen species

DOI:  https://doi.org/10.1016/j.bbrc.2019.10.039
Eur J Pharmacol. 2019 Oct 12. pii: S0014-2999(19)30687-9. [Epub ahead of print] 172735

Neurotensin receptors regulate transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner.

Terry W Moody, Lingaku Lee, Irene Ramos-Alvarez, Robert T Jensen.

  Neurotensin is a 13 amino acid peptide which is present in many lung cancer cell lines. Neurotensin binds with high affinity to the neurotensin receptor 1, and functions as an autocrine growth factor in lung cancer cells. Neurotensin increases tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and the neurotensin receptor 1 antagonist SR48692 blocks the transactivation of the EGFR. Here the effects of reactive oxygen species on the transactivation of the EGFR and HER2 were investigated. Using non-small cell lung cancer (NSCLC) cell lines, neurotensin receptor 1 mRNA and protein were present. Using NCI-H838 cells, neurotensin or neurotensin8-13 but not neurotensin1-8 increased EGFR, ERK and HER2 tyrosine phosphorylation which was blocked by SR48692. Neurotensin addition to NCI-H838 cells increased significantly reactive oxygen species which was inhibited by SR48692, Tiron (superoxide scavenger) and diphenylene iodonium (DPI inhibits the ability of NADPH oxidase and dual oxidase enzymes to produce reactive oxygen species). Tiron or DPI impaired the ability of neurotensin to increase EGFR, ERK and HER2 tyrosine phosphorylation. Neurotensin stimulated NSCLC cellular proliferation whereas the growth was inhibited by SR48692, DPI or lapatinib (lapatinib is tyrosine kinase inhibitor of the EGFR and HER2). Lapatinib inhibited the ability of the neurotensin receptor 1 to transactivate the EGFR and HER2. The results indicate that neurotensin receptor 1 regulates the transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner.

Keywords:  EGFR; HER2; Lung cancer; Neurotensin; Reactive oxygen species

DOI:  https://doi.org/10.1016/j.ejphar.2019.172735
Redox Biol. 2019 Oct 09. pii: S2213-2317(19)31111-5. [Epub ahead of print]28 101341

The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production.

Renata L S Goncalves, Mark A Watson, Hoi-Shan Wong, Adam L Orr, Martin D Brand.

  Reactive oxygen species are important signaling molecules crucial for muscle differentiation and adaptation to exercise. However, their uncontrolled generation is associated with an array of pathological conditions. To identify and quantify the sources of superoxide and hydrogen peroxide in skeletal muscle we used site-specific suppressors (S1QELs, S3QELs and NADPH oxidase inhibitors). We measured the rates of hydrogen peroxide release from isolated rat muscle mitochondria incubated in media mimicking the cytosol of intact muscle. By measuring the extent of inhibition caused by the addition of different site-specific suppressors of mitochondrial superoxide/hydrogen peroxide production (S1QELs for site IQ and S3QELs for site IIIQo), we determined the contributions of these sites to the total signal. In media mimicking resting muscle, their contributions were each 12-18%, consistent with a previous method. In C2C12 myoblasts, site IQ contributed 12% of cellular hydrogen peroxide production and site IIIQo contributed about 30%. When C2C12 myoblasts were differentiated to myotubes, hydrogen peroxide release increased five-fold, and the proportional contribution of site IQ doubled. The use of S1QELs and S3QELs is a powerful new way to measure the relative contributions of different mitochondrial sites to muscle hydrogen peroxide production under different conditions. Our results show that mitochondrial sites IQ and IIIQo make a substantial contribution to superoxide/hydrogen peroxide production in muscle mitochondria and C2C12 myoblasts. The total hydrogen peroxide release rate and the relative contribution of site IQ both increase substantially upon differentiation to myotubes.

Keywords:  Hydrogen peroxide; NOX; Reactive oxygen species; S1QEL; S3QEL; Skeletal muscle mitochondria

DOI:  https://doi.org/10.1016/j.redox.2019.101341
Invest Ophthalmol Vis Sci. 2019 Oct 01. 60(13): 4215-4223

miR-590-3p Inhibits Pyroptosis in Diabetic Retinopathy by Targeting NLRP1 and Inactivating the NOX4 Signaling Pathway.

Chufeng Gu, Deji Draga, Chuandi Zhou, Tong Su, Chen Zou, Qing Gu, Tashi Lahm, Zhi Zheng, Qinghua Qiu.

Purpose: To elucidate the mechanism whereby miR-590-3p regulates pyroptosis in diabetic retinopathy (DR).
Methods: Human retinal microvascular endothelial cells (HRMECs) incubated with high glucose (HG) were used to establish cell models, and the expression levels of miR-590-3p, caspase-1, IL-1β, NLRP1, NOX4, TXNIP, NLRP3, and ROS were determined. Additionally, miR-590-3p was altered using a mimic or an inhibitor, and siRNAs targeting NLRP1 and NOX4 were applied to explore the regulatory mechanism of miR-590-3p in DR. The relationships between miR-590-3p and NLRP1/NOX4 also were investigated using a luciferase reporter assay. Furthermore, vitreous tissue samples were collected to confirm pyroptosis in clinical DR.
Results: Downregulated miR-590-3p and upregulated NLRP1/NOX4 levels were observed in a cell culture model of DR. Inhibiting miR-590-3p upregulated NLRP1, the NOX4/ROS/TXNIP/NLRP3 pathway, and caspase-1. NLRP1 and NOX4 were confirmed as direct target genes of miR-590-3p. The overexpression of miR-590-3p or knockdown of NLRP1 and NOX4 increased cell activity and suppressed pyroptosis. Intriguingly, the upregulation of IL-1β induced the downregulation of miR-590-3p by lowering the DNA promoter activity of pri-miR-590.
Conclusions: HG induced pyroptosis in a cell culture model of DR, and the downregulation of miR-590-3p promoted pyroptotic death by targeting NLRP1 and activating the NOX4/ROS/TXNIP/NLRP3 pathway via an IL-1β-mediated positive feedback loop.

DOI: https://doi.org/10.1167/iovs.19-27825
Front Neurosci. 2019 ;13 1036

Potential Neuroprotective Treatment of Stroke: Targeting Excitotoxicity, Oxidative Stress, and Inflammation.

Qianwen Yang, Qianyi Huang, Zhiping Hu, Xiangqi Tang.

  Stroke is a major cause of death and adult disability. However, therapeutic options remain limited. Numerous pathways underlie acute responses of brain tissue to stroke. Early events following ischemic damage include reactive oxygen species (ROS)-mediated oxidative stress and glutamate-induced excitotoxicity, both of which contribute to rapid cell death within the infarct core. A subsequent cascade of inflammatory events escalates damage progression. This review explores potential neuroprotective strategies for targeting key steps in the cascade of ischemia-reperfusion (I/R) injury. NADPH oxidase (NOX) inhibitors and several drugs currently approved by the U.S. Food and Drug Administration including glucose-lowering agents, antibiotics, and immunomodulators, have shown promise in the treatment of stroke in both animal experiments and clinical trials. Ischemic conditioning, a phenomenon by which one or more cycles of a short period of sublethal ischemia to an organ or tissue protects against subsequent ischemic events in another organ, may be another potential neuroprotective strategy for the treatment of stroke by targeting key steps in the I/R injury cascade.

Keywords:  ROS; excitotoxicity; inflammation; neuroprotective; potential treatment; stroke

DOI:  https://doi.org/10.3389/fnins.2019.01036
Antioxidants (Basel). 2019 Oct 12. pii: E482. [Epub ahead of print]8(10):

Salix tetrasperma Roxb. Extract Alleviates Neuropathic Pain in Rats via Modulation of the NF-κB/TNF-α/NOX/iNOS Pathway.

Mansour Sobeh, Mona F Mahmoud, Samar Rezq, Amira E Alsemeh, Omar M Sabry, Islam Mostafa, Mohamed A O Abdelfattah, Khadija Ait El-Allem, Assem M El-Shazly, Aziz Yasri, Michael Wink.

  Patients with neuropathic pain experience chronic painful tingling, burning, and prickling sensations accompanied with hyperalgesia and/or allodynia. In this study, 38 secondary metabolites of a methanol extract from Salix tetrasperma flowers were identified by liquid chromatography-mass spectrometry (HPLC-MS/MS). The extract showed substantial anti-inflammatory, central and peripheral anti-nociceptive, antipyretic, and antioxidant activities in vitro and in different animal models. In the chronic constriction injury (CCI) rat model, the extract was able to attenuate and significantly relieve hyperalgesia and allodynia responses in a dose dependent manner and restore the myelin sheath integrity and Schwann cells average number in the sciatic nerve. The enzyme-linked immunosorbent assay (ELISA) showed that the extract significantly reduced the expression of various pro-inflammatory biomarkers including nuclear factor kabba B (NF-κB), tumor necrosis factor alpha (TNF-α), prostaglandin E2 (PGE2), 5-lipoxygenase (5-LOX), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and the oxidative stress biomarker NADPH oxidase 1 (NOX1), in brain stem and sciatic nerve tissues. These findings were supported by in vitro enzyme inhibition assays (COX-1, COX-2 and 5-LOX). Moreover, the extract significantly reduced p53 expression in the brain stem tissue. These findings support the use of S. tetrasperma in folk medicine to alleviate pain. It could be a promising natural product for further clinical investigations to treat inflammation, nociceptive pain and chronic neuropathic pain.

Keywords:  NF-κB/TNF-α/NOX/iNOS pathway; Salix tetrasperma; chronic constriction injury; neuropathy; p53; polyphenols

DOI:  https://doi.org/10.3390/antiox8100482
Inflammation. 2019 Oct 14.

Inhibition of NADPH Oxidase-Derived Reactive Oxygen Species Decreases Expression of Inflammatory Cytokines in A549 Cells.

Joanna Wieczfinska, Przemyslaw Sitarek, Ewa Skała, Tomasz Kowalczyk, Rafal Pawliczak.

  Various experimental models strongly support the hypothesis that airway inflammation can be caused by oxidative stress. Inflammatory airway diseases like asthma and COPD are characterized by higher levels of ROS and inflammatory cytokines. One of the sources of ROS is NADPH oxidase. Therefore, the aim of the study was to investigate influence of NADPH oxidase inhibition on the expression of IL-6, IL-8, TNF, TSLP, CD59, and PPAR-γ in vitro. A549 cells were incubated with apocynin in three concentrations (0.5 mg/ml, 1 mg/ml, and 3 mg/ml). Cells were trypsinized and RNA isolated after 1 h, 2 h, and 4 h of apocynin incubation at each concentration. Afterwards, reverse transcription was performed to evaluate mRNA expression using real-time PCR. The time-response and dose-response study showed that apocynin significantly influenced the relative expression of chosen genes (IL-6, IL-8, TNF, PPAR-γ, TSLP, and CD59). Apocynin decreased the mRNA expression of TNF-α at all concentrations used, and of IL-6 at concentrations of 1 and 3 mg/ml (p < 0.05). TSLP mRNA expression was also reduced by apocynin after 1 h and 2 h, and CD59 mRNA after 1 h, but only at the highest concentration. The expression of PPAR-γ was reduced after apocynin in the highest concentrations only (p < 0.05). The results might suggest that proinflammatory agents' expression levels are strongly connected to the presence of oxidative stress generated by NADPH oxidase and this might be at least partially eliminated by anti-oxidative action. Apocynin, as an effective inhibitor of NADPH oxidase, seems to be useful in potential anti-oxidative and anti-inflammatory therapy.

Keywords:  apocynin; inflammatory cytokines; oxidative stress

DOI:  https://doi.org/10.1007/s10753-019-01084-0
Biochem Biophys Res Commun. 2019 Oct 10. pii: S0006-291X(19)31916-3. [Epub ahead of print]

Toll-like receptor 4 protects against irradiation-induced hematopoietic injury by promoting granulopoiesis and alleviating marrow adipogenesis.

Zhengxu Sun, Kunpeng Wu, Qiwang Lin, He Fei, Hua Jiang, Tong Chen, Yan Yuan.

  Irradiation induces severe damage in the hematopoietic system, which leads to bone marrow hyperplasia, pancytopenia, and aggravated tissue formation in bone marrow. Studies have shown that Toll-like receptor 4 (TLR4) has a protective effect against irradiation, but the underlying mechanism remains unclear. In this study, we used a TLR4 knockout (TLR4-/-) mouse irradiation model and found that the white blood cell and platelet counts in the peripheral blood of TLR4-/- mice recovered slowly after irradiation, with bone marrow hyperplasia and increased mortality. Additionally, we found that the proportion of CD11b+Gr1+ granulocytes in the peripheral blood and bone marrow of TLR4-/- mice was lower than that of wild-type mice after irradiation. Further, we found that the expression of NADPH Oxidases (NOXs) in the bone marrow was down-regulated after irradiation of TLR4-/- mice, and administration of the NOXs inhibitor VAS2870 reduced the proportion of CD11b+Gr1+ cells in the bone marrow and peripheral blood of wild-type mice after irradiation. Irradiation induced severe marrow adipocytes accumulation in TLR4-/- mice, TLR4 ligand lipopolysaccharide promoted proliferation and inhibited adipogenic differentiation of mesenchymal stromal cells. In summary, our data suggest that TLR4 promotes myeloid hyperplasia by up-regulating the expression of NOXs after irradiation, prohibits marrow adipogensis and increases the tolerance of mice to irradiation.

Keywords:  Adipogenesis; Granulocyte; Irradiation; Toll like receptor 4

DOI:  https://doi.org/10.1016/j.bbrc.2019.10.027