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Activation of Dual Oxidases Duox1 and Duox2

2009; Elsevier BV; Volume: 284; Issue: 11 Linguagem: Inglês

10.1074/jbc.m806893200

1083-351X

Sabrina Rigutto, Candice Hoste, Helmut Grasberger, Milutin Milenkovic, David Communi, Jacques E. Dumont, Bernard Corvilain, Françoise Miot, Xavier De Deken,

Marine Toxins and Detection Methods

Dual oxidases were initially identified as NADPH oxidases producing H2O2 necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic 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 (EC50 = 0.1 μm) 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 H2O2 generation (phorbol 12-myristate 13-acetate EC50 = 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. Dual oxidases were initially identified as NADPH oxidases producing H2O2 necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic 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 (EC50 = 0.1 μm) 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 H2O2 generation (phorbol 12-myristate 13-acetate EC50 = 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. Dual oxidases (Duox1 and Duox2) belong to the family of NADPH oxidases (Nox), which is composed of five additional enzymes: Nox1–5 (1Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 3Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2467) Google Scholar). These transmembrane proteins are characterized by a COOH-terminal NADPH oxidase catalytic core responsible for reactive oxygen species synthesis. The best characterized NADPH oxidase Nox2 is involved in the leukocyte respiratory burst and activated by invading pathogens (4Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar). The mechanisms controlling the Nox-mediated reactive oxygen species production are multiple and complex. The activation of Nox1 (5Banfi B. Clark R.A. Steger K. Krause K.H. J. Biol. Chem. 2003; 278: 3510-3513Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 6Takeya R. Ueno N. Kami K. Taura M. Kohjima M. Izaki T. Nunoi H. Sumimoto H. J. Biol. Chem. 2003; 278: 25234-25246Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), Nox2 (3Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2467) Google Scholar, 7Sumimoto H. Miyano K. Takeya R. Biochem. Biophys. Res. Commun. 2005; 338: 677-686Crossref PubMed Scopus (256) Google Scholar), and Nox3 (8Cheng G. Ritsick D. Lambeth J.D. J. Biol. Chem. 2004; 279: 34250-34255Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 9Banfi B. Malgrange B. Knisz J. Steger K. Dubois-Dauphin M. Krause K.H. J. Biol. Chem. 2004; 279: 46065-46072Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) requires the coordinated assembly of several subunits: the association with the transmembrane protein p22phox and the recruitment of three cytosolic proteins, the small G protein Rac, p47phox (or NOXO1), and p67phox (or NOXA1). Duox1 and Duox2 isoenzymes are large members of the Nox/Duox family. In addition to the catalytic core, Duox1 and Duox2 proteins are NH2-terminally extended by an extracellular peroxidase-like domain followed by a membrane-spanning segment and an intracellular domain comprising two canonical EF-hand motifs (2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Duox1 and Duox2 are the sole proteins directly generating H2O2 (10Dupuy C. Kaniewski J. Deme D. Pommier J. Virion A. Eur. J. Biochem. 1989; 185: 597-603Crossref PubMed Scopus (49) Google Scholar) outside the cells, whereas small Nox homologues are mostly superoxide generators (3Lambeth J.D. Nat. Rev. Immunol. 2004; 4: 181-189Crossref PubMed Scopus (2467) Google Scholar). Duox isoforms do not need to be associated with cytosolic factors to be active but undergo a critical maturation process necessary to acquire their active conformation at the apical cell surface of the thyrocytes (11De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar). The immature nonfunctional form (180 kDa) is not properly glycosylated and maintained in the endoplasmic reticulum compartment. Only the co-expression of the Duox maturation factors (DuoxA1 and DuoxA2) allows functional reconstitution. They are N-glycosylated proteins permitting the endoplasmic reticulum exit of properly folded Duox enzymes (12Grasberger H. Refetoff S. J. Biol. Chem. 2006; 281: 18269-18272Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 13Grasberger H. De Deken X. Miot F. Pohlenz J. Refetoff S. Mol. Endocrinol. 2007; 21: 1408-1421Crossref PubMed Scopus (77) Google Scholar). The DUOXA genes are localized near their respective DUOX gene in a head to head orientation on chromosome 15 and co-expressed with their Duox counterpart in the same tissues (12Grasberger H. Refetoff S. J. Biol. Chem. 2006; 281: 18269-18272Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Dual oxidases expressed at the apical side of surface epithelia exposed to microorganisms, like the airways or the digestive tract, are supposed to function as components of the innate host defense system (14Geiszt M. Witta J. Baffi J. Lekstrom K. Leto T.L. FASEB J. 2003; 17: 1502-1504Crossref PubMed Scopus (418) Google Scholar, 15El Hassani R.A. Benfares N. Caillou B. Talbot M. Sabourin J.C. Belotte V. Morand S. Gnidehou S. Agnandji D. Ohayon R. Kaniewski J. Noel-Hudson M.S. Bidart J.M. Schlumberger M. Virion A. Dupuy C. Am. J. Physiol. 2005; 288: G933-G942Crossref PubMed Scopus (191) Google Scholar, 16Ha E.M. Oh C.T. Bae Y.S. Lee W.J. Science. 2005; 310: 847-850Crossref PubMed Scopus (586) Google Scholar). However, Duox1 and Duox2 isoenzymes were initially identified as H2O2-generating thyroid oxidases (1Dupuy C. Ohayon R. Valent A. Noel-Hudson M.S. Deme D. Virion A. J. Biol. Chem. 1999; 274: 37265-37269Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). The main function of the thyroid is the uptake and concentration of iodide from the bloodstream to synthesize thyroid hormones (T3 and T4) in the follicular lumen (17Dumont J.E. Vitam. Horm. 1971; 29: 287-412Crossref PubMed Scopus (317) Google Scholar). H2O2 produced at the apical pole of the thyrocyte is utilized by thyroperoxidase as an electron acceptor to oxidize iodide, covalently link oxidized iodide to tyrosines of thyroglobulin, and couple iodinated tyrosyl residues to form protein-bound iodothyronines (T3 and T4) (18Nunez J. Pommier J. Vitam. Horm. 1982; 39: 175-229Crossref PubMed Scopus (108) Google Scholar). Under physiological iodide supply, hormonogenesis is rate-limited by the availability of hydrogen peroxide (19Corvilain B. Van Sande J. Laurent E. Dumont J.E. Endocrinology. 1991; 128: 779-785Crossref PubMed Scopus (152) Google Scholar). Patients presenting iodide organification defect caused by mutations in their DUOX2 gene suffer from transient or permanent hypothyroidism depending on the mono- or bi-allelic character of the mutation, demonstrating the crucial function of Duox2 in thyroid hormone synthesis (20Moreno J.C. Bikker H. Kempers M.J. van Trotsenburg A.S. Baas F. de Vijlder J.J. Vulsma T. Ris-Stalpers C. N. Engl. J. Med. 2002; 347: 95-102Crossref PubMed Scopus (394) Google Scholar, 21Vigone M.C. Fugazzola L. Zamproni I. Passoni A. Di Candia S. Chiumello G. Persani L. Weber G. Hum. Mutat. 2005; 26: 395Crossref PubMed Scopus (101) Google Scholar, 22Varela V. Rivolta C.M. Esperante S.A. Gruneiro-Papendieck L. Chiesa A. Targovnik H.M. Clin. Chem. 2006; 52: 182-191Crossref PubMed Scopus (62) Google Scholar, 23Pfarr N. Korsch E. Kaspers S. Herbst A. Stach A. Zimmer C. Pohlenz J. Clin. Endocrinol. 2006; 65: 810-815Crossref PubMed Scopus (50) Google Scholar, 24Ohye H. Fukata S. Hishinuma A. Kudo T. Nishihara E. Ito M. Kubota S. Amino N. Ieiri T. Kuma K. Miyauchi A. Thyroid. 2008; 18: 561-566Crossref PubMed Scopus (41) Google Scholar, 25Maruo Y. Takahashi H. Soeda I. Nishikura N. Matsui K. Ota Y. Mimura Y. Mori A. Sato H. Takeuchi Y. J. Clin. Endocrinol. Metab. 2008; 93: 4261-4267Crossref PubMed Scopus (102) Google Scholar). However, the physiological meaning of the co-existence of the two dual oxidases and their respective maturation factors in the thyroid tissue remains an open question. In this study, we analyzed the mechanisms of activation of Duox1 and Duox2 using Duox/DuoxA co-transfected cells stimulated by agonists known to regulate the thyroid metabolism (26Raspe E. Laurent E. Andry G. Dumont J.E. Mol. Cell. Endocrinol. 1991; 81: 175-183Crossref PubMed Scopus (62) Google Scholar, 27Corvilain B. Laurent E. Lecomte M. Vansande J. Dumont J.E. J. Clin. Endocrinol. Metab. 1994; 79: 152-159Crossref PubMed Google Scholar). Our results indicate that Duox1 and Duox2 activities are mainly calcium-dependent NADPH oxidases. Moreover, additional mechanisms governing their intrinsic activity are different; Duox1 is positively regulated by the cAMP-dependent protein kinase A (PKA) 6The abbreviations used are: PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; WT, wild type; HA, hemagglutinin; TSH, thyroid-stimulating hormone; FACS, fluorescence-activated cell sorter; Fsk, forskolin; 6-MB-cAMP, N6-monobutyryladenosine-3′,5′-cyclic monophosphate. cascade, whereas Duox2 is highly induced by activation of protein kinase C (PKC) with very low concentrations of PMA. Plasmids and Mutagenesis-The wild type untagged versions of human Duox1 (accession number AF230495) and Duox2 (accession number AF230496) cDNA were cloned from ATG to stop codon into the vector pcDNA3 (Invitrogen). The NH2-terminal hemagglutinin epitope-tagged human Duox2 (HA-Duox2-pcDNA3.1) and the COOH-terminal c-Myc epitope-tagged human DuoxA1 and DuoxA2 (DuoxA1-Myc-pcDNA3.1 and DuoxA2-Myc-pcDNA3.1) were described elsewhere (12Grasberger H. Refetoff S. J. Biol. Chem. 2006; 281: 18269-18272Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). We added an additional Rho tag to Duox1 to be able to distinguish it from HA-Duox2 in future experiments. The Duox1 native signal peptide was replaced by the TSH receptor signal peptide to facilitate the cloning step. The Rho-HA-Duox1-pcDNA3 construct was generated as follows. The 23 first amino acids (residue 1 corresponding to the initiation methionine) of the human Duox1 protein (accession number AAF73921) were replaced by the signal peptide of the human TSH receptor (MRPADLLQLVLLLDLPRDLGG) (accession number CAA02195) and the first 19 residues of the bovine rhodopsin (Rho) (accession number P02699) (MNGTEGPNFYVPFSNKTGVV; two putative glycosylation sites are underlined) (28Vlaeminck-Guillem V. Ho S.C. Rodien P. Vassart G. Costagliola S. Mol. Endocrinol. 2002; 16: 736-746Crossref PubMed Scopus (174) Google Scholar). The cDNA corresponding to the NH2-terminal HA-tagged Duox1 protein (24–1551 amino acids) was cloned just downstream of the Rho tag by insertion of an EcoRI site. The presence of the TSH receptor signal peptide, Rho, and HA tags at the NH2 terminus of Duox1 did not affect its expression nor its peroxide generating activity (supplemental Fig. S1). Furthermore, the extra N-linked sugars present on the Rho tag have been very useful to separate the two glycosylated forms of Duox1 for mass spectrometry analysis, because it is mainly the mature highly glycosylated form of Duox1 that is phosphorylated upon stimulation. Mutations in Duox1–2 were introduced by directed mutagenesis with the QuikChange system (Stratagene, La Jolla, CA) (primers are described in supplemental Table S1). All of the constructs were verified by Big Dye Terminator cycle sequencing on an automated ABI Prism 3100 sequencer (Applied Biosystems, Foster City, CA). Cell Culture and Transfection-Cos-7 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Invitrogen), 2% streptomycin-penicillin, 1% fungizone, and 1% sodium pyruvate. For H2O2 assay, adherent cells at 50–60% confluence were transfected in 6-well plates using FuGENE 6 reagent (Roche Applied Science) according to the manufacture's protocol (ratio: 1 μg of DNA for 3 μl of FuGENE 6) with 500 ng of Rho-HA-Duox1-pcDNA3 and 500 ng of DuoxA1-Myc-pcDNA3.1 or with 500 ng of HA-Duox2-pcDNA3.1 and 500 ng of DuoxA2-Myc-pcDNA3.1. Under optimal conditions, the transfection efficiency reached 20–30% of cells expressing Duox proteins at the cell surface detected by FACS. For immunoprecipitation experiments, the cells seeded in 10-cm-diameter dishes were transfected with 8 μg of DNA and 24 μl of FuGENE 6. Human Thyroid Primary Culture-Human thyroid tissue was obtained from patients undergoing partial or total thyroidectomy for resection of solitary cold nodules or multinodular goiters. Only healthy, normal-looking, non-nodular tissue was used within 30 min after surgical removal. Thyrocytes in primary culture obtained from follicles isolated by collagenase digestion and differential centrifugation were cultured in Dulbecco's modified Eagle's medium/Ham's F-12/MCDB104 (2:1:1) medium (Invitrogen) with 1% sodium pyruvate, 40 μg/ml ascorbic acid, 5 μg/ml insulin, 2% streptomycin-penicillin, and 1% fungizone (29Roger P.P. Hotimsky A. Moreau C. Dumont J.E. Mol. Cell. Endocrinol. 1982; 26: 165-176Crossref PubMed Scopus (108) Google Scholar, 30Roger P. Taton M. van Sande J. Dumont J.E. J. Clin. Endocrinol. Metab. 1988; 66: 1158-1165Crossref PubMed Scopus (184) Google Scholar). Thyrocytes were seeded 5 days before the assay. The protocol has been approved by the hospital ethics committee. H2O2 Measurement and Flow Immunocytometry Analysis-Production of H2O2 was determined by the sensitive fluorimetric method of Bénard and Brault (31Benard B. Brault J. Union Med. Can. 1971; 100: 701-705PubMed Google Scholar) slightly modified as previously described (11De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar). The peroxide released from transfected cells (Duox1/DuoxA1 or Duox2/DuoxA2) into Krebs-Ringer-Hepes medium was accumulated in the presence of stimulating agents for 2.5 h at 37 °C. After removing the medium, cell surface expression of Duox1–2 proteins was measured by flow cytometry (FACS). Briefly, the cells detached with phosphate-buffered saline EDTA/EGTA (5 mm) were incubated sequentially with anti-HA antibody (clone 3F10; Roche Applied Science) and fluorescein-conjugated anti-rat IgG, both diluted 1/100 in phosphate-buffered saline, 0.1% bovine serum albumin. Propidium iodide (5 μg/ml) staining in the second incubation step was used to exclude damaged cells from subsequent analysis. Fluorescence was analyzed using cell sorting (FACS-can; Becton Dickinson, Erembodegem, Belgium) counting 20,000 events/sample. Relative protein expression was determined by calculating the differences in total fluorescence intensity (Arbitrary Unit) between the samples and an equal-sized population of control cells expressing only Duox1 or Duox2 constructs without their respective maturation factors. Without the latter, no cell surface expression of Duox could be detected (11De Deken X. Wang D. Dumont J.E. Miot F. Exp. Cell Res. 2002; 273: 187-196Crossref PubMed Scopus (157) Google Scholar). H2O2 production was normalized to cell surface expression of each construct and reported as pg of H2O2/FACS. For cultures of human thyrocytes, H2O2 released over 90 min in the presence of various agents was normalized to total proteins extracted in Laemmli buffer and quantified by paper dye binding assay (ng of H2O2/μg of protein) (32Minamide L.S. Bamburg J.R. Anal. Biochem. 1990; 190: 66-70Crossref PubMed Scopus (237) Google Scholar). Immunoprecipitation and Western Blot Analysis-Proteins were extracted in lysis buffer (10 mm Tris-HCl, pH 7.5, 150 mm KCl, 0.5% Nonidet P-40, 120 mm β-mercaptoethanol, 100 mm NaF, 2 mm EDTA, pH 8.0, 50 nm okadaic acid, 1 mm vanadate) supplemented with a mixture of protease inhibitors (Complete; Roche Applied Science) for 1 h at 4 °C. The lysate was centrifuged 15 min at 10,000 rpm, and the supernatant was pre-cleared with Sepharose beads (GE Healthcare). Duox1 complexes were immunoprecipitated with anti-Duox antibody (1/100) conjugated to Sepharose beads (2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar) and Duox2 proteins with monoclonal anti-HA antibody precoated on agarose beads (Clone HA-7; Sigma-Aldrich). Proteins of the immunoprecipitate were separated by SDS/PAGE and transferred to nitrocellulose as previously described (2De Deken X. Wang D. Many M.C. Costagliola S. Libert F. Vassart G. Dumont J.E. Miot F. J. Biol. Chem. 2000; 275: 23227-23233Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). Phosphorylated Duox proteins were detected using a rabbit polyclonal antibody raised against phospho-(Ser/Thr) PKA substrate (1/1,000; Cell Signaling, Danvers, MA), which is directed to the phospho motif RXX(S/T). Fluorescent secondary antibodies (1/10,000; IRDye 800 anti-rabbit from LI-COR, Lincoln, NE) were used for image acquisition and quantification with the Odyssey infrared imaging system (LI-COR). The membrane was stripped and immunoblotted with the anti-Duox antibody (1/16,000) and fluorescent secondary antibodies (1/10,000; IRDye 680 anti-rabbit) to quantify total Duox proteins. Radioactive Phosphorus Incorporation-Cells maintained 24 h in serum-free phosphate-depleted medium were incubated 2 h with 500 μCi (Cos-7) or 1mCi (thyrocytes) of [32P]orthophosphate. The proteins were prepared as described above, and phosphorylated proteins were detected and quantified with a Storage Phosphor Screen (GE Healthcare) scanned with Typhoon Trio+ (GE Healthcare). Total Duox proteins for each condition were measured by Odyssey infrared imaging system coupled with the polyclonal anti-Duox antibody. In Vitro PKA Phosphorylation Assay-Cos-7 Duox1/DuoxA1 transfected cells were cultured 24 h without serum. The proteins were prepared in lysis buffer, and Duox1 complexes were immunoprecipitated overnight with monoclonal anti-HA antibody precoated on agarose beads as described above. Immunoprecipitated proteins were incubated for 10 min at 37 °C in a 50-μl final volume that contained 20 mm Hepes, pH 7.4, 0.1 mm dithiothreitol, 10 mm MgCl2, 0.1 mm [γ-32P]ATP (5 μCi/tube), and 100 ng of purified catalytic subunit of protein kinase A (Calbiochem, Gibbstown, NJ). After SDS/PAGE and Western blotting, radioactive signals were quantified with Typhoon Trio+; Duox proteins were immunodetected with the anti-Duox antibody and visualized with Odyssey imaging system. Mass Spectrometry Analysis-Cos-7 cells transfected with wild type Duox1 and DuoxA1 constructs were cultured 24 h without serum and stimulated 30 min with 10 μm forskolin. Duox1 complexes were immunoprecipitated with anti-Duox antibody as described above. Duox1 proteins were separated by 7%-polyacrylamide gel electrophoresis and stained with colloidal Coomassie Blue. After excision of the Duox gel bands, the proteins were in-gel digested with trypsin or chymotrypsin, and the resulting peptides were extracted from the gel (33Shevchenko A. Tomas H. Havlis J. Olsen J.V. Mann M. Nat. Protoc. 2006; 1: 2856-2860Crossref PubMed Scopus (3554) Google Scholar). The digested peptides were separated onto a C18 reverse phase 1 × 50 mm column (Vydac; Alltech Associates, Lokeren, Belgium) and deposited onto a stainless steel target. Mass spectrometry analysis was performed on a Quadrupole-time of flight Ultima Global mass spectrometer equipped with a matrix-assisted laser desorption ionization source (Micromass, Waters, Zellik, Belgium) calibrated using the monoisotopic masses of tryptic and chymotryptic peptides from bovine serum albumin. Statistical Analysis-The data are presented as the means ± S.D. The results were analyzed using the unpaired Student's t test, and p < 0.05 was considered statistically significant (**, p < 0.01; ***, p < 0.001). DuoxA-based Functional Assay to Study Duox-mediated H2O2 Generation-Heterologous systems combining DuoxA expression have already been successfully used to characterize Duox activity of human Duox2 natural mutants (13Grasberger H. De Deken X. Miot F. Pohlenz J. Refetoff S. Mol. Endocrinol. 2007; 21: 1408-1421Crossref PubMed Scopus (77) Google Scholar) and to reconstitute a functional H2O2-generating system in a lung cancer cell line (34Luxen S. Belinsky S.A. Knaus U.G. Cancer Res. 2008; 68: 1037-1045Crossref PubMed Scopus (119) Google Scholar). The originality of our study is to analyze the specific activity of Duox1 and Duox2 isoenzymes by normalizing the hydrogen peroxide production to the cell surface expression of the respective proteins. Insertion of an HA tag in the Duox1–2 ectodomain provides an effective means to reliably estimate Duox membrane expression and compare the activities of the two enzymes. We first validated our heterologous system: 1) Measurement of H2O2 produced after 1 μm ionomycin stimulation of cells transfected with Duox/DuoxA (constant DuoxA quantity, 50 ng) was proportional to the quantity of transfected Duox plasmids (25–500 ng) with a plateau reached at 250–500 ng of Duox DNA probably caused by a limited amount of DuoxA (Fig. 1A). 2) We observed a linear relationship between Duox-mediated H2O2 generation and the membrane expression of Duox as measured by FACS (Fig. 1B). The specific activity of both Duox enzymes was comparable, and no H2O2 was detected in cells expressing Duox or DuoxA alone (Fig. 1A). 3) We verified that the addition of the tags did not modify the expression and activity of Duox1–2 proteins (supplemental Fig. S1). In all experiments, the specific activity of the Duox enzymes is represented as the amount of H2O2 produced normalized to their surface expression (pg H2O2/FACS). In humans, the thyroid metabolism is under the control of the phosphatidylinositol 4,5-bisphosphate cascade and the cAMP cascade (26Raspe E. Laurent E. Andry G. Dumont J.E. Mol. Cell. Endocrinol. 1991; 81: 175-183Crossref PubMed Scopus (62) Google Scholar, 27Corvilain B. Laurent E. Lecomte M. Vansande J. Dumont J.E. J. Clin. Endocrinol. Metab. 1994; 79: 152-159Crossref PubMed Google Scholar). In our functional assay, 1 μm ionomycin increased the activity of both Duox1 and Duox2 enzymes to the same level, but raising concentrations of ionomycin from 2 to 4 μm resulted in higher activity of Duox1 than Duox2 (Fig. 2A). The adenylate cyclase agonist, forskolin (Fsk), raised the amount of H2O2 generated by Duox1 but not by Duox2 with a 50% effective concentration (EC50) of 0.1 μm (Fig. 2B). Moreover PMA, a PKC activator, differentially regulated activities of Duox1 and Duox2 (Fig. 2, C and D). Although micromolar concentrations of PMA were needed to increase Duox1 activity with an EC50 of 1.8 μm, a maximal activation of Duox2 enzyme was already observed with nanomolar PMA concentrations (EC50 = 0.8 nm). The addition of Fsk or PMA to ionomycin in the incubation medium provoked a release of H2O2 corresponding to the sum of the amounts generated by the cells treated with the agents separately (Iono versus Iono+Fsk for Duox1: p < 0.01; Iono versus Iono+PMA for Duox1: p < 0.001; Iono versus Iono+PMA for Duox2: p < 0.01) (Fig. 2E). In contrast to Duox1, raising calcium concentration was not sufficient to activate Duox2 by Fsk. In all of the conditions, 1 μm diphenyleneiodonium, a flavoprotein inhibitor, reduced the H2O2 level produced by the two enzymes, indicating that it derives from an NADPH oxidase (data not shown). Regulation of Duox Activity by Calcium-Duox-mediated H2O2 generation is presumed to be regulated by binding of Ca2+ to the two EF-hand motifs, located in the cytosolic portion spanning transmembrane domain 1 and 2 (13Grasberger H. De Deken X. Miot F. Pohlenz J. Refetoff S. Mol. Endocrinol. 2007; 21: 1408-1421Crossref PubMed Scopus (77) Google Scholar, 34Luxen S. Belinsky S.A. Knaus U.G. Cancer Res. 2008; 68: 1037-1045Crossref PubMed Scopus (119) Google Scholar, 35Dupuy C. Deme D. Kaniewski J. Pommier J. Virion A. FEBS Lett. 1988; 233: 74-78Crossref PubMed Scopus (31) Google Scholar). Indeed, the absence of calcium ions in the H2O2 measurement assay abolished the Duox1–2 activity (data not shown). The other calcium-dependent NADPH oxidase, NOX5, possesses four calcium-binding sites arranged in two functional pairs (36Banfi B. Tirone F. Durussel I. Knisz J. Moskwa P. Molnar G.Z. Krause K.H. Cox J.A. J. Biol. Chem. 2004; 279: 18583-18591Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). To study the role of the two Duox EF-hands, the crucial glutamate residue was replaced by a glutamine in the twelfth position of the EF-hand sequence. Duox2 mutants E843Q and E879Q completely lost the ability to produce H2O2 in basal or stimulated conditions but maintained a cell surface expression similar to the wild type (WT) protein (Fig. 3). Similarly, inactivation of one of the two EF-hands in Duox1 (E839Q and E875Q Duox1 mutants) was sufficient to inactivate Duox1, although a correct processing to the membrane occurred as for Duox2 (supplemental Fig. S2). These results strongly suggest that the two EF-hand motifs operate as one functional pair necessary for Duox activation in response to an increase of intracellular calcium concentration. Duox1 Activity Is Positively Modulated through the cAMP Pathway-As shown in Fig. 2, Duox1-dependent H2O2 generation was positively controlled by Fsk, contrary to Duox2. To establish the role of the protein kinase A, N6-monobutyryladenosine-3′,5′-cyclic monophosphate (6-MB-cAMP), a site-selective PKA agonist, was used. H2O2 produced by Duox1 was significantly increased with either 50 μm 6-MB-cAMP or 1 μm Fsk (Fig. 4A). We also co-transfected a vector encoding the α isoform of the PKA catalytic subunit with WT Duox/DuoxA constructs (37Uhler M.D. McKnight G.S. J. Biol. Chem. 1987; 262: 15202-15207Abstract Full Text PDF PubMed Google Scholar, 38Buchler W. Meinecke M. Chakraborty T. Jahnsen T. Walter U. Lohmann S.M. Eur. J. Biochem. 1990; 188: 253-259Crossref PubMed Scopus (12) Google Scholar). Overexpression of PKA for 48 h, verified by Western blotting (data not shown), also induced an increase of Duox1 activity. Co-expression of the PKA subunit had only a minor effect on Duox2 activity, and treatment with the 6-MB-cAMP did not increase H2O2 production by Duox2. Motif scanning for PKA substrates based on the consensus sequence (R/K)2X(S/T) identifies three potential PKA phosphorylation sites in Duox1 (Ser955, Thr1007, and Ser1217) (supplemental Fig. S3). We analyzed the Duox1 phosphorylation state with an anti-RXX(pS/pT) antibody that could potentially recognize phosphorylation on Ser955 and Ser1217 but not on Thr1007. Under basal conditions, the mature form of Duox1 (200 kDa) was already phosphorylated in Cos-7 cells (supplemental Fig. S4). It is noteworthy that the slower migrating form of Duox1 (mature form) and the immature form were shifted to 200 and 190 kDa, respectively, instead of the wild type 190- and 180-kDa proteins. This phenomenon can be explained by the addition of extra N-linked sugars on the two putative N-glycosylation sites present in the Rho tag sequence as previously described for similar TSH receptor constructs (28Vlaeminck-Guillem V. Ho S.C. Rodien P. Vassart G. Costagliola S. Mol. Endocrinol. 2002;