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Unité 486, Institut National de la Santé et de la Recherche Médicale, Université Paris 11, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France
Address all correspondence and requests for reprints to: C. Dupuy, Unité 486, Institut National de la Santé et de la Recherche Médicale, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France. E-mail: corinne.dupuy{at}cep.u-psud.fr.
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Introduction |
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The recent discovery of a biallele-inactivating mutation on the DUOX2 gene in a patient with total iodide organification defect combined with severe and permanent congenital hypothyroidism has confirmed the major role of Duox2 in Tg iodination, in all probability as a supplier of H2O2 for Tpo, but has raised the question of whether Duox1 is involved in the thyroid H2O2 generator (10). A functional H2O2-generating system based on Duox1 or Duox2 proteins will have to be reconstituted to determine the role of each protein. This has not yet been achieved, probably because other unidentified components are also required for the maturation of the Duox proteins and/or their targeting to the plasma membrane (11).
Iodide is an important modulator of thyroid gland activity that exerts its mainly inhibitory effects at various stages during its metabolism (12). The inhibition by iodide of its own organification by Tpo is known as the Wolff-Chaikoff effect (13), and it is caused by the inhibition by iodide of the H2O2 generator stimulated by various agonists (14, 15). This inhibition is suppressed by antithyroid drugs, which inhibit Tpo-catalyzed iodide organification, indicating that iodide becomes inhibitory only after it has been oxidized (14, 15).
In the work reported in this article, we analyzed the effect of iodide on the expression and activity of the H2O2 generator in pig thyroid follicles cultured under stimulatory conditions. We observed that a low concentration of iodide decreased the ability of thyrocytes to release H2O2 into the medium and inhibited to the same extent the activity of NADPH oxidase present in the particulate fraction of these cells. The simultaneous decrease in the expression of the mature form of Duox2 observed under these conditions could explain the inhibitory effect of iodide on NADPH oxidase.
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Materials and Methods |
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Treatment of pig thyroid follicles
Follicles were washed twice by sedimenting for 10 min (1 x g) in 20 ml PBS solution and resuspended in fresh DMEM supplemented with antibiotics, Fungizone, and 5% fetal calf serum. The follicles were cultured for another 48 h with or without 10 µM forskolin or 0.5 mM 8-(4-chlorophenylthio)-cAMP(pCPT-cAMP). KI (potassium iodide) was added to this medium with or without 100 µM methimazole (MMI). Mechanical treatment was adjusted to produce open follicles and follicle segments made of polarized thyrocytes, as previously described (16).
Measurement of H2O2 release from open follicles
H2O2 released by porcine cells in the medium was quantified by measuring the oxidation of homovanilic acid into its fluorescent derivative in the presence of horseradish peroxidase (17). Follicles (2 x 107 cells) were washed twice with HEPES-buffered Earle’s solution and resuspended in 1 ml of the same buffer. Aliquots of the suspension (
2 x 106 cells), were incubated at 37 C for 30 min in 0.5 ml HEPES-buffered Earle’s solution containing 0.44 mM homovanilic acid, 0.5 mg/ml (2 U/ml) horseradish peroxidase, 1 mM sodium azide, and 0.5 µM ionomycin with or without 1 mM EGTA. At the end of the incubation, 200 µl medium from each tube were collected, and fluorescence was measured (315 and 425 nm excitation and emission, respectively). A standard curve obtained by incubating increasing amounts of H2O2 (0–25 µM) in the same solution was used to measure the concentration of H2O2 released into the medium. After solubilization of protein for 1 h in 1 N NaOH, the amount of protein was estimated by the Bradford technique (18).
Preparation of the follicle particulate fraction
Cells from two Petri dishes (about 2 x 107 cells) were collected by centrifuging at 200 x g for 7 min, resuspended in Earle’s solution buffered with 20 mM HEPES (pH 7.2), and centrifuged again. The cells were then suspended in 1 ml 50 mM sodium phosphate buffer containing 0.25 M sucrose, 0.1 mM dithiothreitol, 1 mM EGTA (pH 7.2), and a cocktail of protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin, 157 µg/ml benzamidine) and homogenized using a motor-driven Teflon pestle homogenizer. After centrifuging at 3000 x g for 15 min, the pellet was resuspended in 2 ml 50 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 10 µM flavin adenine dinucleotide (FAD), 1 mM MgCl2, and the cocktail of protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 157 µg/ml benzamidine, and 1 µg/ml pepstatin). After centrifuging under the same conditions as before, the particles were resuspended in 2 ml of the same buffer and centrifuged again. The final pellet was resuspended in the same buffer but without flavin adenine dinucleotide, quick-frozen in liquid nitrogen, and stored at -80 C until used.
Measurement of NADPH oxidase activity in the follicle particulate fraction
NADPH oxidase activity in cell particulate fraction was measured using the scopoletin method (5) modified as follows. Particles were incubated at 30 C in 1 ml of 160 mM sodium phosphate buffer pH 7.4 containing 1 mM sodium azide, 1.5 mM CaCl2, 1 mM EGTA, 0.4 mM MgCl2, 50 mM sucrose, and 0.1 mM NADPH. Eight aliquots (100 µl) from each sample were collected at intervals from 0 to 20 min and mixed with 20 µl 3 N HCl to stop the reaction and destroy the remaining NADPH. The fluorescence (ex.: 360 nm; em.: 460 nm) of 2-ml aliquots was measured using an MPF 43 A spectrofluorometer (Perkin-Elmer Corp., Les Ulis, France) after adding to each 200 mM sodium phosphate buffer, pH 7.8, containing 0.25 µM scopoletin and 5 µg/ml horseradish peroxidase. The concentration of H2O2 was directly correlated to the concentration of oxidized (nonfluorescent) scopoletin.
Northern blot analysis
RNA was extracted from the primary culture of pig follicles by the method of Chomczynski and Sacchi (19). Northern blot analyses were performed as previously described (7). Final washes were carried out at 60 C in 0.1x SSC (1x SSC = 0.15 M NaCl, 15 mM sodium citrate), 0.1% sodium dodecyl sulfate (SDS). The pig cDNA probes used were prepared by RT-PCR, using total RNA from pig thyrocytes. The sense and antisense pig 3'-UTR DUOX2 PCR primers (MGW Biotechnology, Les Ulis, France) were 5'-CACTTCAGGCCTTAGCTGGA-3' and 5'-GACCAAACGAATCTAGAGCA-3', respectively. The sense and antisense pig 3'-UTR NIS PCR primers were 5'-GGACAGACATCACACATGCTCT-3' and 5'-GCAAGTTTATTCTTTGCAGGCT-3', respectively. The sense and antisense pig TPO PCR primers were 5'-GCATCCGGATAACATTGACGTC-3' and 5'-TGTCTCGTCTTCACACTCGTTGA-3', respectively (20). The cDNA probes were 32P-labeled by random priming extension using a kit (Amersham Pharmacia Bio-Tek, Saclay, France). The 32P-radioactivity bound to membranes was counted using an INSTANTIMAGER (Packard Instrument SA, Rungis, France).
Nylon membranes were washed clean of the probes in 0.5% SDS at 95 C. To ensure that the same amount of RNA was present in each lane, the membranes were hybridized with a 28S rRNA probe (5'-GAGATTTACACCCTCTCCCCCGGATTTTCA-3') labeled by [32P]-
ATP (SA > 3000 Ci/mmol; Perkin-Elmer). The results were normalized relative to the 28S rRNA labeling.
Cloning of porcine DUOX1 and DUOX2 cDNAs
The amplifications of the porcine DUOX1 and DUOX2 cDNAs (GenBank accession AF547266 and AF547267, respectively) were achieved by rapid amplification of cDNA ends using the SMART RACE cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA) and applying the manufacturer’s protocol with 1 µg total thyroid mRNA. DUOX1 and DUOX2 PCR products (5.8 kb and 6.1 kb, respectively) were cloned into pCR-XL-TOPO vectors (Invitrogen, Cergy Pontoise, France). Sequencing was performed three times in the 5' to 3' direction.
Transient expression of porcine Duox1 and Duox2 in CHO cell line
For expression studies, the complete coding sequences of pig DUOX1 and DUOX2 were subcloned into pcDNA3.1D-TOPO (Invitrogen). Chinese hamster ovary (CHO) cells were maintained in F-12 nutrient mixture (Ham) medium (Invitrogen-Life Technologies, Cergy Pontoise, France) supplemented with 10% fetal calf serum, antibiotics, and Fungizone. At 50–60% of confluence, CHO cells were transfected with pcDNA3.1D-DUOX1 or pcDNA3.1D-DUOX2 by using the FuGENE (Roche, Meylan, France) transfection reagent. After 24 h, cells were washed twice with PBS and scraped in the same solution supplemented with a cocktail of protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin, 157 µg/ml benzamidine). After a centrifuging at 200 x g for 10 min at 4 C, cell pellets from two flasks (75 cm2) were homogenized using a motor-driven Teflon pestle homogenizer in 1 ml 50 mM sodium phosphate buffer containing 0.25 M sucrose, 0.1 mM dithiothreitol, 1 mM EGTA (pH 7.2), and a cocktail of protease inhibitors (5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin, 157 µg/ml benzamidine). After centrifuging at 3000 x g for 30 min, the pellet was resuspended in 0.5 ml 50 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 1 mM MgCl2, and the cocktail of protease inhibitors.
Deglycosylation experiments
Deglycosylation of pig protein was performed as follows: Follicle particulate fraction (120 µg) was treated in 50 mM phosphate buffer, pH 7.4 (60 µl final volume), containing the antiprotease cocktail, 2.5 mM EDTA, 0.1% SDS, before adding 50 U/ml N-glycosidase F (Roche). After a first incubation lasting 1 h, an additional 50 U/ml N-glycosidase F was added and the incubation continued for another hour. Controls were performed without adding N-glycosidase F. The reaction was stopped by adding 12 µl concentrated Laemmli sample buffer before performing electrophoresis.
Western blot analysis
SDS-PAGE and immunoblot analyses were performed as previously described (21). Duox proteins were detected using the antipeptide raised against the 14-amino-acid peptide encompassing the L410-T423 portion of human and porcine Duox2 (21). For control experiments, a rabbit polyclonal antibody was raised against the first intracellular portion of human Duox2 (E636–R1039) produced in Escherichia coli by the pTrcHis-TOPO vector system (Invitrogen). Duox1/2 proteins were detected using the serum at a dilution of 1:2500.
Statistical methods
Data were statistically analyzed using the t test. A P value greater than 5% was not considered significant.
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shows that follicles generated different amounts of H2O2 in the presence of ionomycin and calcium (without EGTA), depending on the treatment of the cells. Thyrocytes cultured in the presence of forskolin generated approximately 2–2.5 times more H2O2 than cells cultured in control medium. The presence of 1 µM KI induced a 50–60% inhibition of the activity of the forskolin-treated cells. In agreement with studies on the acute effect of iodide on the dog thyroid H2O2 generator (14, 15), the inhibitory effect of iodide activity of porcine follicles was suppressed by MMI, an inhibitor of the Tpo-catalyzed iodide organification.
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shows that, unlike follicles reconstituted from trypsin-isolated thyrocytes (5), some NADPH oxidase activity persisted in particles from collagenase-isolated follicles cultured without forskolin. However, it was equivalent to only 40–50% of the activity of follicles cultured with forskolin or the membrane-permeable cAMP analog pCPT-cAMP. Depending on the follicle preparation, the NADPH oxidase activity in follicles cultures under stimulatory conditions varied in the range of 100–300 nmol H2O2 x h-1 x mg protein-1.
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, KI at concentrations of 10 nM and 100 nM had no significant effect, whereas 1 µM KI induced a 50% decrease of the NADPH oxidase activity. Again, the inhibitory effect of KI was prevented by MMI (Fig. 2C), indicating the involvement of an oxidized form of iodide, or an iodocompound (XI). One major well-known effect of XI is the inhibition of both TSH- and forskolin-induced accumulation of cAMP in thyroid slices from various species (22, 23) and porcine thyrocytes (24). Because the expression of NADPH oxidase depends on cAMP (4, 5), we evaluated the effect of 1 µM KI when a high level of nucleotide was maintained by incubating follicles with pCPT-cAMP. As shown in Fig. 2D, the inhibitory effect of KI was not suppressed by the presence of the cyclic nucleotide analog, indicating that KI, in addition to its probable effect on adenylate cyclase, which was not investigated in this study, also acted on a more distal target. A distal effect of KI of this type was also noted on the release of H2O2 from dog thyroid slices stimulated by various agonists (14). To evaluate the possibility that iodide could decrease the concentration of active NADPH oxidase by modulating the rate of Duox2 synthesis and/or degradation, we analyzed the expression of DUOX2 mRNA and Duox2 protein by Northern and Western blots, respectively.
Effect of iodide on DUOX2 mRNA expression in forskolin-stimulated follicles
The effect of iodide on DUOX2 mRNA expression was compared with that on the mRNA expression level of NIS and TPO, both of which are known to be down-regulated in vivo by moderate doses of KI (25). Cells were cultured under conditions described in Fig. 2. Forskolin (Fig. 3A) and CPT-cAMP (Fig. 3B) stimulated the expression of the 6.1-kb DUOX2 mRNA. The expression of the 3.2-kb TPO mRNA (20) was more sensitive to cAMP than that of DUOX2 mRNA, but the greatest impact of cAMP was observed on the expression of the two NIS mRNA species detected with an NIS-specific 3'-UTR probe and which probably corresponded to the 3.5-kb and the 3-kb porcine NIS transcripts described in GenBank (accession nos. AJ487855 and AJ277989, respectively). KI clearly reduced the expression of the NIS gene in the presence of forskolin or CPT-cAMP, and to a lesser extent that of TPO, but had no significant effect on the level of DUOX2 mRNA. The effect of KI on NIS and TPO gene expression was abolished by MMI. Figure 4 shows the combined results for three to seven different cultures. The mean mRNA levels, normalized with the 28S rRNA, were expressed as an x-fold difference, relative to the basal sample. Figure 4 confirmed the inhibition by KI of NIS and TPO gene expression by about 60% and 30%, respectively. These experiments reproduced in our cultured cells model the inhibition by iodide of NIS and TPO gene expression obtained in vivo after dogs had been treated for 48 h with KI (25). The quantification of Northern blots (Fig. 4) also confirmed that DUOX2 gene expression in the presence of cAMP was not significantly modified by KI. This observation led us to investigate the expression of Duox2 at the protein level.
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). The antipeptide (Anti-P) essentially recognized the pig Duox2 protein (Fig. 5A). The cloning and sequencing of DUOX1 cDNA showed that it encodes a 1553-amino-acid protein displaying 77% sequence identity with porcine Duox2. The corresponding V407-T420 peptide in porcine Duox1 is VQDFWPGPLKFSRT, which differs by four amino acids (bold italic) from Duox2 peptide. The presence of an excess of synthetic Duox1 peptide did not prevent the labeling of Duox2 by the antipeptide (Fig. 5B). In contrast, preincubating the antibody with an excess of synthetic Duox2 peptide did prevent the labeling of Duox2 by the antipeptide (Fig. 5B). In the absence of a specific antibody to Duox1, it was not possible to determine the relative proportion of each Duox in pig thyroid, so we used the antipeptide antibody to specifically monitor the expression of Duox2 in pig thyrocytes by Western blotting.
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). After the membrane proteins had been exposed to N-glycosidase F, only one band persisted at 150 kDa (Fig. 6), as with human thyroid particles in a previous study (21). Consequently, we concluded that the 165- and 175-kDa proteins are two differently N-glycosylated forms of Duox2, with N-glycosylation motifs accounting for 15 and 25 kDa, respectively, of their molecular mass.
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). The addition of forskolin increased the labeling of both bands but had a greater effect on the more highly glycosylated form (Fig. 7A), showing that cAMP increased the amount of Duox2, either through the inhibition of its degradation or, more probably, by an increase in its synthesis and glycosylation.
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). Figure 7B shows that iodide decreased the expression of the 175-kDa form of Duox2 independently of its inhibitory effect on the adenylate cyclase because it persisted when CPT-cAMP was used instead of forskolin. |
Discussion |
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Longer exposure of the thyrocytes to iodide could also regulate the expression of the H2O2 generator at the mRNA stage, as it does in vivo for NIS and TPO gene expression (25). Alternatively, a posttranscriptional KI effect could not be excluded. We demonstrated in this study that when pig thyrocytes were exposed to 1 µM KI for 48 h under stimulating conditions, the release of H2O2 by thyrocytes and the NADPH oxidase activity measured in a cell-free system using their particulate fraction were inhibited. This inhibitory concentration of KI is 2- to 10-fold higher than what is generally considered to be the physiological range (0.1–0.5 µM). Similar concentrations of KI have also been reported to activate the cellular production of H2O2 in thyroid slices from various species, but these were acute effects of KI observed under nonstimulating conditions (27) and could reflect the effects of KI on NADPH oxidase activation or H2O2 metabolism.
Acute effects of KI required 50-fold higher concentrations to inhibit the H2O2 generation by carbamylcholine-stimulated dog thyrocytes (15) and of carbamylcholine-, TSH-, ionomycin-, or 12-O-tetradecanoylphorbol 13-acetate-stimulated dog thyroid slices (14). Because it is very likely that the cellular production of H2O2 is catalyzed by NADPH oxidase, these discrepancies probably are due to differences in experimental design and/or cell model. In particular, the chronic exposure of porcine follicles (48 h) to KI could induce inhibition of NADPH oxidase activity at lower concentrations than in studies involving short exposure times (1–2 h) of cells and thyroid slices to KI (14, 15).
Although KI, used at 1 µM, down-regulated the expression of TPO and NIS mRNAs in our cell system, it did not affect the level of DUOX2 mRNA. However, during the early stages, the decrease in TPO and NIS gene expression, could also contribute to the Wolff-Chaikoff effect by reducing the ability of the cells to take up and oxidize iodide, in synergy with the inhibition of NADPH oxidase. The resulting decrease in XI formation would then subsequently allow the normal iodide organification capacities of the thyrocytes to be restored.
Western blot analyses of Duox2 expression detected two proteins with different oligosaccharide contents. Sequencing of the full-length pig DUOX2 cDNA showed that porcine Duox2 displays six putative sites of N-glycosylation, whereas five were identified in the human protein (11). Three of these sites (N100, N348, and N455) occur in both human and porcine Duox2 proteins. The sites that are actually N-glycosylated have not yet been identified, but the total Duox1/2 content of oligosaccharide motifs has been estimated to account for 15 kDa in the 165-kDa form and 25 kDa in the 175-kDa form. A 165-kDa Duox1/2 protein had already been observed in the 100,000 x g pellet from normal human thyroid and after transient transfection of nonthyroid cells (21). N-deglycosylation experiments indicated that human Duox1/2 have a nonglycosylated apparent molecular mass of 148 kDa (21), close to that found in this study for pig Duox2 (150 kDa).
The 175-kDa form, which is probably the flavoprotein solubilized, purified, and microsequenced from porcine thyroid plasma membrane (7), was not detectable using our antipeptide antibody in the 100,000 x g particulate fraction from human tissue (21). It is likely that the concentration of the 175-kDa protein in human thyroid tissue is much lower than in membrane fractions from pig thyroid cells or tissue and that an antibody with a higher affinity is required for its detection. The specific detection of the 175-kDa form in a porcine thyroid fraction containing 10–20 times more NADPH oxidase activity than the human particulate fraction (3) suggests that it constitutes the mature and active form of Duox2 and that the 165-kDa form is its precursor. These data are in close agreement with those obtained with dog and human thyrocytes cultured with forskolin, which also displayed two bands of N-glycosylated Duox1/2 of 180 and 190 kDa, respectively, with an apparent molecular mass of 160 kDa for the nonglycosylated proteins (11). Only the more highly glycosylated form was resistant to endoglycosidase H digestion, indicating its passage through the Golgi apparatus and also suggesting that it constitutes the mature form involved in the active NADPH oxidase at the plasma membrane (11).
The decrease in NADPH oxidase activity observed after 48-h treatment with KI was concomitant with the reduction in the 175-kDa form of Duox2, suggesting that it could result from accelerated degradation of the active form of Duox2 or the inhibition of its synthesis, rather than from a direct inhibitory effect on the enzyme. Assuming that the 175-kDa protein is in fact the mature, active form of Duox2 at the apical membrane of thyrocytes, this iodide effect could protect the cell against oxidative stress resulting from the overexpression and overactivity of the H2O2/Tpo system under stimulatory conditions. It could contribute to the paradoxical decrease in H2O2-generating activity observed in toxic adenomas (28) and the decrease in Duox immunolabeling generally observed in hyperfunctioning tissues (21). The mechanism by which KI regulates the proportion of 175-kDa protein, by either accelerating its degradation or blocking the maturation of the 165-kDa form, remains to be established. It also remains to be confirmed whether the Wolff-Chaikoff effect can be fully accounted for by the KI-induced decrease in the concentration of the 175-kDa Duox2 protein. In particular, the exact role of the 175-kDa form of Duox2 in NADPH oxidase and its cellular location in thyrocytes remains to be established. It also would be interesting to find out whether KI regulates the amount of the highly glycosylated form of Duox2 in vivo, as it does in vitro. It has been reported that NADPH oxidase activity is very low or absent in thyroid tissue from patients with diffuse toxic goiter treated with KI before surgery (29). Because all but one of these patients had also been given MMI or propylthiouracil, either KI induced inhibition of NADPH through a different mechanism or the doses given was not high enough to prevent XI formation. Further experiments with animal models are therefore required to elucidate the effect of KI on Duox2 expression in vivo.
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Acknowledgments |
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Footnotes |
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Abbreviations: Abbreviations: Anti-P, Antipeptide; CHO, Chinese hamster ovary; DUOX, dual oxidase gene; Duox, dual oxidase protein; KI, potassium iodide; MMI, methimazole; NADPH, nicotinamide adenine dinucleotide phosphate; NIS, Na+/I- symporter; pCPT-cAMP, 8-(4-chlorophenylthio)-cAMP; SDS, sodium dodecyl sulfate; Tg, thyroglobulin; THOX, thyroid oxidase flavoprotein; Tpo, thyroid peroxidase; XI, iodocompound.
Received September 18, 2002.
Accepted for publication December 19, 2002.
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