Molecular and Cellular Biology

NADPH Oxidases

Publications

Publications

Main Representative Publications

Xavier De Deken:    

  • Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin.
    Related Articles

    Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin.

    Thyroid. 2016 Oct;26(10):1499-1512

    Authors: Eskalli Z, Achouri Y, Hahn S, Many MC, Craps J, Refetoff S, Liao XH, Dumont JE, Van Sande J, Corvilain B, Miot F, De Deken X

    Abstract
    BACKGROUND: The dual oxidases (Duox) are involved in hydrogen peroxide generation, which is essential for thyroid hormone synthesis, and therefore they are markers of thyroid function. During inflammation, cytokines upregulate DUOX gene expression in the airway and the intestine, suggesting a role for these proteins in innate immunity. It was previously demonstrated that interleukin-4 (IL-4) upregulates DUOX gene expression in thyrocytes. Although the role of IL-4 in autoimmune thyroid diseases has been studied extensively, the effects of IL-4 on thyroid physiology remain largely unknown. Therefore, a new animal model was generated to study the impact of IL-4 on thyroid function.
    METHODS: Transgenic (Thyr-IL-4) mice with thyroid-targeted expression of murine IL-4 were generated. Transgene expression was verified at the mRNA and protein level in thyroid tissues and primary cultures. The phenotype of the Thyr-IL-4 animals was characterized by measuring serum thyroxine (T4) and thyrotropin levels and performing thyroid morphometric analysis, immunohistochemistry, whole transcriptome sequencing, quantitative reverse transcription polymerase chain reaction, and ex vivo thyroid function assays.
    RESULTS: Thyrocytes from two Thyr-IL-4 mouse lines (#30 and #52) expressed IL-4, which was secreted into the extracellular space. Although 10-month-old transgenic animals had T4 and thyrotropin serum levels in the normal range, they had altered thyroid follicular structure with enlarged follicles composed of elongated thyrocytes containing numerous endocytic vesicles. These follicles were positive for T4 staining the colloid, indicating their capacity to produce thyroid hormones. RNA profiling of Thyr-IL-4 thyroid samples revealed modulation of multiple genes involved in inflammation, while no major leukocyte infiltration could be detected. Upregulated expression of Duox1, Duoxa1, and the pendrin anion exchanger gene (Slc26a4) was detected. In contrast, the iodide symporter gene Slc5a5 was markedly downregulated resulting in impaired iodide uptake and reduced thyroid hormone levels in transgenic thyroid tissue. Hydrogen peroxide production was increased in Thyr-IL-4 thyroid tissue compared with wild-type animals, but no significant oxidative stress could be detected.
    CONCLUSIONS: This is the first study to show that ectopic expression of IL-4 in thyroid tissue upregulates Duox1/Duoxa1 and Slc26a4 expression in the thyroid. The present data demonstrate that IL-4 could affect thyroid morphology and function, mainly by downregulating Slc5a5 expression, while maintaining a normal euthyroid phenotype.

    PMID: 27599561 [PubMed - in process]

  • NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.
    Related Articles

    NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.

    Proc Natl Acad Sci U S A. 2015 Apr 21;112(16):5051-6

    Authors: Ameziane-El-Hassani R, Talbot M, de Souza Dos Santos MC, Al Ghuzlan A, Hartl D, Bidart JM, De Deken X, Miot F, Diallo I, de Vathaire F, Schlumberger M, Dupuy C

    Abstract
    Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.

    PMID: 25848056 [PubMed - indexed for MEDLINE]

  • Roles of DUOX-mediated hydrogen peroxide in metabolism, host defense, and signaling.
    Related Articles

    Roles of DUOX-mediated hydrogen peroxide in metabolism, host defense, and signaling.

    Antioxid Redox Signal. 2014 Jun 10;20(17):2776-93

    Authors: De Deken X, Corvilain B, Dumont JE, Miot F

    Abstract
    SIGNIFICANCE: Among the NADPH oxidases, the dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially called thyroid oxidases, based on their high level of expression in thyroid tissue. Genetic alterations causing inherited hypothyroidism clearly demonstrate their physiological implication in thyroid hormonogenesis. However, a growing list of biological functions triggered by DUOX-dependent reactive oxygen species (ROS) in highly differentiated mucosae have recently emerged.
    RECENT ADVANCES: A role of DUOX enzymes as ROS providers for lactoperoxidase-mediated killing of invading pathogens has been well established and a role in bacteria chemorepulsion has been proposed. Control of DUOX expression and activity by inflammatory molecules and immune receptor activation consolidates their contributions to innate immune defense of mucosal surfaces. Recent studies conducted in ancestral organisms have identified effectors of DUOX redox signaling involved in wound healing including epithelium regeneration and leukocyte recruitment. Moreover, local generation of hydrogen peroxide (H2O2) by DUOX has also been suggested to constitute a positive feedback loop to promote receptor signaling activation.
    CRITICAL ISSUES: A correct balance between H2O2 generation and detoxification mechanisms must be properly maintained to avoid oxidative damages. Overexpression of DUOX genes has been associated with an increasing number of chronic inflammatory diseases. Furthermore, H2O2-mediated DNA damage supports a mutagenic function promoting tumor development.
    FUTURE DIRECTIONS: Despite the high sequence similarity shared between DUOX1 and DUOX2, the two isoforms present distinct regulations, tissue expression and catalytic functions. The phenotypic characterization of novel DUOX/DUOXA invalidated animal models will be very useful for defining their medical importance in pathological conditions.

    PMID: 24161126 [PubMed - indexed for MEDLINE]

  • IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response.
    Related Articles

    IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response.

    Cell Res. 2013 May;23(5):673-90

    Authors: Fink K, Martin L, Mukawera E, Chartier S, De Deken X, Brochiero E, Miot F, Grandvaux N

    Abstract
    Airway epithelial cells are key initial innate immune responders in the fight against respiratory viruses, primarily via the secretion of antiviral and proinflammatory cytokines that act in an autocrine/paracrine fashion to trigger the establishment of an antiviral state. It is currently thought that the early antiviral state in airway epithelial cells primarily relies on IFNβ secretion and the subsequent activation of the interferon-stimulated gene factor 3 (ISGF3) transcription factor complex, composed of STAT1, STAT2 and IRF9, which regulates the expression of a panoply of interferon-stimulated genes encoding proteins with antiviral activities. However, the specific pathways engaged by the synergistic action of different cytokines during viral infections, and the resulting physiological outcomes are still ill-defined. Here, we unveil a novel delayed antiviral response in the airways, which is initiated by the synergistic autocrine/paracrine action of IFNβ and TNFα, and signals through a non-canonical STAT2- and IRF9-dependent, but STAT1-independent cascade. This pathway ultimately leads to the late induction of the DUOX2 NADPH oxidase expression. Importantly, our study uncovers that the development of the antiviral state relies on DUOX2-dependent H2O2 production. Key antiviral pathways are often targeted by evasion strategies evolved by various pathogenic viruses. In this regard, the importance of the novel DUOX2-dependent antiviral pathway is further underlined by the observation that the human respiratory syncytial virus is able to subvert DUOX2 induction.

    PMID: 23545780 [PubMed - indexed for MEDLINE]

  • Stroma cell-derived factor-1α signaling enhances calcium transients and beating frequency in rat neonatal cardiomyocytes.
    Related Articles

    Stroma cell-derived factor-1α signaling enhances calcium transients and beating frequency in rat neonatal cardiomyocytes.

    PLoS One. 2013;8(2):e56007

    Authors: Hadad I, Veithen A, Springael JY, Sotiropoulou PA, Mendes Da Costa A, Miot F, Naeije R, De Deken X, Entee KM

    Abstract
    Stroma cell-derived factor-1α (SDF-1α) is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP3R) blocker, but not with a ryanodine receptor (RyR) antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP3R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP3-gated fashion leading to a positive chronotropic and inotropic effect.

    PMID: 23460790 [PubMed - indexed for MEDLINE]

 

 

Françoise Miot:        

  • Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin.
    Related Articles

    Overexpression of Interleukin-4 in the Thyroid of Transgenic Mice Upregulates the Expression of Duox1 and the Anion Transporter Pendrin.

    Thyroid. 2016 Oct;26(10):1499-1512

    Authors: Eskalli Z, Achouri Y, Hahn S, Many MC, Craps J, Refetoff S, Liao XH, Dumont JE, Van Sande J, Corvilain B, Miot F, De Deken X

    Abstract
    BACKGROUND: The dual oxidases (Duox) are involved in hydrogen peroxide generation, which is essential for thyroid hormone synthesis, and therefore they are markers of thyroid function. During inflammation, cytokines upregulate DUOX gene expression in the airway and the intestine, suggesting a role for these proteins in innate immunity. It was previously demonstrated that interleukin-4 (IL-4) upregulates DUOX gene expression in thyrocytes. Although the role of IL-4 in autoimmune thyroid diseases has been studied extensively, the effects of IL-4 on thyroid physiology remain largely unknown. Therefore, a new animal model was generated to study the impact of IL-4 on thyroid function.
    METHODS: Transgenic (Thyr-IL-4) mice with thyroid-targeted expression of murine IL-4 were generated. Transgene expression was verified at the mRNA and protein level in thyroid tissues and primary cultures. The phenotype of the Thyr-IL-4 animals was characterized by measuring serum thyroxine (T4) and thyrotropin levels and performing thyroid morphometric analysis, immunohistochemistry, whole transcriptome sequencing, quantitative reverse transcription polymerase chain reaction, and ex vivo thyroid function assays.
    RESULTS: Thyrocytes from two Thyr-IL-4 mouse lines (#30 and #52) expressed IL-4, which was secreted into the extracellular space. Although 10-month-old transgenic animals had T4 and thyrotropin serum levels in the normal range, they had altered thyroid follicular structure with enlarged follicles composed of elongated thyrocytes containing numerous endocytic vesicles. These follicles were positive for T4 staining the colloid, indicating their capacity to produce thyroid hormones. RNA profiling of Thyr-IL-4 thyroid samples revealed modulation of multiple genes involved in inflammation, while no major leukocyte infiltration could be detected. Upregulated expression of Duox1, Duoxa1, and the pendrin anion exchanger gene (Slc26a4) was detected. In contrast, the iodide symporter gene Slc5a5 was markedly downregulated resulting in impaired iodide uptake and reduced thyroid hormone levels in transgenic thyroid tissue. Hydrogen peroxide production was increased in Thyr-IL-4 thyroid tissue compared with wild-type animals, but no significant oxidative stress could be detected.
    CONCLUSIONS: This is the first study to show that ectopic expression of IL-4 in thyroid tissue upregulates Duox1/Duoxa1 and Slc26a4 expression in the thyroid. The present data demonstrate that IL-4 could affect thyroid morphology and function, mainly by downregulating Slc5a5 expression, while maintaining a normal euthyroid phenotype.

    PMID: 27599561 [PubMed - in process]

  • NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.
    Related Articles

    NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation.

    Proc Natl Acad Sci U S A. 2015 Apr 21;112(16):5051-6

    Authors: Ameziane-El-Hassani R, Talbot M, de Souza Dos Santos MC, Al Ghuzlan A, Hartl D, Bidart JM, De Deken X, Miot F, Diallo I, de Vathaire F, Schlumberger M, Dupuy C

    Abstract
    Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.

    PMID: 25848056 [PubMed - indexed for MEDLINE]

  • Roles of DUOX-mediated hydrogen peroxide in metabolism, host defense, and signaling.
    Related Articles

    Roles of DUOX-mediated hydrogen peroxide in metabolism, host defense, and signaling.

    Antioxid Redox Signal. 2014 Jun 10;20(17):2776-93

    Authors: De Deken X, Corvilain B, Dumont JE, Miot F

    Abstract
    SIGNIFICANCE: Among the NADPH oxidases, the dual oxidases, DUOX1 and DUOX2, constitute a distinct subfamily initially called thyroid oxidases, based on their high level of expression in thyroid tissue. Genetic alterations causing inherited hypothyroidism clearly demonstrate their physiological implication in thyroid hormonogenesis. However, a growing list of biological functions triggered by DUOX-dependent reactive oxygen species (ROS) in highly differentiated mucosae have recently emerged.
    RECENT ADVANCES: A role of DUOX enzymes as ROS providers for lactoperoxidase-mediated killing of invading pathogens has been well established and a role in bacteria chemorepulsion has been proposed. Control of DUOX expression and activity by inflammatory molecules and immune receptor activation consolidates their contributions to innate immune defense of mucosal surfaces. Recent studies conducted in ancestral organisms have identified effectors of DUOX redox signaling involved in wound healing including epithelium regeneration and leukocyte recruitment. Moreover, local generation of hydrogen peroxide (H2O2) by DUOX has also been suggested to constitute a positive feedback loop to promote receptor signaling activation.
    CRITICAL ISSUES: A correct balance between H2O2 generation and detoxification mechanisms must be properly maintained to avoid oxidative damages. Overexpression of DUOX genes has been associated with an increasing number of chronic inflammatory diseases. Furthermore, H2O2-mediated DNA damage supports a mutagenic function promoting tumor development.
    FUTURE DIRECTIONS: Despite the high sequence similarity shared between DUOX1 and DUOX2, the two isoforms present distinct regulations, tissue expression and catalytic functions. The phenotypic characterization of novel DUOX/DUOXA invalidated animal models will be very useful for defining their medical importance in pathological conditions.

    PMID: 24161126 [PubMed - indexed for MEDLINE]

  • Comparative analysis of the thyrocytes and T cells: responses to H2O2 and radiation reveals an H2O2-induced antioxidant transcriptional program in thyrocytes.
    Related Articles

    Comparative analysis of the thyrocytes and T cells: responses to H2O2 and radiation reveals an H2O2-induced antioxidant transcriptional program in thyrocytes.

    J Clin Endocrinol Metab. 2013 Oct;98(10):E1645-54

    Authors: Versteyhe S, Driessens N, Ghaddhab C, Tarabichi M, Hoste C, Dumont JE, Miot F, Corvilain B, Detours V

    Abstract
    CONTEXT: Radiation is an established cause of thyroid cancer, and growing evidence supports a role for hydrogen peroxide (H2O2) in spontaneous thyroid carcinogenesis. Little is known about the molecular programs activated by these agents in thyrocytes.
    OBJECTIVE: The purpose of this study was to compare the responses of thyrocytes and T cells to H2O2 and radiation.
    METHODS: We profiled the DNA damage and cell death induced by γ-radiation (0.1-5 Gy) and H2O2 (0.0025-0.3 mM) in primary human thyrocytes and T cells. We next prepared thyroid and T-cell primary cultures from 8 donors operated for noncancerous thyroid pathological conditions and profiled their genome-wide transcriptional response 4 hours after (1) exposure to 1-Gy radiation, (2) treatment with H2O2 and (3) no treatment. Two H2O2 concentrations were investigated, calibrated in each cell type to elicit levels of single- and double-strand breaks equivalent to 1-Gy γ-radiation.
    RESULTS: Although thyrocytes and T cells had comparable radiation responses, 3- to 10-fold more H2O2 was needed to induce detectable DNA damage in thyrocytes. At H2O2 and radiation doses inducing double-strand breaks, cell death occurred after 24 hours in T cells but not in thyrocytes. The transcriptional responses of thyrocytes and T cells to radiation were similar, involving DNA repair and cell death genes. In addition to this transcriptional program, H2O2 also up-regulated antioxidant genes in thyrocytes, including glutathione peroxidases and heme oxygenase at the double-strand breaks-inducing concentration. In contrast, a transcriptional storm involving thousands of genes was raised in T cells. Finally, we showed that inhibiting glutathione peroxidases activity increased the DNA damaging effect of H2O2 in thyrocytes.
    CONCLUSION: We propose that high H2O2 production in thyrocytes is matched with specific transcriptionally regulated antioxidant protection.

    PMID: 23666977 [PubMed - indexed for MEDLINE]

  • Endotext
    Related Articles

    Endotext

    Book. 2000

    Authors: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A

    Abstract
    The main function of the thyroid gland is to make hormones, T4 and T3, which are essential for the regulation of metabolic processes throughout the body. As at any factory, effective production depends on three key components – adequate raw material, efficient machinery, and appropriate controls. Iodine is the critical raw material, because 65% of T4 weight is iodine. Ingested iodine is absorbed and carried in the circulation as iodide. The thyroid actively concentrates the iodide across the basolateral plasma membrane of thyrocytes by the sodium/iodide symporter, NIS. Intracellular iodide is then transported in the lumen of thyroid follicles. Meanwhile, the thyrocyte endoplasmic reticulum synthesizes two key proteins, TPO and Tg. Tg is a 660kDa glycoprotein secreted into the lumen of follicles, whose tyrosyls serve as substrate for iodination and hormone formation. TPO sits at the apical plasma membrane, where it reduces H2O2, elevating the oxidation state of iodide to an iodinating species, and attaches the iodine to tyrosyls in Tg. H2O2 is generated at the apex of the thyrocyte by Duox, a NADPH oxydase. Initial iodination of Tg produces MIT and DIT. Further iodination couples two residues of DIT, both still in peptide linkage, to produce T4, principally at residues 5 in the Tg polypeptide chain. When thyroid hormone is needed, Tg is internalized at the apical pole of thyrocytes, conveyed to endosomes and lysosomes and digested by proteases, particularly the endopeptidases cathepsins B, L, D and exopeptidases. After Tg digestion, T4 and T3 are released into the circulation. Nonhormonal iodine, about 70% of Tg iodine, is retrieved intrathyroidally by DEHAL1, an iodotyrosine deiodinase and made available for recycling within the gland. TSH is the stimulator that affects virtually every stage of thyroid hormone synthesis and release. Early control involves the direct activation of the cellular and enzymatic machineries while delayed and chronic controls are on gene expression of key proteins. Iodine supply, either too much or too little, impairs adequate synthesis. Antithyroid drugs act by interfering with iodide oxidation. Genetic abnormalities in any of the key proteins, particularly NIS, TPO, Duox and Tg, can produce goiter and hypothyroidism. For complete coverage of this and related areas in Endocrinology, please visit our free web-book, www.endotext.org.


    PMID: 25905405

 

Bernard Corvilain:  

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