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 (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.
Rigutto
Sabrina
S
IRIBHM, Université Libre de Bruxelles, Campus Erasme, Bat.C., 808, route de Lennik, B-1070 Bruxelles, Belgium. sabrina.rigutto@ulb.ac.be
Hoste
Candice
C
Dumont
Jacques E
JE
Corvilain
Bernard
B
Miot
Françoise
F
De Deken
Xavier
X
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D000074623:Dual Oxidases; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007454:Iodides / Q000378:metabolism; D016086:Lentivirus; D019255:NADPH Oxidases; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D013961:Thyroid Gland / Q000166:cytology* / Q000378:metabolism*
D005420:Flavoproteins; D007454:Iodides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C524752:Duox1 protein, rat; C486611:Duox2 protein, rat