bims-noxint 2021-03-28 papers

bims-noxint

Biomed News

on NADPH oxidases in tumorigenesis

Issue of 2021–03–28
eight papers selected by
Laia Caja Puigsubira, Uppsala University

Nuclear factor-kappaB regulates the transcription of NADPH oxidase 1 in human alveolar epithelial cells.
Inhibition of NADPH oxidase 4 attenuates lymphangiogenesis and tumor metastasis in breast cancer.
microRNA-363-3p reduces endothelial cell inflammatory responses in coronary heart disease via inactivation of the NOX4-dependent p38 MAPK axis.
Mechanisms underlying the hormetic effect of conjugated linoleic acid: focus on Nrf2, mitochondria and NADPH oxidases.
Regulation of dual oxidase hydrogen peroxide synthesis results in an epithelial respiratory burst.
Fibronectin regulates anoikis resistance via cell aggregate formation.
LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4.
Pharmacological Regulation of Cytochrome P450 metabolites of arachidonic acid attenuates Cardiac Injury in Diabetic Rats: The Role of Cytochromes P450 Metabolites in Diabetic Cardiomyopathy.

BMC Pulm Med. 2021 Mar 23. 21(1): 98

Nuclear factor-kappaB regulates the transcription of NADPH oxidase 1 in human alveolar epithelial cells.

Weijing Wu, Li Li, Xiaoshan Su, Zhixing Zhu, Xiaoping Lin, Jiamin Zhang, Zesen Zhuang, Hongyi Cai, Wenjie Huang.

   OBJECTIVE: Acute lung injury (ALI) is characterized by inflammation and oxidative stress. Nuclear factor-kappaB (NF-κB) mediates the expression of various inflammation-related genes, including the NADPH oxidase family. This study aimed to identify the potential regulatory role of NF-κB on NADPH oxidases in tumor necrosis factor-α (TNF-α)-induced oxidative stress in human alveolar epithelial cells.
METHODS: A549 cells were treated with TNF-α for 24 h to establish ALI cell models. RT-PCR, western blot, assessment of oxidative stress, Alibaba 2.1 online analysis, electrophoretic mobility shift assays and luciferase reporter analysis were employed to identify the potential regulatory role of NF-κB on NADPH oxidases in TNF-α-induced oxidative stress in human alveolar epithelial cells.
RESULTS: The expression of NF-κB/p65 was notably upregulated in TNF-α-stimulated A549 cells. NF-κB knockdown by siRNA significantly inhibited the TNF-α-induced oxidative stress. Moreover, NF-κB/p65 siRNA could inhibit the activation of NOX1, NOX2 and NOX4 mRNA and protein expression in TNF-α-stimulated A549 cells. The next study demonstrated that NF-κB activated the transcription of NOX1 by binding to the -261 to -252 bp (NOX1/κB2, TAAAAATCCC) region of NOX1 promoter in TNF-α-stimulated A549 cells.
CONCLUSION: Our data demonstrated that NF-κB can aggravate TNF-α-induced ALI by regulating the oxidative stress response and the expression of NOX1, NOX2 and NOX4. Moreover, NF-κB could promote the NOX1 transcriptional activity via binding its promoter in TNF-α-stimulated A549 cells.

Keywords:  Acute lung injury; NADPH oxidase; NOX1; Nuclear factor-kappaB; Reactive oxygen species; Tumor necrosis factor-α

DOI:  https://doi.org/10.1186/s12890-021-01464-z
FASEB J. 2021 Apr;35(4): e21531

Inhibition of NADPH oxidase 4 attenuates lymphangiogenesis and tumor metastasis in breast cancer.

Xinzhao Wang, Zhaoyun Liu, Jujie Sun, Xiang Song, Mengxue Bian, Fukai Wang, Feng Yan, Zhiyong Yu.

  Lymphangiogenesis is thought to contribute to promote tumor cells to enter lymphatic vessels and plant at a secondary site. Endothelial cells are the cornerstone of the generation of new lymphatic vessels. NADPH oxidase 4 (Nox4) is the most abundant one of NADPH oxidases in endothelial cells and the most studied one in relevance with cancer. Our purpose is to analyze the relationship between Nox4 and lymphangiogenesis and find out whether the newborn lymphatic vessels lead to cancer metastasis. We first explored the expression of Nox4 in lymphatic endothelial cells of primary invasive breast tumors and human normal mammary glands using GEO databases and found that Nox4 was upregulated in primary invasive breast tumors samples. In addition, its high expression correlated with lymph node metastasis in breast cancer patients. Nox4 could increase the tube formation and lymphatic vessel sprouting in a three-dimensional setting. In vivo, inhibition of Nox4 in 4T1 tumor-bearing mice could significantly decrease the tumor lymphangiogenesis and metastasis. Nox4 may increase tumor lymphangiogenesis via ROS/ERK/CCL21 pathway and attract CCR7-positive breast cancer cells to entry lymphatic vessels and distant organs. In conclusion, our results show that Nox4 is a factor that promotes lymphangiogenesis and is a potential target of antitumor metastasis.

Keywords:  Nox4; breast cancer; lymphangiogenesis; metastasis

DOI:  https://doi.org/10.1096/fj.202002533R
Aging (Albany NY). 2021 03 19. 13

microRNA-363-3p reduces endothelial cell inflammatory responses in coronary heart disease via inactivation of the NOX4-dependent p38 MAPK axis.

Tao Zhou, Suining Li, Liehong Yang, Daokang Xiang.

  Coronary heart disease (CHD) is one of the leading causes of heart-associated deaths worldwide. This study aimed to investigate the mechanism by which microRNA-363-3p (miR-363-3p) regulates endothelial injury induced by inflammatory responses in CHD. The expression patterns of miR-363-3p, NADPH oxidase 4 (NOX4), and p38 MAPK/p-p38 MAPK were examined in an established atherosclerosis (AS) model in C57BL/6 mice and in isolated coronary arterial endothelial cells (CAECs) after gain- or loss-of-function experiments. We also measured the levels of inflammatory factors (IL-6, ICAM-1, IL-10 and IL-1β), hydrogen peroxide (H2O2), and catalase (CAT) activity, followed by detection of cell viability and apoptosis. In AS, miR-363-3p was downregulated and NOX4 was upregulated, while miR-363-3p was identified as targeting NOX4 and negatively regulating its expression. The AS progression was reduced in NOX4 knockout mice. Furthermore, miR-363-3p resulted in a decreased inflammatory response, oxidative stress, and cell apoptosis in CAECs while augmenting their viability via blockade of the p38 MAPK signaling pathway. Overall, miR-363-3p hampers the NOX4-dependent p38 MAPK axis to attenuate apoptosis, oxidative stress injury, and the inflammatory reaction in CAECs, thus protecting CAECs against CHD. This finding suggests the miR-363-3p-dependent NOX4 p38 MAPK axis as a promising therapeutic target for CHD.

Keywords:  NADPH oxidase 4; coronary heart disease; endothelial cell; microRNA-363-3p; p38 MAPK

DOI:  https://doi.org/10.18632/aging.202721
Free Radic Biol Med. 2021 Mar 19. pii: S0891-5849(21)00167-2. [Epub ahead of print]

Mechanisms underlying the hormetic effect of conjugated linoleic acid: focus on Nrf2, mitochondria and NADPH oxidases.

Di Cristofano M, Ferramosca A, Di Giacomo M, Fusco C, Boscaino F, Luongo D, V Lombardi-Aufiero, Maurano F, Cocca E, Mazzarella G, Zara V, Rossi M, Bergamo P.

  Nuclear factor erythroid 2-related factor2 (Nrf2) is a redox-sensitive transcription factor. Its activation by low dietary intake of ligands leads to antioxidant effects (eustress), while pro-oxidant effects (oxidative distress) may be associated with high doses. NADPH oxidases (NOXs) and the mitochondrial electron transport chain are the main sources of intracellular ROS, but their involvement in the biphasic/hormetic activity elicited by Nrf2 ligands is not fully understood. In this study, we investigated the involvement of NOX expression and mitochondrial function in the hormetic properties of omega-3 typically present in fish oil (FO) and conjugated linoleic acid (CLA) in the mouse liver. Four-week administration of FO, at both low and high doses (L-FO and H-FO) improves Nrf2-activated cyto-protection (by phase 2 enzymes), while a significant increase in respiration efficiency occurs in the liver mitochondria of H-FO BALB/c mice. Eustress conditions elicited by low dose CLA (L-CLA) are associated with increased activity of phase 2 enzymes, and with higher NOX1-2, mitochondrial defences, mitochondrial uncoupling protein 2 (UCP2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression, compared with controls. Steatogenic effects (lipid accumulation and alteration of lipid metabolism) elicited by high CLA (H-CLA) elicited that are associated with oxidative distress, increased mitochondrial complex I/III activity and reduced levels of phase 2 enzymes, in comparison with L-CLA-treated mice. Our results confirm the steatogenic activity of H-CLA and first demonstrate the role of NOX1 and NOX2 in the eustress conditions elicited by L-CLA. Notably, the negative association of the Nrf2/PGC-1α axis with the different CLA doses provides new insight into the mechanisms underlying the hormetic effect triggered by this Nrf2 ligand.

Keywords:  Conjugated Linoleic Acid; hormetic effect; mitochondrial function; redox homeostasis

DOI:  https://doi.org/10.1016/j.freeradbiomed.2021.03.015
Redox Biol. 2021 Mar 05. pii: S2213-2317(21)00079-3. [Epub ahead of print]41 101931

Regulation of dual oxidase hydrogen peroxide synthesis results in an epithelial respiratory burst.

Gregory E Conner.

  Redox status is a central determinant of cellular activities and redox imbalance is correlated with numerous diseases. NADPH oxidase activity results in formation of H2O2, that, in turn, sets cellular redox status, a key regulator of cellular homeostasis and responses to external stimuli. Hydrogen peroxide metabolism regulates cell redox status by driving changes in protein cysteine oxidation often via cycling of thioredoxin/peroxiredoxin and glutathione; however, regulation of enzymes controlling synthesis and utilization of H2O2 is not understood beyond broad outlines. The data presented here show that calcium-stimulated epithelial Duox H2O2 synthesis is transient, independent of intracellular calcium renormalization, H2O2 scavenging by antioxidant enzymes, or substrate depletion. The data support existence of a separate mechanism that restricts epithelial H2O2 synthesis to a burst and prevents harmful changes in redox tone following continuous stimulation. Elucidation of this H2O2 synthesis tempering mechanism is key to understanding cellular redox regulation and control of downstream effectors, and this observation provides a starting point for investigation of the mechanism that controls H2O2-mediated increases in redox tone.

Keywords:  Cell redox status; Duox; Hydrogen peroxide; NADPH oxidase

DOI:  https://doi.org/10.1016/j.redox.2021.101931
Cancer Lett. 2021 Mar 23. pii: S0304-3835(21)00116-6. [Epub ahead of print]

Fibronectin regulates anoikis resistance via cell aggregate formation.

Hyeong-Jun Han, Jee Young Sung, Su-Hyeon Kim, Un-Jung Yun, Hyeryeong Kim, Eun Ju Jang, Ha-Eun Yoo, Eun Kyung Hong, Sung-Ho Goh, Aree Moon, Jae-Seon Lee, Sang-Ku Ye, Jaegal Shim, Yong-Nyun Kim.

  The loss of cell-matrix interactions induces apoptosis, known as anoikis. For successful distant metastasis, circulating tumor cells (CTCs) that have lost matrix attachment need to acquire anoikis resistance in order to survive. Cell aggregate formation confers anoikis resistance, and CTC clusters are more highly metastatic compared to single cells; however, the molecular mechanisms underlying this aggregation are not well understood. In this study, we demonstrated that cell detachment increased cell aggregation and upregulated fibronectin (FN) levels in lung and breast cancer cells, but not in their normal counterparts. FN knockdown decreased cell aggregation and increased anoikis. In addition, cell detachment induced cell-cell adhesion proteins, including E-cadherin, desmoglein-2, desmocollin-2/3, and plakoglobin. Interestingly, FN knockdown decreased the levels of desmoglein-2, desmocollin-2/3, and plakoglobin, but not E-cadherin, suggesting the involvement of desmosomal junction in cell aggregation. Accordingly, knockdown of desmoglein-2, desmocollin-2, or plakoglobin reduced cell aggregation and increased cell sensitivity to anoikis. Previously, we reported that NADPH oxidase 4 (Nox4) upregulation is important for anoikis resistance. Nox4 inhibition by siRNA or apocynin decreased cell aggregation and increased anoikis with the downregulation of FN, and, consequently, decreased desmoglein-2, desmocollin-2/3, or plakoglobin. The coexpression of Nox4 and FN was found to be significant in lung and breast cancer patients, based on cBioPortal data. In vivo mouse lung metastasis model showed that FN knockdown suppressed lung metastasis and thus enhanced survival. FN staining of micro tissue array revealed that FN expression was positive for human lung cancer (61%) and breast cancer (58%) patients. Furthermore, the expression levels of FN, desmoglein-2, desmocollin-2, and plakoglobin were significantly correlated with the poor survival of lung and breast cancer patients, as per the Kaplan-Meier plotter analysis. Altogether, our data suggest that FN upregulation and enhanced desmosomal interactions are critical for cell aggregation and anoikis resistance upon cell detachment.

Keywords:  Anoikis resistance; Circulating tumor cells; Fibronectin

DOI:  https://doi.org/10.1016/j.canlet.2021.03.011
Int J Ophthalmol. 2021 ;14(3): 349-355

LRG1 promotes epithelial-mesenchymal transition of retinal pigment epithelium cells by activating NOX4.

Li Zhou, De-Peng Shi, Wen-Juan Chu, Ling-Ling Yang, Hai-Feng Xu.

   AIM: To investigate the effect of leucine-rich-alpha-2-glycoprotein 1 (LRG1) on epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells, and to explore the role of NADPH oxidase 4 (NOX4).
METHODS: RPE cells (ARPE-19 cell line) were treated with transforming growth factor-β1 (TGF-β1) to induce EMT. Changes of the mRNA and protein expression levels of LRG1 were tested in the TGF-β1 treated cells. The recombinant human LRG1 protein (rLRG1) and siRNA of LRG1 were used to establish accumulation of exogenous LRG1 model and the down-regulation of LRG1 model in ARPE-19 cells respectively, and to detect EMT-related markers including fibronectin, α-smooth muscle actin (α-SMA) and zonula occludens-1 (ZO-1). The mRNA and protein expression level of NOX4 were measured according to the above treatments. VAS2870 was used as a NOX4 inhibitor in rLRG1-treated cells. EMT-related markers were detected to verify the effect of NOX4 in the process of EMT.
RESULTS: TGF-β1 promoted the expression of LRG1 at both the mRNA and protein levels during the process of EMT which showed the up-regulation of fibronectin and α-SMA, as well as the down-regulation of ZO-1. Furthermore, the rLRG1 promoted EMT of ARPE-19 cells, which manifested high levels of fibronectin and α-SMA and low level of ZO-1, whereas knockdown of LRG1 prevented EMT by decreasing the expressions of fibronectin and α-SMA and increasing the expression of ZO-1 in ARPE-19 cells. Besides, the rLRG1 activated and LRG1 siRNA suppressed NOX4 expression. EMT was inhibited when VAS2870 was used in the rLRG1-treated cells.
CONCLUSION: These results for the first time demonstrate that LRG1 promotes EMT of RPE cells by activating NOX4, which may provide a novel direction to explore the mechanisms of subretinal fibrosis.

Keywords:  NADPH oxidase 4; epithelial-mesenchymal transition; leucine-rich-alpha-2-glycoprotein 1; retinal pigment epithelium cells; subretinal fibrosis

DOI:  https://doi.org/10.18240/ijo.2021.03.03
Transl Res. 2021 Mar 18. pii: S1931-5244(21)00073-6. [Epub ahead of print]

Pharmacological Regulation of Cytochrome P450 metabolites of arachidonic acid attenuates Cardiac Injury in Diabetic Rats: The Role of Cytochromes P450 Metabolites in Diabetic Cardiomyopathy.

Lynn M Alaeddine, Frederic Harb, Maysaa Hamza, Batoul Dia, Nahed Mogharbil, Nadim S Azar, Mohamed H Noureldein, Mirella El Khoury, Ramzi Sabra, Assaad A Eid.

  Diabetic cardiomyopathy (DCM) is a well-established complication of type 1 and type 2 diabetes associated with a high rate of morbidity and mortality. DCM is diagnosed at advanced and irreversible stages. Therefore, it is of utmost need to identify novel mechanistic pathways involved at early stages to prevent or reverse the development of DCM. In vivo experiments were performed on type 1 diabetic rats (T1DM). Functional and structural studies of the heart were executed and correlated with mechanistic assessments exploring the role of cytochromes P450 metabolites, the 20-hydroxyeicosatetraenoic acids (20-HETEs) and epoxyeicosatrienoic acids (EETs), and their crosstalk with other homeostatic signaling molecules. Our data displays that hyperglycemia results in CYP4A upregulation and CYP2C11 downregulation in the left ventricles (LV) of T1DM rats, paralleled by a differential alteration in their metabolites 20-HETEs (increased) and EETs (decreased). These changes are concomitant with reductions in cardiac outputs, LV hypertrophy, fibrosis, and increased activation of cardiac fetal and hypertrophic genes. Besides, pro-fibrotic cytokine TGF-ß overexpression and NADPH (Nox4) dependent-ROS overproduction are also correlated with the observed cardiac functional and structural modifications. Of interest, these observations are attenuated when T1DM rats are treated with 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), which blocks EETs metabolism, or N-hydroxy-N'-(4-butyl-2-methylphenol)Formamidine (HET0016), which inhibits 20-HETEs formation. Taken together, our findings confer pioneering evidence about a potential interplay between CYP450-derived metabolites and Nox4/TGF-β axis leading to DCM. Pharmacologic interventions targeting the inhibition of 20-HETEs synthesis or the activation of EETs synthesis may offer novel therapeutic approaches to treat DCM.

Keywords:  CYP450 Metabolites; Diabetic Cardiomyopathy; NADPH oxidases; Reactive oxygen species; TGF-ß

DOI:  https://doi.org/10.1016/j.trsl.2021.03.010