Regulation of Dual Oxidase Expression and H₂O₂Production 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 generation was quantified by a fluorescent dye, 5-(and-6)-chloromethyl-2¢,7¢-dichlorodihydrofluorescein diacetate, and acetyl ester (CM-H 2 DCFDA). 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 H 2 O 2 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.

Follicular thyroglobulin enhances gene expression necessary for thyroid hormone secretion

“…Indeed, Tg inhibited TSH-stimulated iodine uptake and H 2 O 2 generation, which are essential for TH synthesis [2,5,6]. Such negative-feedback mechanisms have also been observed in primary cultures of normal human thyrocytes [7].…”

“…Preparation of cell protein and Western blot analysis were performed as described [2,6,7]. Briefly, cells were lysed in a buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM EDTA, 0.1% NP40 and 20% glycerol, followed by addition of protease inhibitor cocktail tablets (Complete Mini; Roche Diagnostics, Basel, Switzerland) at 4°C for 1 h. The mixture was centrifuged at 4°C for 20 min to recover cell protein.…”

“…The protein concentration was determined using DC protein assay reagents (BIO-RAD, Hercules, CA, USA) and a VMax Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) according to the manufacturer's instructions. Western blotting was performed using 10 μg of protein extracts separated on NuPage 4-12% BisTris gels (Invitrogen) and transferred to PVDF membranes as described previously [2,6,7].…”

“…Rat thyroid FRTL-5 cells were grown in Coon's modified Ham's F-12 medium supplemented with 5% bovine serum (Invitrogen, Waltham, MA, USA) and a six-hormone mixture (1 mU/mL TSH, 10 μg/mL insulin, 10 ng/mL somatostatin, 0.36 ng/mL hydrocortisone, 5 μg/mL transferrin, and 2 ng/mL glycyl-L-histidyl-L-lysine acetate) as described [2,6,15]. Culture medium that did not contain TSH was also used in some experiments.…”

“…receptor (Tshr), thyroid transcription factor 1 (TTF1; Nkx2-1), thyroid transcription factor 2 (TTF2; Foxe1), and paired box gene 8 (Pax8), all of which are essential participants in the 'assembly line' of TH production [2][3][4][5][6]. Indeed, Tg inhibited TSH-stimulated iodine uptake and H 2 O 2 generation, which are essential for TH synthesis [2,5,6].…”