Duox expression and related H2O2 measurement in mouse thyroid: onset in embryonic development and regulation by TSH in adult

Abstract

In the thyroid, H 2 O 2 is produced at the apical pole of thyrocytes by one or two NADPH oxidases (NOX), Duox1/2 proteins. The onset of Duox expression was analysed by immunohistochemistry in the developing mouse thyroid in parallel with thyroglobulin (Tg) iodination and the expression of other thyroid differentiation markers. Duox proteins were found at embryonic day (E) 15 . 5 and were mainly localised at the apical pole of thyrocytes. Tg was detected 1 day before (E14 . 5) and Tg iodination was concomitant with the expression of both Duox and NaE15 . 5). The role of TSH in regulating Duox expression and H 2 O 2 accumulation was evaluated in thyroids of adult mice with reduced (Tshr hyt/hyt or mice treated with thyroxine) or increased (methimazole or perchlorate treatment) TSH/Tshr activity. In mice with suppressed TSH/Tshr activity, Duox expression was only partially decreased when compared with wild-type, as observed by western blot. In Tshr hyt/hyt strain, Duox was still expressed at the apical pole and H 2 O 2 measurements were normal. On the other hand, chronic TSH stimulation of the gland led to a decrease of H 2 O 2 measurements without affecting Duox expression. The onset of Duox protein expression is compatible with their proposed function in thyroid hormone synthesis and it can be considered as a functional marker of the developing thyroid. However, Duox expression in adult is much less regulated by TSH than NIS and thyroperoxidase. It is not always correlated with the overall thyroid H 2 O 2 accumulation, highlighting the importance of additional regulatory mechanisms which control either the production or H 2 O 2 degradation.

Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte

“…3, C and D). This correlates with increased serum TSH level (20). ANO1 localization in rat thyroid is therefore compatible with a role in iodide export from thyrocytes to the follicle lumen.…”

“…Thyroids were quickly dissected and further fixed by overnight immersion in 4% (wt/vol) paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The tissue was then transferred to successive graded sucrose solutions (10,20, and 30%, overnight each) and finally embedded in Tissue-Tek OCT compound, snap-frozen in cold 2-methylbutane, and stored at a temperature of Ϫ80°C. Cryosections (10 m) were cut on a cryostat (Leitz), mounted on slides coated with 0.1% poly-L-lysine (Sigma), and stored at Ϫ20°C until use.…”

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1

“…Anti-rabbit-horseradish peroxidase and anti-mousehorseradish peroxidase were from Amersham Biosciences (Piscataway, NJ, USA). Polyclonal anti-Duox antibody was raised against the Arg618-His1044 fragment of human Duox1 (Milenkovic et al, 2007). EZ-Link™ Maleimide-PEG11-Biotin reagent was from Thermo Fisher Scientific, Life Technologies.…”

“…Antibody binding was detected using enhanced chemiluminescence and Fuji Super RX medical X-ray films. Importantly samples were never boiled when processed for western blotting with the Duox1 antibody (11). The blots were analyzed on a GBox-Chemi XX6 (Syngene, Cambridge, UK) gel doc system and relative band densities of Western blots were quantified using ImageJ software.…”

Oxygen in the Evolution of Complex Life and the Price We Pay

“…In addition to cellular signaling, there are a number of diverse biological contexts in which extracellular H 2 O 2 , via peroxidase-catalyzed reactions, mediates tyrosine crosslinking of extracellular matrix (ECM) proteins; examples include the pathogen-evoked hypersensitivity response to restrict spread of pathogens in plants (30), protection of the freshly fertilized egg from polyspermy in sea urchins (31,32), stabilization of cuticular extracellular matrix (ECM) in Caenorhabditis elegans (33), and the conferring of ''resilient'' mechanical properties to resilin found in joints and tendons of insects (34). In mammals, the biosynthesis of thyroxine involves the activity of an H 2 O 2 -generating enzyme, a member of the NOX/ DUOX gene family (35). The ability of the pro-fibrotic cytokine, transforming growth factor-b1 (TGF-b1), to induce crosslinking of (myo)fibroblast-derived extracellular matrix proteins is dependent on H 2 O 2 in the presence of an extracellular heme peroxidase (36); the specific mammalian peroxidase(s) that mediate such reactions in physiologic/pathophysiologic contexts in vivo requires further study.…”