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NADPH Oxidase DUOX1 Promotes Long-Term Persistence of Oxidative Stress After an Exposure to Irradiation

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NADPH Oxidase DUOX1 Promotes Long-Term Persistence of Oxidative Stress After an Exposure to Irradiation NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation Rabii Ameziane-El-Hassania,b,c, Monique Talbota,b, Maria Carolina de Souza Dos Santosa,b,d, Abir Al Ghuzlana,b,d, Dana Hartlb, Jean-Michel Bidarta,b,d, Xavier De Dekene, Françoise Miote, Ibrahima Diallob,d,f, Florent de Vathaireb,d,f, Martin Schlumbergera,b,d, and Corinne Dupuya,b,d,1 aUMR 8200, CNRS, Villejuif F-94805, France; bInstitut Gustave Roussy, Villejuif F-94805, France; cUnité de Biologie et de Recherche Médicale, Centre National de l’Energie, des Sciences et des Techniques Nucléaires, Rabat M-10001, Morocco; dUniversity Paris-Sud, Orsay F-91400, France; eInstitut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Université Libre de Bruxelles, 1050 Brussels, Belgium; and fUMR 1018, INSERM, Villejuif F-94805, France Edited by James E. Cleaver, University of California, San Francisco, CA, and approved March 16, 2015 (received for review November 4, 2014) Ionizing radiation (IR) causes not only acute tissue damage, but oxidative stress could be responsible for the RET/PTC rearrange- also late effects in several cell generations after the initial expo- ment frequently found in radiation-induced thyroid tumors (6). sure. The thyroid gland is one of the most sensitive organs to the Cells can produce ROS through activation and/or induction of carcinogenic effects of IR, and we have recently highlighted that NADPH oxidases, which constitute a family of enzymes known an oxidative stress is responsible for the chromosomal rearrange- as NOX/DUOX (7). Unlike other oxidoreductases, NADPH ments found in radio-induced papillary thyroid carcinoma. Using oxidases are “professional” ROS producers, whereas the other both a human thyroid cell line and primary thyrocytes, we inves- enzymes produce ROS only as by-products along with their tigated the mechanism by which IR induces the generation of re- specific catalytic pathways. ROS produced by NOXs participate active oxygen species (ROS) several days after irradiation. We in the regulation of many cell functions and have been implicated focused on NADPH oxidases, which are specialized ROS-generating in various pathological conditions, including the late side effects enzymes known as NOX/DUOX. Our results show that IR induces induced by IR and chemotherapy (8–10). Thyroid cells express delayed NADPH oxidase DUOX1-dependent H O production in a 2 2 three of these NADPH oxidases, including two H2O2-generating dose-dependent manner, which is sustained for several days. We systems located at the apical plasma membrane of the thyroid report that p38 MAPK, activated after IR, increased DUOX1 via cells: DUOX2, which is implicated in thyroid hormone bio- IL-13 expression, leading to persistent DNA damage and growth synthesis, and DUOX1, whose role in the thyroid is still unknown arrest. Pretreatment of cells with catalase, a scavenger of H2O2, (11, 12). Furthermore, recently NOX4 was found to be expressed or DUOX1 down-regulation by siRNA abrogated IR-induced DNA inside these cells (13). damage. Analysis of human thyroid tissues showed that DUOX1 is Because ROS may contribute to the late effects observed after elevated not only in human radio-induced thyroid tumors, but also radiation exposure, we hypothesized that IR induces a delayed in sporadic thyroid tumors. Taken together, our data reveal a key oxidative stress in thyroid cells via the activation and/or in- role of DUOX1-dependent H2O2 production in long-term persistent duction of NADPH oxidase. In the present study, we demon- radio-induced DNA damage. Our data also show that DUOX1- strate that DUOX1 expression, induced via the IL-13 pathway in dependent H O production, which induces DNA double-strand 2 2 response to IR, is the primary source of sustained ROS pro- breaks, can cause genomic instability and promote the generation duction that causes persistent DNA damage. We show that p38 of neoplastic cells through its mutagenic effect. MAPK activation is required for the increased radio-induced ionizing radiation | oxidative stress | NADPH oxidase | thyroid | DNA damage Significance onizing radiation (IR) can cause various delayed effects in Increasing evidence supports the role of chronic oxidative Icells, including genomic instability that leads to the accumula- stress in late radiation-induced effects, including malignancy tion of gene mutations and chromosomal rearrangements, which and genetic instability. To date, elevated levels of reactive are thought to play a pivotal role in radiation-induced carcino- oxygen species (ROS) have been considered a cause of per- genesis. The persistence of such effects in progeny cells has pro- sistent instability, but until now the mechanism(s) underlying found implications for long-term health risks, including emergence the perpetuation of ROS generation in irradiated cells and their of a second malignancy after radiotherapy (1). The thyroid gland is progeny was undetermined. Cells can produce ROS through ac- tivation and/or induction of NADPH oxidases. The present in- one of the most sensitive organs to the carcinogenetic effects of vestigation identifies the DUOX1-based NADPH oxidase as a ROS- IR. The risk of thyroid tumors is maximal for exposure at a generating system induced after irradiation, causing delayed DNA younger age and increases linearly with radiation dose (2). More breakage. Overexpression of DUOX1inradio-induced thyroid than 90% of these cancers are papillary, presenting a RET/PTC tumors suggests that DUOX1 may contribute to a chronic oxida- chromosomal rearrangement in 70% of cases. Thus, the thyroid CELL BIOLOGY tive stress promoting genomic instability and tumorigenesis. can serve as a paradigm for analyzing the long-term delayed ef- fects of IR. Author contributions: C.D. designed research; R.A.-E.-H., M.T., and M.C.d.S.D.S. performed The mechanism by which radiation exposure is memorized and research; A.A.G., D.H., X.D.D., and F.M. contributed new reagents/analytic tools; R.A.-E.-H., leads to delayed DNA breakage remains to be determined. Hyp- J.-M.B., I.D., F.d.V., M.S., and C.D. analyzed data; and C.D. wrote the paper. oxia and antioxidant therapy reduce the X-ray–induced delayed The authors declare no conflict of interest. effects, suggesting that radio-induced oxidative stress plays a sig- This article is a PNAS Direct Submission. nificant role in determining the susceptibility of irradiated cells to 1To whom correspondence should be addressed. Email: [email protected]. – genetic instability (3 5). We recently showed that H2O2 is able to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cause RET/PTC1 rearrangement in thyroid cells, indicating that 1073/pnas.1420707112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1420707112 PNAS | April 21, 2015 | vol. 112 | no. 16 | 5051–5056 Downloaded by guest on September 23, 2021 DUOX1 expression. Finally, our analysis of human thyroid tissues production via DUOX1 up-regulation. Irradiation induced an + shows that DUOX1 is overexpressed in both radio-induced and increase in cytosolic [Ca2 ] in thyroid cells at day 4, consistent sporadic tumors, suggesting that radiation exposure by inducing with activation of the calcium-dependent H2O2-generating ac- DUOX1-based oxidative stress might favor a neoplastic process tivity of DUOX1 (16) (Fig. S1D). that can occur naturally. Our findings assign the NADPH oxidase DUOX1 a previously unidentified role in radio-induced genetic Radiation Induces IL-13 in Human Thyroid Cells. To identify the instability. molecular mechanism underlying chronic DUOX1 expression after irradiation, we analyzed the gene expression profile of Results immune-related genes in HThy-ori cells at different intervals γ Radiation Exposure Induces Chronic DUOX1-Dependent H2O2 Production after -ray exposure at 10 Gy. Among the cytokine genes ana- β α in Human Thyroid Cells. The concentration of extracellular H2O2 lyzed, IL-1 , IL-13, IL-6, IL-8, and TNF- were found to be up- produced by thyroid cells (HThy-ori) after γ-ray irradiation at 10 regulated after irradiation in a time-dependent manner (Fig. S1 Gy increased from day 3 up to day 4, and then remained stable E and F). The induction of IL-13 mRNA was correlated with an until day 7 (Fig. 1A). DUOX1 protein level also increased from increased level of IL-13 protein in HThy-ori cells assayed by day 1 to day 10 after irradiation (Fig. 1B). Strikingly, irradiation Western blot analysis at days 4 and 7 after irradiation (Fig. 2A). (10 Gy) of HThy-ori cells preferentially resulted in the up-reg- A neutralizing IL-13 monoclonal antibody (clone 32116) that ulation of DUOX1 mRNA level (1- to 14-fold) compared not blocks the binding of IL-13 to its receptor abrogated radiation- only with levels of NOX4 and DUOX2 mRNA, two NADPH induced H2O2 production (Fig. 2B). In addition, IL-13 down- oxidases expressed in the normal thyroid gland (Fig. 1C), but also regulation by RNA interference resulted in a significant reduction with the other NOXs (Table S1).TheincreaseinDUOX1 of DUOX1 mRNA level (Fig. S1G). Our data indicate that IL-13 mRNA level was dose-dependent (Fig. S1B). regulates the radiation-induced increase in DUOX1 expression. DUOX1 needs the maturation factor DUOXA1 to exit the endoplasmic reticulum and be active on cell surface. The DUOX1/ p38 MAPK Regulates DUOX1 Expression. To define the upstream DUOXA1 genes are aligned head-to-head in a compressed genomic mechanisms that regulate IL-13–induced DUOX1 expression in locus on chromosome 15, suggesting that expression of DUOX1 irradiated cells, we tested the effect of pharmacologic inhibitors oxidase and its maturation factor are coordinated by a common of NF-KB and canonical mitogen-associated protein kinase bidirectional promoter (14). Several alternative splicing variants (MAPK) pathways. Of these inhibitors, only SB203580 (SB), which of DUOXA1 mRNA have been identified, and the lack of coding specifically inhibits p38 MAPK, attenuated the induction of exon 6 has been shown to generate inactive forms of DUOXA1 (15).
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