Dual oxidases were initially identified as NADPH oxidases producing H 2 O 2 necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by biallelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC 50 ‫؍‬ 0.1 M) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H 2 O 2 generation (phorbol 12-myristate 13-acetate EC 50 ‫؍‬ 0.8 nM). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.

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.

TSH, mainly acting through cAMP, is the principal physiological regulator of thyroid gland function, differentiation expression, and cell proliferation. Both cAMP-dependent protein kinases [protein kinase A (PKA)] and the guanine-nucleotide-exchange factors for Rap proteins, exchange proteins directly activated by cAMP (Epac) 1 and Epac2, are known to mediate a broad range of effects of cAMP in various cell systems. In the present study, we found a high expression of Epac1 in dog thyrocytes, which was further increased in response to TSH stimulation. Epac1 was localized in the perinuclear region. Epac2 showed little or no expression. The TSH-induced activation of Rap1 was presumably mediated by Epac1 because it was mimicked by the Epac-selective cAMP analog (8-p-chloro-phenyl-thio-2'-O-methyl-cAMP) and not by PKA-selective cAMP analogs. Surprisingly, in view of the high Epac1 expression and its TSH responsiveness, all the cAMP-dependent functions of TSH in cultures or tissue incubations of dog thyroid, including acute stimulation of thyroid hormone secretion, H(2)O(2) generation, actin cytoskeleton reorganization, p70(S6K1) activity, delayed stimulation of differentiation expression, and mitogenesis, were induced only by PKA-selective cAMP analogs. The Epac activator 8-p-chloro-phenyl-thio-2'-O-methyl-cAMP, used alone or combined with PKA-selective cAMP analogs, had no measurable effect on any of these TSH targets. Therefore, PKA activation seems to mediate all the recognized cAMP-dependent effects of TSH and is thus presumably responsible for the pathological consequences of its deregulation. The role of Epac1 and TSH-stimulated Rap1 activation in thyrocytes is still elusive.

. The enzymatic activity has been purified as a Ca 2ϩ -dependent flavoprotein presenting an oxidase activity using NADPH as coenzyme (8 -13). Recently the corresponding cDNA has been cloned starting from this purified enzymatic activity (14). It encoded a protein of 138 kDa, which was the truncated form of Duox2. At the same time, based on the functional homology existing between the leukocyte and the thyroid H 2 O 2 generating systems, two cDNAs encoding two proteins of 1551 and 1548 amino acids, respectively, called ThOX1 and ThOX2 were cloned (15). These ThOX proteins belong to the family of NADPH oxidases and were later called Duox1/Duox2 because their expression is not restricted to the thyroid tissue and because they present peroxidase like domain at their N-terminal ends.In the thyroid Duox1/Duox2 show two N-glycosylated states: the fully glycosylated mature form (190 kDa) expressed at the plasma membrane and the high mannose glycosylated immature form (180 kDa) expressed exclusively inside the cell in the endoplasmic reticulum (16,17). Duox cDNA-transfected cell lines express only the immature form of the protein and no H 2 O 2 production can be detected (16) suggesting the involvement of other proteins in the maturation process of Duoxs to permit the capacity of the oxidase system.It is well known that in the leukocyte gp91 phox (also referred to as NOX2), the NADPH oxidase moiety of the superoxide anions generator, needs p22 phox to be processed to the membrane (18 -20 . production mainly in the colon. The unsuccessful reconstitution of the H 2 O 2 -generating capacity in PLB-XCGD (gp91 phox -knock-out PLB) cells with Duox1 and Duox2 suggests that other thyroid-specific factors are required to permit H 2 O 2 production in thyroid (16).