Duox1 is the main source of hydrogen peroxide in the rat thyroid cell line PCCl3

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 (H2O2) 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 H2O2 in the thyroid were unfruitful. Using the RNA interference technique, we demonstrated in this study that Duox1 is the main source of H2O2 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 H2O2 production; (3) the re-expression of Duox1 in silenced cells by a lentivirus based method rescued totally H2O2 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.