DUOX1 has been originally identified in the thyroid gland (12)

DUOX1 has been originally identified in the thyroid gland (12). part in determining the susceptibility of irradiated cells to genetic instability (3C5). We recently showed that H2O2 is able to cause rearrangement in thyroid cells, indicating that oxidative stress could be responsible for the rearrangement regularly found in radiation-induced thyroid tumors (6). Cells can produce ROS through activation and/or induction of NADPH oxidases, which constitute a family of enzymes known as NOX/DUOX (7). Unlike additional oxidoreductases, NADPH oxidases are professional ROS makers, whereas the additional enzymes create ROS only as by-products along with their specific catalytic pathways. ROS produced by NOXs participate in the rules of many cell functions and have been implicated in various pathological conditions, including the late side effects induced by IR and chemotherapy KPT-6566 (8C10). Thyroid cells communicate three of these NADPH oxidases, including two H2O2-generating systems located in the apical plasma membrane of the thyroid cells: DUOX2, which is definitely implicated in thyroid hormone biosynthesis, and DUOX1, whose part in the thyroid is still unfamiliar (11, 12). Furthermore, recently NOX4 was found to be indicated inside these cells (13). Because ROS may contribute to the late effects observed after radiation exposure, we hypothesized that IR induces a delayed oxidative stress in thyroid cells via the activation and/or induction of NADPH oxidase. In the KPT-6566 present study, we demonstrate that DUOX1 manifestation, induced via the IL-13 pathway in response to IR, is the primary source of sustained ROS production that causes prolonged DNA damage. We display that p38 MAPK activation is required for the improved radio-induced DUOX1 manifestation. Finally, our analysis of human being thyroid cells demonstrates DUOX1 is definitely overexpressed in both radio-induced and sporadic tumors, suggesting that radiation exposure by inducing DUOX1-based oxidative stress KPT-6566 might favor a neoplastic process that can occur naturally. Our findings assign the NADPH oxidase DUOX1 a previously unidentified role in radio-induced genetic instability. Results Radiation Exposure Induces Chronic DUOX1-Dependent H2O2 Production in Human Thyroid Cells. The concentration of extracellular H2O2 produced by thyroid cells (HThy-ori) after -ray irradiation at 10 Gy increased from day 3 up to day 4, CDC47 and then remained stable until day 7 (Fig. KPT-6566 1genes in irradiated HThy-ori cells, analyzed by real-time qRT-PCR. ( 0.01. DUOX1 needs the maturation factor DUOXA1 to exit KPT-6566 the endoplasmic reticulum and be active on cell surface. The genes are aligned head-to-head in a compressed genomic locus on chromosome 15, suggesting that expression of DUOX1 oxidase and its maturation factor are coordinated by a common bidirectional promoter (14). Several alternative splicing variants of DUOXA1 mRNA have been identified, and the lack of coding exon 6 has been shown to generate inactive forms of DUOXA1 (15). We designed an oligonucleotide primer set in the DUOXA1 mRNA region made up of exon 6. Real time quantitative RT-PCR (qRT-PCR) analysis performed at 4 d after a 10-Gy exposure of HThy-ori cells showed that a spliced variant of DUOXA1 mRNA encoding an active form was selectively increased in this condition. This mRNA variant was up-regulated in a dose-dependent manner (Fig. S1and 0.05; ** 0.01; *** 0.001. p38 MAPK Regulates DUOX1 Expression. To define the upstream mechanisms that regulate IL-13Cinduced DUOX1 expression in irradiated cells, we tested the effect of pharmacologic inhibitors of NF-KB and canonical mitogen-associated protein kinase (MAPK) pathways. Of these inhibitors, only SB203580 (SB), which specifically inhibits p38 MAPK, attenuated the induction of DUOX1 mRNA expression at day 4 after irradiation (Fig. S1 0.05; ** 0.01; *** 0.001. A genetically encoded highly specific fluorescent probe has been developed for detecting H2O2 inside living cells (19). This biosensor,.

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