bims-noxint 2022-07-17 papers

bims-noxint

Biomed News

on NADPH oxidases in tumorigenesis

Issue of 2022–07–17
seven papers selected by
Laia Caja Puigsubira, Uppsala University

Bioluminescence-Based Complementation Assay to Correlate Conformational Changes in Membrane-Bound Complexes with Enzymatic Function.
Nox4 promotes endothelial differentiation through chromatin remodeling.
Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction.
NOX2-mediated reactive oxygen species are double-edged swords in focal cerebral ischemia in mice.
Plumbagin protects H9c2 cardiomyocytes against TBHP-induced cytotoxicity by alleviating ROS-induced apoptosis and modulating autophagy.
LncRNA MALAT1 Aggravates Renal Tubular Injury via Activating LIN28A and the Nox4/AMPK/mTOR Signaling Axis in Diabetic Nephropathy.
NOX4 promotes Kupffer cell inflammatory response via ROS-NLRP3 to aggravate liver inflammatory injury in acute liver injury.

Methods Mol Biol. 2022 ;2525 123-137

Bioluminescence-Based Complementation Assay to Correlate Conformational Changes in Membrane-Bound Complexes with Enzymatic Function.

Sharon O'Neill, Ulla G Knaus.

  The proteomics field has undergone tremendous development with the introduction of many innovative methods for the identification and characterization of protein-protein interactions (PPIs). Sensitive and quantitative protein association-based techniques represent a versatile tool to probe the architecture of receptor complexes and receptor-ligand interactions and expand the drug discovery toolbox by facilitating high-throughput screening (HTS) approaches. These novel methodologies will be highly enabling for interrogation of structural determinants required for the activity of multimeric membrane-bound enzymes with unresolved crystal structure and for HTS assay development focused on unique characteristics of complex assembly instead of common catalytic features, thereby increasing specificity. We describe here an example of a binary luciferase reporter assay (NanoBiT®) to quantitatively assess the heterodimerization of the catalytically active NADPH oxidase 4 (NOX4) enzyme complex. The catalytic subunit NOX4 requires association with the protein p22phox for stabilization and enzymatic activity, but the precise manner by which these two membrane-bound proteins interact to facilitate hydrogen peroxide (H2O2) generation is currently unknown. The NanoBiT complementation reporter quantitatively determined the accurate, reduced, or failed complex assembly, which can then be confirmed by determining H2O2 release, protein expression, and heterodimer trafficking. Multimeric complex formation differs between NOX enzyme isoforms, facilitating isoform-specific, PPI-based drug screening in the future.

Keywords:  Bioluminescence (BL); Enzyme; Heterodimerization; NADPH oxidase; NOX4; NanoBiT; Protein; Protein interaction (PPI); p22phox

DOI:  https://doi.org/10.1007/978-1-0716-2473-9_9
Redox Biol. 2022 Jul 06. pii: S2213-2317(22)00153-7. [Epub ahead of print]55 102381

Nox4 promotes endothelial differentiation through chromatin remodeling.

F Hahner, F Moll, T Warwick, D M Hebchen, G K Buchmann, J Epah, W Abplanalp, T Schader, S Günther, R Gilsbach, R P Brandes, K Schröder.

   RATIONALE: Nox4 is a constitutively active NADPH oxidase that constantly produces low levels of H2O2. Thereby, Nox4 contributes to cell homeostasis and long-term processes, such as differentiation. The high expression of Nox4 seen in endothelial cells contrasts with the low abundance of Nox4 in stem cells, which are accordingly characterized by low levels of H2O2. We hypothesize that Nox4 is a major contributor to endothelial differentiation, is induced during the process of differentiation, and facilitates homeostasis of the resulting endothelial cells.
OBJECTIVE: To determine the role of No×4 in differentiation of murine inducible pluripotent stem cells (miPSC) into endothelial cells (ECs).
METHODS AND RESULTS: miPSC, generated from mouse embryonic wildtype (WT) and Nox4-/- fibroblasts, were differentiated into endothelial cells (miPSC-EC) by stimulation with BMP4 and VEGF. During this process, Nox4 expression increased and knockout of Nox4 prolonged the abundance of pluripotency markers, while expression of endothelial markers was delayed in differentiating Nox4-depleted iPSCs. Eventually, angiogenic capacity of iPSC-ECs is reduced in Nox4 deficient cells, indicating that an absence of Nox4 diminishes stability of the reached phenotype. As an underlying mechanism, we identified JmjD3 as a redox target of Nox4. iPSC-ECs lacking Nox4 display a lower nuclear abundance of the histone demethylase JmjD3, resulting in an increased triple methylation of histone 3 (H3K27me3), which serves as a repressive mark for several genes involved in differentiation.
CONCLUSIONS: Nox4 promotes differentiation of miPSCs into ECs by oxidation of JmjD3 and subsequent demethylation of H3K27me3, which forced endothelial differentiation and stability.

Keywords:  Differentiation; H3K27me3; JmjD3; Nox4; Redox-switch; iPSC

DOI:  https://doi.org/10.1016/j.redox.2022.102381
Breast Cancer Res. 2022 Jul 14. 24(1): 48

Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction.

Shakeel Mir, Briana D Ormsbee Golden, Brandon J Griess, Raghupathy Vengoji, Eric Tom, Elizabeth A Kosmacek, Rebecca E Oberley-Deegan, Geoffrey A Talmon, Vimla Band, Melissa Lt Teoh-Fitzgerald.

   BACKGROUND: A pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFβ-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer.
METHODS: We targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA.
CONCLUSIONS: We found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.

Keywords:  Adaptive stress response pathway; Autophagy; Breast cancer progression and metastasis; CAF survival; Cancer-associated fibroblasts; NADPH Oxidase 4; Nuclear factor-erythroid factor 2-related factor 2; Pro-oxidant

DOI:  https://doi.org/10.1186/s13058-022-01548-6
J Neuroinflammation. 2022 Jul 14. 19(1): 184

NOX2-mediated reactive oxygen species are double-edged swords in focal cerebral ischemia in mice.

Ye Yingze, Jian Zhihong, Jin Tong, Li Yina, Zeng Zhi, Zhang Xu, Xiong Xiaoxing, Gu Lijuan.

   BACKGROUND: Reactive oxygen species (ROS) often promote acute brain injury after stroke, but their roles in the recovery phase have not been well studied. We tested the hypothesis that ROS activity mediated by NADPH oxidase 2 (NOX2) contributes to acute brain injury but promotes functional recovery during the delayed phase, which is linked with neuroinflammation, autophagy, angiogenesis, and the PI3K/Akt signaling pathway.
METHODS: We used the NOX2 inhibitor apocynin to study the role of NOX2 in brain injury and functional recovery in a middle cerebral artery occlusion (MCAO) stroke mouse model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7, 10 and 14 after reperfusion. In addition, dynamic NOX2-induced ROS levels were measured by dihydroethidium (DHE) staining. Autophagy, inflammasomes, and angiogenesis were measured by immunofluorescence staining and western blotting. RNA sequencing was performed, and bioinformatics technology was used to analyze differentially expressed genes (DEGs), as well as the enrichment of biological functions and signaling pathways in ischemia penumbra at 7 days after reperfusion. Then, Akt pathway-related proteins were further evaluated by western blotting.
RESULTS: Our results showed that apocynin injection attenuated infarct size and mortality 3 days after stroke but promoted mortality and blocked functional recovery from 5 to 14 days after stroke. DHE staining showed that ROS levels were increased at 3 days after reperfusion and then gradually declined in WT mice, and these levels were significantly reduced by the NOX2 inhibitor apocynin. RNA-Seq analysis indicated that apocynin activated the immune response under hypoxic conditions. The immunofluorescence and western blot results demonstrated that apocynin inhibited the NLRP3 inflammasome and promoted angiogenesis at 3 days but promoted the NLRP3 inflammasome and inhibited angiogenesis at 7 and 14 days after stroke, which was mediated by regulating autophagy activation. Furthermore, RNA-Seq and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that apocynin injection resulted in PI3K-Akt signaling pathway enrichment after 7 days of MCAO. We then used an animal model to show that apocynin decreased the protein levels of phosphorylated PI3K and Akt and NF-κB p65, confirming that the PI3K-Akt-NF-κB pathway is involved in apocynin-mediated activation of inflammation and inhibition of angiogenesis.
CONCLUSIONS: NOX2-induced ROS production is a double-edged sword that exacerbates brain injury in the acute phase but promotes functional recovery. This effect appears to be achieved by inhibiting NLRP3 inflammasome activation and promoting angiogenesis via autophagy activation.

Keywords:  Angiogenesis; Autophagy; Ischemic stroke; NADPH; NLRP3 inflammasome; ROS

DOI:  https://doi.org/10.1186/s12974-022-02551-6
Exp Ther Med. 2022 Aug;24(2): 501

Plumbagin protects H9c2 cardiomyocytes against TBHP-induced cytotoxicity by alleviating ROS-induced apoptosis and modulating autophagy.

Qianrui Zhang, Haitan Fu, Wenjuan Gong, Feng Cao, Tao Wu, Fei Hu.

  Plumbagin (PLB) has been previously reported to alleviate myocardial ischemia/reperfusion injury in vivo. In the present study, the potential of plumbagin to protect against hydrogen peroxide-induced injury in cardiomyocytes was analyzed. Specifically, the cytoprotective effects of PLB were evaluated in H9c2 cardiomyocytes, in which oxidative stress was induced by tertiary butyl hydrogen peroxide (TBHP; 75 µM) treatment. After the cardiomyocytes were treated with different concentrations of PLB, cell viability, creatine kinase (CK) activity and lactate dehydrogenase (LDH) release were determined. The apoptosis rate and reactive oxygen species (ROS) levels were evaluated by flow cytometry. Western blot analyses of cleaved caspase-3, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4 (NOX4), and phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK) were performed. PLB pretreatment (5, 10 or 20 µM) restored TBHP-treated H9c2 cell viability (P<0.01). Additionally, PLB significantly decreased CK (P<0.01) and LDH activity (P<0.01). TBHP induced apoptosis and oxidative stress in cardiomyocytes, whereas PLB pretreatment significantly reduced the TBHP-induced apoptosis rate (P<0.01) and ROS levels (P<0.01). Furthermore, PLB resulted in a decrease in the expression of cleaved caspase-3, NOX4, and p-p38 MAPK in TBHP-treated H9c2 cells. The active marker of autophagosomes, LC3-II/LC3-I, was increased following treatment with PLB, indicating the induction of autophagy. The present study revealed the protective role of PLB against TBHP-induced cardiomyocyte injury via the alleviation of ROS-mediated apoptosis and induction of autophagy.

Keywords:  apoptosis; autophagy; myocardial injury; plumbagin; reactive oxygen species

DOI:  https://doi.org/10.3892/etm.2022.11428
Front Endocrinol (Lausanne). 2022 ;13 895360

LncRNA MALAT1 Aggravates Renal Tubular Injury via Activating LIN28A and the Nox4/AMPK/mTOR Signaling Axis in Diabetic Nephropathy.

Panai Song, Yinyin Chen, Zhiwen Liu, Hong Liu, Li Xiao, Lin Sun, Jiali Wei, Liyu He.

   Background: Diabetic nephropathy (DN) is a serious complication among patients with diabetes. Elucidating its pathogenesis is crucial for identifying novel biomarkers and therapeutic targets for DN.
Methods: DN tissues were harvested for examining MALAT1, LIN28A and Nox4. Human kidney-2 (HK-2) cells were treated with high glucose (HG) for establishing a cell model of DN. Cell viability was examined by MTT assay. HG-induced cell apoptosis and secretion of TNF-α and IL-6 were analyzed by TUNEL and ELISA assays, respectively. RIP and RNA pull-down assays were applied to analyze the interaction between MALAT1, LIN28A and Nox4 in HK-2 and human embryonic kidney 293T (HEK-293T) cells. A rat model of DN was established to determine the role of MALAT1 in DN in vivo.
Results: MALAT1, LIN28A and Nox4 were upregulated in DN tissues and HG-treated HK-2 cells. Overexpression of MALAT1, LIN28A or Nox4 reduced cell viability and enhanced cell apoptosis, ROS generation and secretion of inflammatory cytokines in HG-treated HK-2 cells, whereas knockdown of MALAT1, LIN28A or Nox4 exerted opposite effects. Furthermore, MALAT1 directly interacted with LIN28A. Moreover, MALAT1 facilitated the interaction between LIN28A and Nox4 to increase Nox4 stability. Knockdown of Nox4 relieved HG-induced injury by suppressing the AMPK/mTOR signaling in HK-2 cells. Knockdown of MALAT1 alleviated renal tubular epithelial injury by suppressing LIN28A and the Nox4/AMPK/TOR signaling in DN.
Conclusion: MALAT1 activates the AMPK/mTOR signaling via interacting with LIN28A to stabilize Nox4 mRNA, thereby aggravating high glucose-induced renal tubular epithelial injury. Our findings provide potential therapeutic targets for DN.

Keywords:  AMPK/mTOR signaling; LIN28A; MALAT1; diabetic nephropathy; renal tubular injury

DOI:  https://doi.org/10.3389/fendo.2022.895360
Aging (Albany NY). 2022 Jul 13. 14(undefined):

NOX4 promotes Kupffer cell inflammatory response via ROS-NLRP3 to aggravate liver inflammatory injury in acute liver injury.

Liping Zhai, Hongyan Pei, Yi Yang, Yu Zhu, Shuiliang Ruan.

   AIM: This work aimed to investigate the mechanism of NOX4 in promoting Kupffer cells (KCs) activation and tissue inflammatory response in acute liver injury.
METHODS: Initially, the mouse KCs were cultured in vitro. Thereafter, the NOX4 overexpression plasmid was transfected into KCs to construct the overexpression cell line. Then, KCs inflammatory response was induced by LPS + Nigericin treatment. CCK-8 assay was performed to detect cell viability, flow cytometry (FCM) was conducted to measure cell apoptosis, enzyme-linked immunosorbent assay (ELISA) was performed to detect inflammatory factor levels in the culture medium, NLRP3 and ASC expression in cells was detected by immunofluorescence (IF) staining, and ROS expression was detected by the DCFH-DA probe. Furthermore, the expression levels of NLRP3, ASC and Caspase-1 proteins were detected by Western-Blot (WB) assay. Furthermore, cells were pre-treated with NOX inhibitor or NAC to suppress NOX4 expression or ROS production, aiming to further investigate the effect on KCs inflammatory response. In mouse experiments, the NOX4 knockdown mice and wild-type (WT) mice were adopted for carrying out experiments. The mouse model of ALI was constructed with LPS and D-GalN treatment. Thereafter, the changes in tissue samples were detected by H&E staining, NLRP3 expression was measured by histochemical staining, inflammatory factors in tissues were analyzed by ELISA, and the levels of NLRP3, ASC and Caspase-1 proteins in tissues were detected by WB assay.
RESULTS: LPS induced KCs inflammatory response. NOX4 overexpression decreased the mouse viability and increased the apoptosis rate. The levels of inflammatory factors were up-regulated in the culture medium. In addition, ROS were activated, and the positive cell number increased. Moreover, NOX4 promoted NLRP3 activation and significantly increased the expression of NLRP3 and ASC. Pretreatment with NOX4 inhibitor or NAC antagonized the effects of NOX4 and suppressed the KCs inflammatory response. In the mouse model, NOX4 knockdown significantly suppressed the activation and inflammatory response of microglial cells in tissues, reducing the NLRP3 expression in tissues.
CONCLUSION: NOX4 activates the NLRP3 inflammasome via ROS to promote inflammatory response in KCs and the release of inflammatory factors, suppressing NOX4 can improve ALI in mice, and NOX4 is promising as a new target for ALI treatment.

Keywords:  Kupffer; NOX4; ROS; acute liver injury; inflammatory response

DOI:  https://doi.org/10.18632/aging.204173