RESUMO
A deliberate generation of ROS is now recognized to be achieved by specific NADPH oxidases (NOX). Dual oxidases (DUOXs) are Ca(2+)-activated NOXs and operate as H(2)O(2)-generators in various tissues. A tight regulation is however required to avoid ROS overproduction that can rapidly be harmful to biological systems. DUOX activator (DUOXA) proteins act as organizing elements for surface expression and activity of the DUOX enzymes. To study DUOX activation by the maturation factors, chimeric DUOXA proteins were generated by replacing particular domains between DUOXA1 and DUOXA2. Their impact on DUOX function and membrane expression were explored in a reconstituted heterologous cell system composed of COS-7 cells. We have shown that the COOH-terminal end of DUOXA1 is responsible for DUOX1-dependent H(2)O(2) generation. The NH(2)-terminal tail of DUOXA2 is critical to specify the type of ROS released by DUOX2, hydrogen peroxide or superoxide. Native DUOXA2 would constrain DUOX2 to produce H(2)O(2). However, alterations of the DUOXA2 NH(2)-terminal domain modify DUOX2 activity triggering superoxide leaking. Our results demonstrate that specific domains of the DUOX maturation factors promote the activation of DUOXs as well as the type of ROS generated by the oxidases.
Assuntos
Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Oxidases Duais , Proteínas de Membrana/genética , Dados de Sequência Molecular , NADPH Oxidases/genética , Alinhamento de SequênciaRESUMO
Dual oxidases (DUOX) 1 and 2 are components of the thyroid H(2)O(2)-generating system. H(2)O(2) is used by thyroperoxidase to oxidize iodide for thyroid hormonogenesis. Mutations in the DUOX2 gene have been described in transient and permanent congenital thyroid dyshormonogenesis. We report here a novel genetic defect causing congenital hypothyroidism in a French-Canadian patient. At neonatal screening, the patient had high TSH and low total T(4) levels. (99m)Tc scan showed a normally shaped orthotopic but mildly enlarged thyroid gland, suggesting dyshormonogenesis. Thyroxine treatment was given from 1 month to 17 years, after which it was stopped for re-evaluation and the patient remained euthyroid. The transient congenital hypothyroidism phenotype prompted us to screen for mutations in DUOX2 and DUOXA2 genes using the PCR-amplified direct sequencing method. We found complete inactivation of DUOX2 caused by a partial genomic deletion of one allele inherited from the mother associated with a paternally inherited missense mutation (c.4552G>A, p.Gly1518Ser). The deleted fragment encompasses the entire COOH-terminal end which is responsible for the NADPH-oxidase activity. The Gly1518Ser DUOX2 protein is expressed at the cell surface of transfected cells albeit at low level, but it is non-functional. This study provides further evidence that the permanent or transient nature of congenital hypothyroidism is not directly related to the number of inactivated DUOX2 alleles, suggesting the existence of other pathophysiological factors.
Assuntos
Domínio Catalítico/genética , Hipotireoidismo Congênito/genética , Heterozigoto , Peróxido de Hidrogênio/metabolismo , Mutação de Sentido Incorreto/genética , NADPH Oxidases/genética , Deleção de Sequência/genética , Adolescente , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência de Bases , Hipotireoidismo Congênito/enzimologia , Análise Mutacional de DNA , Oxidases Duais , Feminino , Glicosilação , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , NADPH Oxidases/química , NADPH Oxidases/metabolismo , GravidezRESUMO
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.
Assuntos
Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Linfócitos T/efeitos da radiação , Glândula Tireoide/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Células Cultivadas , DNA/efeitos dos fármacos , DNA/genética , DNA/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Raios gama , Humanos , Estresse Oxidativo/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismoRESUMO
Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), have a significant role in hormone production in thyroid tissue. Although recent studies have demonstrated that dual oxidases are responsible for the H(2)O(2) synthesis needed in thyroid hormone production, our data suggest a pivotal role for superoxide dismutase 3 (SOD3) as a major H(2)O(2)-producing enzyme. According to our results, Sod3 is highly expressed in normal thyroid, and becomes even more abundant in rat goiter models. We showed TSH-stimulated expression of Sod3 via phospholipase C-Ca(2+) and cAMP-protein kinase A, a pathway that might be disrupted in thyroid cancer. In line with this finding, we demonstrated an oncogene-dependent decrease in Sod3 mRNA expression synthesis in thyroid cancer cell models that corresponded to a similar decrease in clinical patient samples, suggesting that SOD3 could be used as a differentiation marker in thyroid cancer. Finally, the functional analysis in thyroid models indicated a moderate role for SOD3 in regulating normal thyroid cell proliferation being in line with our previous observations.
Assuntos
Antígenos de Diferenciação/metabolismo , Diferenciação Celular , Superóxido Dismutase/metabolismo , Neoplasias da Glândula Tireoide/enzimologia , Animais , Western Blotting , Cálcio/metabolismo , Carcinoma , Carcinoma Papilar , Proliferação de Células , Regulação para Baixo , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxidos/metabolismo , Câncer Papilífero da Tireoide , Carcinoma Anaplásico da Tireoide , Glândula Tireoide/enzimologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
Among the host defense mechanisms against bacteria, leukocyte phagocytosis leads to their hydrogen peroxide (H(2)O(2))-mediated destruction. The recent discovery of dual oxidase (DUOX)-dependent H(2)O(2) generation associated with peroxidase and thiocyanate secretion at the apex of mucosal cells has been similarly interpreted as a killing mechanism. However, the rapid degradation of H(2)O(2) would be expected to reduce the efficiency of this system. It has been demonstrated that H(2)O(2) acts as a chemorepellent for bacteria, and such an effect might be sufficient to block cellular infection. Therefore, H(2)O(2) generation might represent one of the mechanisms that allows the coexistence of mucosae with potentially harmful bacteria. Here, we discuss the possible role of DUOXes and H(2)O(2) in interactions between host mucosae and bacteria to maintain mucosal homeostasis.
Assuntos
Bactérias/metabolismo , Peróxido de Hidrogênio/metabolismo , Mucosa Intestinal , NADPH Oxidases/metabolismo , Bactérias/crescimento & desenvolvimento , Infecções Bacterianas/metabolismo , Infecções Bacterianas/microbiologia , Oxidases Duais , Regulação da Expressão Gênica , Homeostase , Interações Hospedeiro-Patógeno , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , NADPH Oxidases/genética , Peroxidases/metabolismo , Tiocianatos/metabolismoRESUMO
Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.
Assuntos
Membrana Celular/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , NADPH Oxidases/metabolismo , Proteína Quinase C/metabolismo , Glândula Tireoide/enzimologia , Animais , Células COS , Carcinógenos/farmacologia , Membrana Celular/genética , Chlorocebus aethiops , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Oxidases Duais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Iodetos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , NADPH Oxidases/genética , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteína Quinase C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Doenças da Glândula Tireoide/enzimologia , Doenças da Glândula Tireoide/genética , Hormônios Tireóideos/biossínteseRESUMO
Duox1 and Duox2 proteins are particular members of the NADPH oxidase (Nox) family and were first characterized as the thyroid NADPH oxidases. These proteins are responsible for the hydrogen peroxide (H(2)O(2)) production necessary for the synthesis of thyroid hormones. Although mutations in the Duox2 gene have been discovered in hypothyroid patients with iodide organification defects, attempts to confirm the role of one or both proteins in the generation of H(2)O(2) in the thyroid were unfruitful. Using the RNA interference technique, we demonstrated in this study that Duox1 is the main source of H(2)O(2) in the rat thyroid cell line PCCl3. We showed that (1) Duox1 was abundantly expressed in PCCl3 in regard to Duox2, contrary to what was observed in the rat thyroid tissue; (2) the expression of a siRNA specifically targeting Duox1-induced silencing of its transcript and the corresponding protein with a parallel decrease of H(2)O(2) production; (3) the re-expression of Duox1 in silenced cells by a lentivirus based method rescued totally H(2)O(2) production with rat Duox1 and partially with human Duox1. Western blotting analysis confirmed the synthesis of the mature N-linked glycosylated protein responsible for this enzymatic activity.