Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

Abstract

In the thyroid gland Duox2-derived H2O2 is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutations in genes for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2thyd) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in non-thyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T>G transversion in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found the valine→glycine mutant Duox proteins fail to produce H2O2, loose their plasma membrane localization pattern and are retained within the endoplasmic reticulum. Duox2 mutant binds to DuoxA2, but appears to be unstable due to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed Duox2 in mutant mice looses its condensed apical plasma membrane localization pattern characteristic of wild type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and ROS release required for hormonogenesis, resulting in congenital hypothyroidism.

Deficiency in Duox2 activity alleviates ileitis in GPx1- and GPx2-knockout mice without affecting apoptosis incidence in the crypt epithelium

“…In the inflamed colon biopsies, many epithelial cells are stained at the apical and lateral surface of plasma membrane as well as intracellular perinuclear region along the entire gland axis. In the colon biopsies from normal-looking areas, very few epithelial cells are stained by the DUOX Ab, and the staining is present in both apical membrane and intracellular membranes as reported previously [38]. The expression of DUOX2 at the base of inflamed colon glands suggests that Duox2 can have a direct effect in crypt epithelium.…”

“…NOX1 can form an enzyme complex in endosomes to generate ROS intracellularly in addition to extracellularly [39], [40]. DUOX2 is expressed in the apical surface of un-inflamed ileum and colon also to generate ROS intracellularly and extracellularly [4], [38]. We explored whether DUOX2 also contributes to DKO gut pathology.…”

“…The 4th complete 129 allele variant was generated by combining all three individual SNPs. The Duox2 cDNAs in pcDNA5/FRT plasmid were transfected into mouse rectum cancer CMT93 cells, which were co-transfected with a mouse Duoxa2 cDNA and GFP [38]. Forty-eight hours after transfection, cells were distributed in 96-well white-luminescence plates in 200 µl HBSS plus Mg ++ and Ca ++ and stimulated with 1 µM ionomycin or 1 µM phorbol myristate acetate to elicit activity at 37 °C.…”

Paradoxical roles of dual oxidases in cancer biology

“…Another important gap in this field is the lack of information regarding existing DUOX1 polymorphisms and lung cancer. Whereas abundant literature has connected DUOX2 SNPs to hypothyroidism[265, 266], and several reports suggest that the development of hypothyroidism is a risk factor for the onset of cancer[267–269], information on DUOX1 SNPs are scarce, with only one report identifying two SNPs on exons 27 and 28, although no association with lung disease was observed[270]. Potential associations of DUOX1 SNPs with increased cancer incidence or progression could offer important additional insights into the overall importance of DUOX1 in cancer biology.…”

Nox/Duox Family of NADPH Oxidases: Lessons from Knockout Mouse Models

“…Homozygous nonsense mutation in Duox2 causing congenital hypothyroidism demonstrated its obligate function in thyroid hormone biosynthesis [6,53]. Mice with a spontaneously developed missense Duox2 mutation develop the phenotype characteristic for congenital hypothyroidism [54] due to the loss of Duox2-mediated ROS production [55]. Simultaneous knockout of the two genes encoding DuoxA1 and DuoxA2 led to the development of the same phenotype [56].…”