title: Regulation of dual oxidase expression and H2O2 production by thyroglobulin.
abstract: BACKGROUND
  Thyroglobulin (Tg) is a macromolecular precursor in thyroid hormone synthesis to which iodine is stably bound. Tg, which is stored in the follicular space, is also a potent negative feedback regulator of follicular function, and this is achieved by suppressing mRNA levels of thyroid-specific genes such as the sodium/iodide symporter (Slc5a5), Tg, and thyroid peroxidase. Dual oxidase 1 (DUOX1) and DUOX2, originally identified in the thyroid, are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are necessary to produce the H2O2 required for thyroid hormone biosynthesis. Since follicular Tg regulates the expression of genes that are essential for thyroid hormone synthesis, we hypothesized that Tg might also regulate DUOX expression and H2O2 production.
  
  
  METHODS
  Rat thyroid FRTL-5 cells were treated with Tg, and the mRNA expression of Duox1 and Duox2 and their corresponding maturation factors Duoxa1 and Duoxa2 were evaluated by DNA microarray and real-time PCR. Duox2 promoter activity was examined by luciferase reporter gene assay. Protein levels of DUOX2 were also examined by Western blot analysis. Intracellular H2O2 generation was quantified by a fluorescent dye, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, and acetyl ester (CM-H2DCFDA).
  
  
  RESULTS
  mRNA levels of Duox2 and its activation factor Duoxa2 (but not Duox1 or Duoxa1) were significantly suppressed by Tg in a dose-dependent manner and a time-dependent fashion in rat thyroid FRTL-5 cells. DUOX2 promoter activity was significantly suppressed by Tg in a dose-dependent manner. Protein levels of DUOX2 and H2O2 generation in cells were also reduced by Tg treatment.
  
  
  CONCLUSIONS
  We show that physiological concentrations of Tg suppressed the expression and function of DUOX2 in thyroid cells. These results suggest that Tg is a strong suppressor of the expression and the activity of DUOX2/DUOXA2, thereby regulating iodide organification and hormone synthesis in the thyroid. The evidence supports a reported model in which accumulated Tg in thyroid follicles plays important roles in autoregulating the function of individual follicles, which produces the basis of follicular heterogeneity.
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Yoshihara
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hara
authors.forename: Akira
authors.initials: A
authors.lastname: Kawashima
authors.forename: Takeshi
authors.initials: T
authors.lastname: Akama
authors.forename: Kazunari
authors.initials: K
authors.lastname: Tanigawa
authors.forename: Huhehasi
authors.initials: H
authors.lastname: Wu
authors.forename: Mariko
authors.initials: M
authors.lastname: Sue
authors.forename: Yuko
authors.initials: Y
authors.lastname: Ishido
authors.forename: Naoki
authors.initials: N
authors.lastname: Hiroi
authors.forename: Norihisa
authors.initials: N
authors.lastname: Ishii
authors.forename: Gen
authors.initials: G
authors.lastname: Yoshino
authors.forename: Koichi
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000074623:Dual Oxidases; D005420:Flavoproteins / Q000096:biosynthesis*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis*; D019255:NADPH Oxidases / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013954:Thyroglobulin / Q000494:pharmacology*; D013961:Thyroid Gland / Q000502:physiology
chemical_list: C578449:DUOXA2 protein, rat; D005420:Flavoproteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D013954:Thyroglobulin; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C486611:Duox2 protein, rat