Mice Deficient in Dual Oxidase Maturation Factors Are Severely Hypothyroid

Abstract

Dual oxidases (DUOX1 and DUOX2) are evolutionary conserved reduced nicotinamide adenine dinucleotide phosphate oxidases responsible for regulated hydrogen peroxide (H(2)O(2)) release of epithelial cells. Specific maturation factors (DUOXA1 and DUOXA2) are required for targeting of functional DUOX enzymes to the cell surface. Mutations in the single-copy Duox and Duoxa genes of invertebrates cause developmental defects with reduced survival, whereas knockdown in later life impairs intestinal epithelial immune homeostasis. In humans, mutations in both DUOX2 and DUOXA2 can cause congenital hypothyroidism with partial iodide organification defects compatible with a role of DUOX2-generated H(2)O(2) in driving thyroid peroxidase activity. The DUOX1/DUOXA1 system may account for residual iodide organification in patients with loss of DUOX2, but its physiological function is less clear. To provide a murine model recapitulating complete DUOX deficiency, we simultaneously targeted both Duoxa genes by homologous recombination. Knockout of Duoxa genes (Duoxa(-/-) mice) led to a maturation defect of DUOX proteins lacking Golgi processing of N-glycans and to loss of H(2)O(2) release from thyroid tissue. Postnatally, Duoxa(-/-) mice developed severe goitreous congenital hypothyroidism with undetectable serum T4 and maximally disinhibited TSH levels. Heterozygous mice had normal thyroid function parameters. (125)I uptake and discharge studies and probing of iodinated TG epitopes corroborated the iodide organification defect in Duoxa(-/-) mice. Duoxa(-/-) mice on continuous T4 replacement from P6 showed normal growth without an overt phenotype. Our results confirm in vivo the requirement of DUOXA for functional expression of DUOX-based reduced nicotinamide adenine dinucleotide phosphate oxidases and the role of DUOX isoenzymes as sole source of hormonogenic H(2)O(2).

GLIS3 is indispensable for TSH/TSHR-dependent thyroid hormone biosynthesis and follicular cell proliferation

“…Moreover, levels of both free and thyroglobulin-bound T3 and T4 were dramatically decreased in the thyroid glands of Glis3KO mice compared with those of WT mice ( Figure 1B), suggesting a major role for GLIS3 in the regulation of TH biosynthesis. TH plays a critical role in growth (20,38,39); therefore, the reduction in TH levels might, at least in part, be responsible for the observed growth retardation in 3-weekold Glis3KO mice (Supplemental Figure 1; supplemental mate- , and TSH levels between 1-month-old male and female WT (MWT and FWT, respectively) and Glis3KO (MKO and FKO, respectively) mice (n = 7-16). (B) The content of free T3, free T4, and thyroglobulinbound T3 and T4 (Tg T3 and Tg T4, respectively) were analyzed in thyroid tissues from 1-month-old male and female WT and Glis3KO mice (n = 4).…”

“…About 15% of the cases with congenital hypothyroidism are related to defects in TH biosynthesis (dyshormonogenesis) and include genetic alterations in genes critical for TH synthesis, such as TPO, TG, NIS, dual oxidase 2 and A2 (DUOX2 and DUOXA2), PDS, iodotyrosine deiodinase (IYD), and TSHR (17,(19)(20)(21)(22)(23).…”

American Thyroid Association Guide to Investigating Thyroid Hormone Economy and Action in Rodent and Cell Models

“…Thyroid radioactive iodide uptake (RAIU) and other aspects of iodine kinetics can be studied in rodents using different iodine isotopes, most commonly 125 I, which are injected intraperitoneally (2-10 lCi 125 I). The thyroid gland is subsequently studied at different time points either with a gamma probe (used, for example, for the identification of parathyroid tissue in minimally invasive surgery) under anesthesia (45,46) or dissected postmortem with the trachea en bloc under a microscope and processed for radiometry for 1 minute in a gamma counter. The results may be expressed as a function of 125 I in the serum (47) or as percentage of the total injected dose (46).…”

“…Protein-bound 125 I (i.e., the total radioactive thyroid hormones bound to serum transport proteins) is determined in all blood samples after trichloroacetic acid (TCA) precipitation (47). Others have been able to trace iodide uptake and discharge in mice directly using gamma probes (45). Potassium perchlorate (KClO 4 ) has been used in rats 6-18 hours following injection of 125 I and shown to reduce the 125 I thyroid/blood ratio when thyroid peroxidase is inhibited (51,52).…”

“…This mouse model of congenital hypothyroidism can be made more severe by simultaneous targeting of DUOXA1 and DUOXA2, which are specific maturation factors required for targeting of functional DUOX enzymes to the cell surface. Homozygous males and females exhibit goiter and congenital hypothyroidism with undetectable serum T 4 and 500-to 2500-fold elevated TSH levels, respectively (45).…”