Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1–3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1–NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.

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Olfactomedin 4 contributes to hydrogen peroxide-induced NADPH oxidase activation and apoptosis in mouse neutrophils

AJP Cell Physiology ◽

10.1152/ajpcell.00336.2017 ◽

2018 ◽

Vol 315 (4) ◽

pp. C494-C501 ◽

Cited By ~ 3

Author(s):

Wenli Liu ◽

Yueqin Liu ◽

Hongzhen Li ◽

Griffin P. Rodgers

Keyword(s):

Hydrogen Peroxide ◽

Nadph Oxidase ◽

Molecular Mechanisms ◽

Oxidase Inhibitor ◽

Superoxide Production ◽

Wild Type ◽

Escherichia Coli Bacteria ◽

Induced Apoptosis ◽

Nadph Oxidase Activation ◽

Deficient Mice

Neutrophils increase production of reactive oxygen species, including superoxide, hydrogen peroxide (H2O2), and hydroxyl radical, to destroy invading microorganisms under pathological conditions. Conversely, oxidative stress conditions, such as the presence of H2O2, induce neutrophil apoptosis, which helps to remove neutrophils after inflammation. However, the detailed molecular mechanisms that are involved in the latter process have not been elucidated. In this study, we investigated the potential role of olfactomedin 4 (Olfm4) in H2O2-induced superoxide production and apoptosis in mouse neutrophils. We have demonstrated that Olfm4 is not required for maximal-dosage PMA- and Escherichia coli bacteria-induced superoxide production, but Olfm4 contributes to suboptimal-dosage PMA- and H2O2-induced superoxide production. Using an NADPH oxidase inhibitor and gp91phox-deficient mouse neutrophils, we found that NAPDH oxidase was required for PMA-stimulated superoxide production and that Olfm4 mediated H2O2-induced superoxide production through NADPH oxidase, in mouse neutrophils. We have shown that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-induced apoptosis compared with neutrophils from wild-type mice. We also demonstrated that neutrophils from Olfm4-deficient mice exhibited reduced H2O2-stimulated mitochondrial damage and membrane permeability, and as well as reduced caspase-3 and caspase-9 activity, compared with neutrophils from wild-type mice. Moreover, the cytoplasmic translocation of the proapoptotic mitochondrial proteins Omi/HtrA2 and Smac/DIABLO in response to H2O2was reduced in neutrophils from Olfm4-deficient mice compared with neutrophils from wild-type mice. Our study demonstrates that Olfm4 contributes to H2O2-induced NADPH oxidase activation and apoptosis in mouse neutrophils. Olfactomedin 4 might prove to be a potential target for future studies on inflammatory neutrophil biology and for inflammatory disease treatment.

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Reciprocal interactions between protein kinase C and components of the NADPH oxidase complex may regulate superoxide production by neutrophils stimulated with a phorbol ester.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)34132-7 ◽

1994 ◽

Vol 269 (14) ◽

pp. 10813-10819 ◽

Cited By ~ 2

Author(s):

J.T. Curnutte ◽

R.W. Erickson ◽

J. Ding ◽

J.A. Badwey

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Nadph Oxidase ◽

Phorbol Ester ◽

Superoxide Production ◽

Reciprocal Interactions ◽

Kinase C ◽

Nadph Oxidase Complex

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Leishmania pifanoi Amastigotes Avoid Macrophage Production of Superoxide by Inducing Heme Degradation

Infection and Immunity ◽

10.1128/iai.73.12.8322-8333.2005 ◽

2005 ◽

Vol 73 (12) ◽

pp. 8322-8333 ◽

Cited By ~ 48

Author(s):

Nam-Kha Pham ◽

Jennifer Mouriz ◽

Peter E. Kima

Keyword(s):

Nadph Oxidase ◽

Subcellular Fractions ◽

Superoxide Production ◽

Heme Oxygenase 1 ◽

Heme Degradation ◽

Nadph Oxidase Complex ◽

Immature Form ◽

Oxidase Enzyme ◽

Infected Cells ◽

Rate Limiting

ABSTRACT Whereas infections of macrophages by promastigote forms of Leishmania mexicana pifanoi induce the production of superoxide, infections by amastigotes barely induce superoxide production. Several approaches were employed to gain insight into the mechanism by which amastigotes avoid eliciting superoxide production. First, in experiments with nitroblue tetrazolium, we found that 25% of parasitophorous vacuoles (PVs) that harbor promastigotes are positive for the NADPH oxidase complex, in contrast to only 2% of PVs that harbor amastigotes. Second, confocal microscope analyses of infected cells labeled with antibodies to gp91phox revealed that this enzyme subunit is found in PVs that harbor amastigotes. Third, in immunoblots of subcellular fractions enriched with PVs from amastigote-infected cells and probed with antibodies to gp91phox, only the 65-kDa premature form of gp91phox was found. In contrast, subcellular fractions from macrophages that ingested zymosan particles contained both the 91- and 65-kDa forms of gp91phox. This suggested that only the immature form of gp91phox is recruited to PVs that harbor amastigotes. Given that gp91phox maturation is dependent on the availability of heme, we found that infections by Leishmania parasites induce an increase in heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation. Infections by amastigotes performed in the presence of metalloporphyrins, which are inhibitors of HO-1, resulted in superoxide production by infected macrophages. Taken together, we propose that Leishmania amastigotes avoid superoxide production by inducing an increase in heme degradation, which results in blockage of the maturation of gp91phox, which prevents assembly of the NADPH oxidase enzyme complex.

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Glutamine plays a role in superoxide production and the expression of p47phox, p22phox and gp91phox in rat neutrophils

Clinical Science ◽

10.1042/cs1030403 ◽

2002 ◽

Vol 103 (4) ◽

pp. 403-408 ◽

Cited By ~ 44

Author(s):

Tania Cristina PITHON-CURI ◽

Adriana C. LEVADA ◽

Lúcia R. LOPES ◽

Sonia Q. DOI ◽

Rui CURI

Keyword(s):

Amino Acid ◽

Cytochrome C ◽

Nadph Oxidase ◽

Superoxide Anion ◽

Scintillation Counting ◽

Superoxide Production ◽

Glutamine Metabolism ◽

Nadph Oxidase Complex ◽

O2 Production

The effect of glutamine on the activity of the NADPH oxidase complex from rat neutrophils was investigated. Superoxide anion (O2-) production was assessed: (1) by scintillation counting by using lucigenin, and (2) by reduction of cytochrome c over 10min. The effects of glutamine and PMA on the expression of the NADPH oxidase components p22phox, gp91phox and p47phox were also determined. Glutamine at 1 and 2mM increased O2- generation in the presence of PMA by 100% and 74% respectively, in neutrophils maintained previously for 3h in medium deprived of this amino acid. DON (6-diazo-5-oxo-l-norleucine), an inhibitor of phosphate-dependent glutaminase and thus of glutamine metabolism, caused a significant decrease in O2- production by neutrophils stimulated with PMA both in the absence (44%) and in the presence (66%) of glutamine. PMA markedly increased the expression of gp91phox, p22phox and p47phox mRNAs. Glutamine (2mM) increased the expression of these three proteins both in the absence and in the presence of PMA. We postulate that glutamine leads to O2- production in neutrophils, probably via the generation of ATP and regulation of the expression of components of NADPH oxidase.

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Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils

Infection and Immunity ◽

10.1128/iai.73.7.4161-4170.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4161-4170 ◽

Cited By ~ 146

Author(s):

David Bergin ◽

Emer P. Reeves ◽

Julie Renwick ◽

Frans B. Wientjes ◽

Kevin Kavanagh

Keyword(s):

Immune Response ◽

Nadph Oxidase ◽

Galleria Mellonella ◽

Cell Types ◽

Oxidase Inhibitor ◽

Superoxide Production ◽

Human Neutrophils ◽

Greater Wax Moth ◽

Nadph Oxidase Complex ◽

Insect Hemocytes

ABSTRACT The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils. Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins homologous to gp91phox, p67phox, p47phox, and the GTP-binding protein rac 2. A protein equivalent to p40phox was not detected in insect hemocytes. Immunofluorescence analysis localized insect 47-kDa and 67-kDa proteins throughout the cytosol and in the perinuclear region. Hemocyte 67-kDa and 47-kDa proteins were immunoprecipitated and analyzed by matrix-assisted laser desorption ionization—time of flight analysis. The results revealed that the hemocyte 67-kDa and 47-kDa proteins contained peptides matching those of p67phox and p47phox of human neutrophils. The results presented here indicate that insect hemocytes phagocytose and kill bacterial and fungal cells by a mechanism similar to the mechanism used by human neutrophils via the production of superoxide. We identified proteins homologous to a number of proteins essential for superoxide production in human neutrophils and demonstrated that significant regions of the 67-kDa and 47-kDa insect proteins are identical to regions of the p67phox and p47phox proteins of neutrophils.

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Superoxide generation and tyrosine kinase

Biochemistry and Cell Biology ◽

10.1139/o99-068 ◽

2000 ◽

Vol 78 (1) ◽

pp. 11-17 ◽

Cited By ~ 10

Author(s):

Su Yang ◽

Martha Hardaway ◽

George Sun ◽

William L Ries ◽

L Lyndon Key Jr

Keyword(s):

Cell Surface ◽

Nadph Oxidase ◽

Tyrosine Kinase ◽

B Lymphocytes ◽

Specific Inhibitor ◽

Superoxide Production ◽

Regulatory Subunit ◽

Superoxide Generation ◽

Herbimycin A ◽

Nadph Oxidase Complex

NADPH oxidase is a multi-subunit enzyme complex responsible for superoxide generation in many cells, for example, B-lymphocytes and osteoclasts. NADPH oxidase is localized on the cell surface and generates superoxide extracellularly. After synthesis, components of this oxidase are transported to the cell membrane where the functional NADPH oxidase complex is assembled. The mechanism by which the membrane-bound components are transported to the cell surface of osteoclasts remains unclear. In this study, we examined the role of tyrosine kinase activity in the transport of NADPH oxidase components. When B-lymphocytes and osteoclasts were treated with herbimycin A, a specific inhibitor of tyrosine kinase, superoxide production was significantly decreased. The amount of p91, the catalytic subunit of NADPH oxidase, was decreased in the cellular membrane of herbimycin A treated cells compared to untreated controls. Similar results were obtained for the movement of a regulatory subunit of the NADPH oxidase complex, p47, in B-lymphocytes. Thus, inhibition of tyrosine kinase decreases superoxide production by disrupting the translocation of the NADPH oxidase complex.

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Nano-Curcumin Regulates p53 Phosphorylation and PAI-1 Expression during Bleomycin Induced Injury in Alveolar Basal Epithelial Cells

Current Bioactive Compounds ◽

10.2174/1573407214666180727093612 ◽

2020 ◽

Vol 16 (1) ◽

pp. 85-89

Author(s):

Mahesh M. Gouda ◽

Ashwini Prabhu ◽

Varsha Reddy S.V. ◽

Rafa Jahan ◽

Yashodhar P. Bhandary

Keyword(s):

Epithelial Cells ◽

Lung Injury ◽

Molecular Mechanisms ◽

A549 Cells ◽

Plasminogen Activator Inhibitor ◽

Underlying Mechanism ◽

Protective Role ◽

Alveolar Epithelial Cells ◽

Cellular Damage

Background: Bleomycin (BLM) is known to cause DNA damage in the Alveolar Epithelial Cells (AECs). It is reported that BLM is involved in the up-regulation of inflammatory molecules such as neutrophils, macrophages, chemokines and cytokines. The complex underlying mechanism for inflammation mediated progression of lung injury is still unclear. This investigation was designed to understand the molecular mechanisms associated with p53 mediated modulation of Plasminogen Activator Inhibitor-I (PAI-I) expression and its regulation by nano-curcumin formulation. Methods: A549 cells were treated with BLM to cause the cellular damage in vitro and commercially available nano-curcumin formulation was used as an intervention. Cytotoxic effect of nano-curcumin was analyzed using Methyl Thiazolyl Tetrazolium (MTT) assay. Protein expressions were analyzed using western blot to evaluate the p53 mediated changes in PAI-I expression. Results: Nano-curcumin showed cytotoxicity up to 88.5 % at a concentration of 20 μg/ml after 48 h of treatment. BLM exposure to the cells activated the phosphorylation of p53, which in turn increased PAII expression. Nano-curcumin treatment showed a protective role against phosphorylation of p53 and PAI-I expression, which in turn regulated the fibro-proliferative phase of injury induced by bleomycin. Conclusion: Nano-curcumin could be used as an effective intervention to regulate the severity of lung injury, apoptosis of AECs and fibro-proliferation during pulmonary injury.

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Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

BMC Cardiovascular Disorders ◽

10.1186/s12872-021-02128-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Na Wu ◽

Chengying Li ◽

Bin Xu ◽

Ying Xiang ◽

Xiaoyue Jia ◽

...

Keyword(s):

Molecular Mechanisms ◽

Cardiac Fibroblasts ◽

Circular Rna ◽

Protective Role ◽

Luciferase Reporter ◽

Loss Of Function ◽

Candidate Target ◽

Paired Control ◽

Reporter Assays ◽

And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

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Analysis of MCM Proteins’ Role as a Potential Target of Statins in Patients with Acute Type A Aortic Dissection through Bioinformatics

Genes ◽

10.3390/genes12030387 ◽

2021 ◽

Vol 12 (3) ◽

pp. 387

Author(s):

Zheyong Liang ◽

Yongjian Zhang ◽

Qiang Chen ◽

Junjun Hao ◽

Haichen Wang ◽

...

Keyword(s):

Aortic Dissection ◽

Molecular Mechanisms ◽

Vascular Diseases ◽

Enrichment Analysis ◽

Type A ◽

Protective Role ◽

Gene Expression Omnibus ◽

Pathway Enrichment Analysis ◽

Acute Type ◽

Type A Aortic Dissection

Acute aortic dissection is one of the most severe vascular diseases. The molecular mechanisms of aortic expansion and dissection are unclear. Clinical studies have found that statins play a protective role in aortic dissection development and therapy; however, the mechanism of statins’ effects on the aorta is unknown. The Gene Expression Omnibus (GEO) dataset GSE52093, GSE2450and GSE8686 were analyzed, and genes expressed differentially between aortic dissection samples and normal samples were determined using the Networkanalyst and iDEP tools. Weight gene correlation network analysis (WGCNA), functional annotation, pathway enrichment analysis, and the analysis of the regional variations of genomic features were then performed. We found that the minichromosome maintenance proteins (MCMs), a family of proteins targeted by statins, were upregulated in dissected aortic wall tissues and play a central role in cell-cycle and mitosis regulation in aortic dissection patients. Our results indicate a potential molecular target and mechanism for statins’ effects in patients with acute type A aortic dissection.

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