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).

Fig. 1.

Generation of Duoxa^−/− mice. A, Structure of the Duoxa1 and Duoxa2 genes, the targeting construct, and the final knockout allele. A unique PmeI endonuclease site was used to linearize the vector before electroporation. Homologous recombination of ES cell DNA with the targeting construct replaced an approximately 2.9-kbp region with the floxed ACN cassette modified by the addition of terminal stop codons in frame of the Duoxa genes. The ACN cassette contained the Neo^R-positive selection marker and encodes Cre recombinase expressed specifically during spermatogenesis from a testis-specific promoter (tACE). Passage of the targeted locus through the germ line of male chimeras resulted in the self-excision of the ACN cassette with only a single loxP site flanked by stop codons remaining. B, Strategy of genotyping. The location of the three primers and the amplicons from WT and knockout alleles are indicated. C and D, Expression of Duox and Duoxa mRNA in WT (n = 6) and Duoxa^−/− (n = 5) thyroid glands (C) and colon (D). E, DUOX protein expression in thyroid (40 μg total protein) and descending colon (70 μg) of 3-month-old WT and Duoxa^−/− mice. Protein disulfide isomerase (PDI) was immunodetected to validate equal protein loading. F, Determination of H₂O₂ release from thyroid tissue samples of mice (pure 129S6/SvEvTac genetic background) with the indicated Duoxa genotypes. H₂O₂ concentrations accumulating in the medium were normalized for DNA content of the tissue samples. G, Maturation of DUOX N-glycosylation in WT and Duoxa^−/− mice. Cecal protein extracts were analyzed on Western blots with or without prior digestion with Endoglycosidase H (EndoH). Maturation of DUOX N-glycosylation in the Golgi apparatus produces Endo H-resistant glycans (R). DUOX protein from Duoxa^−/− mice is only detectable as EndoH-sensitive glycoform (S) indicating complete retention of DUOX in the endoplasmic reticulum (ER). *, P < 0.05; **, P < 0.01; ns, nonsignificant. PMA, phorbol 12-myristate-13-acetate.

Fig. 2.

Hypothyroid phenotype of Duoxa^−/− mice. A, Exemplar 4-wk-old Duoxa^−/− and WT littermates. B, Delayed eye opening of Duoxa^−/− pups. The curves depict the fraction of mice with bilateral open eyes at the indicated postnatal age. WT, n = 25; Duoxa^−/−, n = 22; Duoxa^−/−/l-T₄, n = 13. C, Body weight (bw) of male WT (in black), heterozygous (gray), Duoxa^−/− mice (red), and Duoxa^−/− mice receiving l-T₄ replacement starting from P6 (brown). Note that Duoxa^−/− without thyroid hormone replacement did not survive weaning at P21 and were, therefore, weaned at P30. D, Serum T₄, T₃, and TSH concentrations in 10- to 12-wk-old WT, heterozygous, and Duoxa^−/− animals. Blue and pink solid symbols denote males and females, respectively. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. E, Skeletal preparations (rib cage, knee joint, forelimb) from WT and Duoxa^−/− mice at P13 stained with Alizarin Red (bone) and Alcian Blue 8GX (cartilage). F, Weights of selected organs in 2-month-old mice normalized for total body weight. Untreated Duoxa^−/− (red bars) displayed thyroid hyperplasia and hyposplenia. In contrast, liver weight was reduced proportionally to total body weight. Note that the absolute brain weights (i.e. not normalized for body weight) were not significant different between the genotypes. ****, P < 0.0001 compared with all other groups. Each bar represents the mean ± sem of n = 7–28 animals.

Fig. 3.

Pathology of thyroid glands and anterior pituitary. A, Exemplar hematoxylin and eosin (H&E)-stained thyrotracheal units of a 2-month-old Duoxa^−/− mouse with manifest inspiratory stridor and a WT littermate. B and C, Immunohistochemical detection of TG on thyroid sections from mice in panel A. D and E, Detection of thyrotrophic cells in the anterior pituitary by immunochemical staining of TSH-β subunit. F, Thyroidal expression of selected TSH-responsive genes determined by real-time RT-PCR. ****, P < 0.0001.

Fig. 4.

Thyroidal ¹²⁵I organification. A, Effect of 2 wk of LID on serum TSH concentrations of WT and Duoxa^−/− mice. B and C, Kinetics of ¹²⁵I distribution in WT and Duoxa^−/− mice. ¹²⁵I activity over the anterior neck (B) and the midsternum (C) was determined using a γ-positioning system. The plots shown are representative curves from individual animals. D, Perchlorate-induced change in ¹²⁵I activity recorded over the anterior neck. Perchlorate was given ip 4 h after administration of ¹²⁵I, and activity over the neck was monitored for 20 min. Representative data from two mice are plotted for each genotype. E, Thyroidal ¹²⁵I activity before (open bars) and 20 min after (black solid bars) administration of ClO₄⁻. Activity of dissected thyrotracheal units was determined using a γ-scintillation counter. Data are expressed in percent of the administered dose (8 μCi/animal). Significant perchlorate-induced loss of thyroidal ¹²⁵I activity was only observed in Duoxa^−/− mice [53.7% washout (95% CI = 22.7–84.8); P = 0.0027], but not WT (+/+) or heterozygous (+/−) animals. F, Perchlorate-induced appearance of ¹²⁵I activity in the circulation. Four hours after injection of ¹²⁵I, blood was collected retroorbitally before and 20 min after administration of KClO₄. Bars represent the perchlorate-induced increase of ¹²⁵I activity in blood, expressed as a percentage of the injected dose. Total blood volume (in milliliters) was estimated to be 7.2% of body weight (in grams) (41). **, P < 0.01; ***, P < 0.001. G, Reducing SDS-PAGE of thyroidal protein extracts (50 μg/lane) from mice on LID. Thyroids were collected at the end of the perchlorate discharge tests. Protein was stained with colloidal Coomassie Brilliant Blue, and ¹²⁵I incorporation was revealed by autoradiography. S and F denote slow and fast migrating forms of TG, respectively (42, 43). Extracts analyzed are from two different WT and Duoxa^−/− mice, respectively. H, Reducing SDS-PAGE of thyroidal protein extracts from mice on an iodine-sufficient diet. Upper panel, Coomassie Blue-stained gel. Lower panel, Western blot probed simultaneously with pan-TG antiserum (green signal) and an antibody specific for iodinated TG (red signal) (38). In the color merge, iodinated and noniodinated forms of TG appear in yellow and green, respectively. Each lane corresponds to protein extract from a different animal.