Cloning of Two Human Thyroid cDNAs Encoding New Members of the NADPH Oxidase Family

Abstract

Two cDNAs encoding NADPH oxidases and constituting the thyroid H 2 O 2 generating system have been cloned. The strategy of cloning was based on the functional similarities between H 2 O 2 generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyroid system would belong to the gp91 Phox /Mox1 gene family and display sequence similarities with gp91Phox . Screening at low stringency with a gp91Phox probe of cDNA libraries from thyroid cells in primary culture yielded two distinct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 1548 amino acids (ThOX2), respectively. The encoded polypeptides display 83% sequence similarity and are clearly related to gp91Phox (53 and 47% similarity). The theoretical molecular mass of 177 kDa is close to the apparent molecular mass of 180 kDa of the native corresponding porcine flavoprotein and the protein(s) detected by Western blot in dog and human thyroid. ThOX1 and ThOX2 display sequence similarities of 53% and 61%, respectively, with a predicted protein of Caenorhabditis elegans over their entire length. They show along their first 500 amino acids a similarity of 43% with thyroperoxidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on chromosome 15q15.3. The dog mRNA expression is thyroid-specific and up-regulated by agents activating the cAMP pathway as is the synthesis of the polypeptides they are coding for. In human thyroid the positive regulation by cAMP is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase.

DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma

“…Cell lysates or lung homogenates were prepared using Western solubilization buffer (50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM PMSF, 2 mM Na 3 VO 4 , 10 μg/ml aprotinin, 10 μg/ml leupeptin; pH 7.4), and samples containing equal amounts of protein (measured using the BCA protein assay kit; Pierce) were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with antibodies against EGFR (C74B9; 1:1,000), pEGFR Tyr845 (D63B4; 1:500), pEGFR Tyr1068 (D7A5; 1:1,000), and Src (L4A1; 1:1,000) (all from Cell Signaling); p-Src Tyr416 (1:1,000; 07-909; Millipore); or DUOX1 (provided by F. Miot, Free University of Brussels, Brussels, Belgium) (62). Primarily antibodies were probed with rabbit-or mouse-specific secondary antibodies conjugated with HRP (Cell Signaling) and detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce).…”

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…This reaction requires H 2 O 2 , which derives from a previously unidentified thyroid NADPH oxidase. Duox was first shown to be this longsought oxidase based on purification and partial cDNA cloning [187]; two forms of this oxidase were subsequently identified by molecular cloning [188][189][190], and both are expressed in thyroid [191]. The essential role for Duox2 in thyroid hormone biosynthesis is demonstrated conclusively by the occurrence of mutations in this gene in genetic variants of hypothyroidism [192,193].…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Molecular biology of DUOX1/2-DUOX1 and DUOX2 were first cloned from thyroid tissues and were originally identified as p138 Thox or ThOX1/2 (11,92). Both DUOX proteins are highly similar N-glycosylated proteins (with 83% homology) with a molecular weight of 180 kD.…”

“…DUOX1 and its maturation factor DUOXA1 are both located on chromosome 15q15.3 in a complex locus in a head-to-head arrangement (92)(93)(94), and may share a bidirectional promoter. The same is true for their paralogs DUOX2/DUOXA2.…”

“…Both lack a TATA box, and the DUOX1 promoter is GC-rich and contains three putative SP1-binding elements, whereas the DUOX2 promoter does not contain any SP1-binding elements (94). Expression of DUOX1 and DUOX2 in thyrocytes was found to be inducible in response to cAMP stimulation by forskolin (12,92), but this was not observed in human epithelial cells ( (90) and unpublished results), and the human DUOX promoters does not appear to contain a cAMP-responsive element (94). The differences in DUOX1/2 promoter regions are consistent with the highly variable transcriptional regulation of airway epithelial DUOX1 and DUOX2 in response to pro-inflammatory cytokines or to bacterial or viral stimuli (36,83,96).…”