Identification of a Truncated Dual Oxidase 2(DUOX2) Messenger Ribonucleic Acid(mRNA) in Two Rat Thyroid Cell Lines. Insulin and Forskolin Regulation
Abstractg$si, 百拇医药
The Duox2 flavoprotein is strongly expressed in the thyroid gland, where it plays a critical role in the synthesis of thyroid hormones likely by providing thyroperoxidase with H2O2. A truncated DUOX2 mRNA was isolated from the rat thyroid cell line FRTL-5. The cDNA sequence predicted an open reading frame of 1458 bp, encoding a polypeptide of 486 amino acids corresponding to the carboxyl fragment of the Duox2 flavoprotein. The truncated form of DUOX2 mRNA, expressed in another rat thyroid cell line, the PC Cl3 cell line, was absent from Fischer rat thyroid glands. Although it was expressed in both cell lines to a greater extent than normal mRNA, it failed to support protein synthesis in an in vitro translation system. Insulin increased the levels of both normal and truncated DUOX2 mRNA in FRTL-5 cells grown in TSH-free medium containing a low concentration of serum. The stimulating effect of insulin on DUOX2 mRNA expression was reproduced in pig thyroid follicles in primary culture. The presence of insulin in the culture medium converted forskolin from a stimulator to an inhibitor in FRTL-5 cells maintained in low serum conditions, but not in porcine thyrocytes in primary culture.
Introductionpa., 百拇医药
THE SYNTHESIS OF thyroid hormone is catalyzed by thyroperoxidase (Tpo) in the presence of H2O2 (1) on the apical membrane of the thyroid follicular cells (2). H2O2 is produced on the apical plasma membrane of rat and pig open follicles (3, 4) by a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in both pig (5, 6, 7) and human thyroid (8), as proposed by Björkman et al. (3). Thyroid NADPH oxidase requires micromolar concentrations of calcium to acquire a functional conformation and to generate H2O2 (7, 8, 9). A functional NADPH oxidase, generating H2O2 in a Ca2+-dependent manner, was solubilized from pig thyroid (10), and a flavoprotein with an apparent molecular mass of about 180 kDa was purified from it (11). Microsequences were used to clone its porcine and human partial cDNAs, making it possible to localize its gene on chromosome 15q15 (11). The full-length cDNA, encoding a 1548- amino-acid protein known as thyroid oxidase gene (Thox)2, has subsequently been cloned from human thyroid (12). Its sequence is 83% similar to that of the Thox1, also expressed in the thyroid gland (12). Thox1,2 proteins and THOX1,2 genes are also known as Duox1,2 and DUOX1,2 genes, according to the recommendations of the Human Gene Nomenclature Committee. Although a functional Duox-based H2O2 generating system has not yet been reconstituted (13), the essential role of Duox2 in thyroid hormone synthesis has been confirmed by the recent observation of permanent and severe congenital hypothyroidism in a patient with a biallelic inactivating mutation in the DUOX2 gene (14).
Little is known about the regulation of DUOX2 expression. In vitro studies have shown that DUOX2 mRNA is up-regulated by cAMP agonists in human (12), porcine (11), and dog thyroid cells in primary culture (12), as well as in the FRTL-5 rat thyroid cell line (15). However, the level of DUOX2 mRNA is reduced in the hyperfunctioning thyroid tissues of rats treated with an antithyroid drug (methimazole; Ref. 15). These findings indicate that the up-regulation of the expression of DUOX2 mRNA by cAMP detected in cultured thyrocytes is strongly modulated in vivo by factors that repress its expression in hyperfunctioning tissues, in contrast to Na+/I- symporter (NIS) and TPO mRNAs, two other markers of thyroid differentiation. Because FRTL-5 cells are often used as a thyrocyte model to investigate thyroid gene expression, we also studied the regulation of DUOX2 expression in these cells and compared it to that observed in thyrocytes in primary culture.n, http://www.100md.com
In the present report, we show that rat FRTL-5 and PC Cl3 thyroid cells, unlike rat thyroid tissue, express relatively high levels of a truncated DUOX2 mRNA compared with the normal DUOX2 transcript. In vitro translation studies show that the synthetic truncated DUOX2 mRNA fails to support protein synthesis. Insulin increased the levels of both species of DUOX2 mRNA in FRTL-5 cells grown in a TSH-free medium containing 0.2% serum. Insulin had a similar effect on the expression of normal DUOX2 mRNA in similarly treated primary cultures of pig thyroid cells. However, in FRTL-5 cells, but not in pig thyrocytes in primary culture, the presence of insulin in the medium converted forskolin from a stimulator to an inhibitor of DUOX2 expression in cells maintained in low serum conditions. Our findings make it clear that caution is required when using FRTL-5 cells to study DUOX gene expression.
Materials and Methods9, 百拇医药
cDNA cloning9, 百拇医药
The amplification of the short DUOX2 cDNA from FRTL-5 cells was achieved by RACE using the SMART RACE cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA) and applying the manufacturer’s protocol with 1 µg total mRNA. PCR products were cloned in pCR-XL-TOPO (Invitrogen, Cergy Pontoise, France) for sequencing. Sequencing was performed three times in the 5' to 3' direction and three times in the reverse direction.9, 百拇医药
Cell culture9, 百拇医药
FRTL-5 cells (CRL no. 8305, American Type Culture Collection, Rockville, MD), a rat thyroid cell line, were a generous gift from Dr. M. Eggo (University of Birmingham, Birmingham, UK). They were grown to 80–90% confluence in Coon’s modified Ham’s F12 medium (Eurobio, Les Ulis, France) supplemented with 5% newborn calf serum, 0.5 mU/ml bovine TSH (Sigma), 10 µg/ml insulin, 5 µg/ml transferrin, 10 ng/ml somatostatin, 10 ng/ml Gly-His-Lys-acetate, and 10 nM hydrocortisone (6H medium). The cells were transferred to TSH-free 5-hormone medium (5H), or in TSH- and insulin-free 4-hormone medium (4H), containing 0.2% newborn calf serum for 6 d; forskolin or insulin was then added to the medium as required.
PC Cl3 cells, developed as previously described (16), were a generous gift from Dr. R. Di Lauro (Stazione Zoologica Anton Dohrn, Napoli, Italy). They were cultured in Coon’s modified Ham’s F12 medium supplemented with 10% fetal calf serum, 10 µU/ml bovine TSH (Sigma), 1 µg/ml insulin, 5 µg/ml transferrin, and 10 nM hydrocortisone.g@y, http://www.100md.com
Isolation of pig thyroid folliclesg@y, http://www.100md.com
Fresh porcine thyroid glands were obtained from a slaughterhouse and immediately transported to the laboratory in cold PBS solution. After the connective tissue was removed, the glands (90 g) were washed briefly in 70% ethanol and rinsed twice with PBS containing antibiotics and fungizone. The glands were minced, and follicles were isolated by collagenase digestion (360 U/ml) for 75 min at 37 C in 200 ml of PBS containing 1 mM calcium, antibiotics, and fungizone. The digest was filtered through a mesh (250 µm) and washed twice by centrifuging (100 x g, 3 min) with 20 ml cold PBS containing antibiotics and fungizone. The final pellet was resuspended in 20 ml of the same solution and sedimented for 10 min on ice. The preparation was suspended in 250 ml DMEM supplemented with antibiotics and fungizone, 5% fetal calf serum, and 1 mU/ml bovine TSH, and cultured in suspension for 3 d in a 95% air/5% CO2 incubator at 37 C. Follicles were then transferred into TSH-free 5H medium or TSH and insulin-free 4H medium, containing 0.2% newborn calf serum for 3 d; forskolin or insulin was then added to the medium as required.
RNA isolation and Northern blot analysis0{9du'{, http://www.100md.com
RNA was extracted from all of the cells and from Fischer rat thyroids by the method of Chomczynski and Sacchi (17). Northern blot analysis was performed as previously described (11). Final washes were carried out at 60 C in 0.1x SSC, 0.1% sodium dodecyl sulfate (1x SSC = 0.15 M NaCl, 15 mM sodium citrate). The rat cDNA probes used were the N-Ter cDNA corresponding to the rat 3'-RACE cDNA EcoRI insert obtained as previously described (15), the rat 3'-untranslated region (UTR) and NIS cDNAs prepared by RT-PCR, using total RNA from Fischer rat thyroids. The sense and antisense 3'-UTR PCR primers (MWG Biotech, Ebersberg, Germany) were 5'-GGGCAGGTAGACAAGGTCAG-3' and 5'-ACAGCTCAGCTTTTTAGTAA-3', respectively. The sense and antisense NIS PCR primers were 5'-CCTTCTGGACTTTCATAGTGC-3' and 5'-TGGGACCAGTAAGGTAGCTGA-3', respectively. The porcine cDNA probes used were DUOX2 cDNA and NIS cDNA prepared by RT-PCR, using total RNA from pig thyroids. The sense and antisense DUOX2 primers, designed on the 3'-UTR of the DUOX2 cDNA, were 5'-CACTTCAGGCCTTAGCTGGA-3' and 5'-GACCAAACGAATCTAGAGCA-3', respectively. The sense and antisense NIS primers were 5'-GGACAGACATCACACATGCTCT-3' and 5'-GCAAGTTTATTCTTTGCAGGCT-3', respectively.
The cDNA probes were {alpha} -32P-labeled by random priming extension using a kit (Amersham Pharmacia Biotech, Saclay, France). Membranes were analyzed by electronic autoradiography using INSTANTIMAGER (Packard Instrument SA, Rungis, France).+4, 百拇医药
RT-PCR experiment+4, 百拇医药
Total RNA (2 µg) was treated with 15 U ThermoScript reverse transcriptase (Invitrogen, Inc.) in 20 µl PCR buffer for 90 min at 60 C according to the manufacturer’s protocol. The control was run without reverse transcriptase. The rat DUOX2 cDNA and truncated DUOX2 cDNA were amplified by 30 temperature cycles (95 C, 5 sec; 62 C, 10 sec; 72 C, 8 min) in a GeneAmp 2400 temperature cycler (Applied Biosystems, Courtaboeuf, France) in 50 µl of prewarmed PCR buffer containing 1 µl Advantage 2 polymerase Mix (CLONTECH Laboratories, Inc.), 250 nM of each sense and antisense oligonucleotide primer, and 200 µM of each deoxynucleoside triphosphate. Glyceraldehyde 3-phosphate dehydrogenase cDNA was amplified in parallel. The sense and antisense PCR primers for rat DUOX2 were designed from the rat DUOX2 cDNA sequence and were 5'-TGAAAGTGCATCCAGGAAGGTGAAGATT-3' and 5'-TACAGCTCAGCTTTTTAGTA-3', respectively. The expected size of the amplified product was 5467 bp. The sense and antisense PCR primers for truncated DUOX2 cDNA were 5'-ATGGCAGAGCTCAGGCACTCTGACTCA-3' and 5'-TACAGCTCAGCTTTTTAGTA-3', respectively; and the expected size of the amplified product was 1880 bp. PCR products were directly sequenced using sense and antisense oligonucleotides.
Translation in vitro5?, 百拇医药
mRNAs used in this study were synthetized using the TNT T7 Quick for PCR DNA kit (Promega, Lyon, France) and PCR DNA as a template, according to the manufacturer’s instructions. PCR-generated DNA for coupled transcription/translation reactions was prepared as follows. First, for PCR DNA containing the 5'-UTR and the N-terminal coding region of the normal rat thyroid gland DUOX2, the 5'-primer that installs the T7 promoter with six additional nucleotides upstream of the T7 promoter was 5'-GGATCCTAATACGACTCACTATAGGAAAGTGGTAACAACGCAGAGTACGAG-3' (the consensus T7 promoter is underlined), the 3'-primer was 5'-TCAATCTCGGCCACTCTGGATACTGCT-3', and the template was the normal rat thyroid gland 5'-RACE cDNA DUOX2 insert (1.8 kb) of pCR4-TOPO previously sequenced (15). Second, for PCR DNA corresponding to the full length of the truncated DUOX2, the 5'-primer was 5'-GGATCCTAATACGACTCACTATAGGAAGCGGGGCCTCAGTGTCTGGATG-3', the 3'-primer was 5'-ACAATCAGCCAAGCCCAGAAAC-3', and the template was the FRTL-5 short DUOX2 cDNA insert (2.7 kb) of pCR-XL-TOPO. Thirdly, for PCR DNA corresponding to the ORF of the truncated DUOX2, the 5'-primer was 5'-GGATCCTAATACGACTCACTATAGGAACAGACCACCATGTTCTCCTACATCCTG-3' (the Kozak consensus sequence and the start codon are in bold type), the 3'-primer was 5'-TCAGAAGTTCTCATAATGATGCACAAA-3', and the template was the FRTL-5 short DUOX2 cDNA insert (2.7 kb) of pCR-XL-TOPO. All of the PCRs were performed with 30 temperature cycles (95 C, 30 sec; 55 C, 30 sec; 72 C, 3 min) in a GeneAmp 2400 temperature cycler (Applied Biosystems) in 50 µl of prewarmed PCR buffer containing 2.5 U of PfuTurbo hotstart DNA polymerase (Stratagene, Amsterdam, The Netherlands), 250 nM of each sense and antisense oligonucleotide primer, and 200 µM of each deoxynucleoside triphosphate. Five microliters from the amplification reaction and 20 µCi [35S]methionine (Amersham Pharmacia Biotech) were mixed with 40 µl of TNT T7 Quick Master Mix and incubated at 30 C for 90 min after adjusting the final volume to 50 µl with water. After 90 min, 2 µl of the reaction mixture was subjected to SDS-PAGE analysis. In the case of full-length short DUOX2, 5 µl and 10 µl of the reaction mixture were also subjected to SDS-PAGE analysis. Gels were analyzed by electronic autoradiography using INSTANTIMAGER (Packard Instrument SA).
Results and Discussion$(rjs, http://www.100md.com
Expression of a truncated DUOX2 mRNA in FRTL-5 cells$(rjs, http://www.100md.com
Total RNA was extracted from the Fischer rat thyroids and FRTL-5 cells and subjected to Northern blot analysis. As previously shown (15), the use of a probe interacting with the 5' region of the DUOX2 mRNA revealed in both the rat thyroid tissue and the rat thyroid cell line an mRNA species approximately 6 kb long corresponding to the full-length DUOX2 mRNA (Fig. 1). Interestingly, by using a specific 3'-UTR probe, we detected a second mRNA species of about 3 kb, which was expressed in FRTL-5 cells to a greater extent than the normal DUOX2 mRNA. Northern blot analysis did not detect this short transcript in the rat thyroid gland (Fig. 1). Because the open reading frame (ORF) probe failed to detect this new mRNA species, it could correspond to a truncated form of DUOX2 mRNA. A 2698-bp cDNA was isolated from FRTL-5 cells. Its sequence was identical to the 3'-end of the normal DUOX2 cDNA. It predicted an ORF of 1458 bp encoding a polypeptide with a theoretical molecular mass of 56 kDa, corresponding to the last 486 amino acids of the C-terminal part of the rat Duox2 (Tyr1032 to Phe1517), which comprises the last five membrane spanning domains (Fig. 2). The 3'-UTR of the normal and short transcripts were identical, whereas the first 102 nucleotides of the truncated transcript were missing from the normal DUOX2 mRNA. There is no evidence that this mRNA can be translated, but if it is, the translation will probably start at Met1063 and not at Tyr1032. Under these conditions, the protein produced will contain neither a signal peptide nor a glycosylation site, and consequently will probably not be adequately targeted to the plasma membrane. We repeated the experiment with RNA prepared from FRTL-5 cells obtained from another source (kindly provided by Dr. G. Vassart, Brussels, Belgium). The same truncated DUOX2 mRNA species was also detected in this cell line (data not shown).
fig.ommitteedz$, 百拇医药
Figure 1. Northern blot analysis of mRNA from Fischer rat thyroid tissue and from FRTL-5 cells. Twenty micrograms of total RNA were analyzed by Northern blot with a cDNA probe corresponding to either the 5'-region of the ORF (N-ter probe) or the 3'-UTR (3'-UTR probe) of the rat DUOX2 mRNA, as described in Materials and Methods.z$, 百拇医药
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Figure 2. Primary structure of the putative truncated Duox2 protein. A, cDNA and deduced amino acid sequences of the truncated Duox2 (GenBank accession no. AF547268). B, Presumed topology and functional domains of the normal and truncated (dark) rat Duox2 proteins. FAD, Flavine adenine dinucleotide.z$, 百拇医药
Absence of the short DUOX2 transcript in rat thyroid tissuez$, 百拇医药
We wanted to find out whether small amounts of the short DUOX2 mRNA are produced in the rat thyroid gland. For this purpose, we used the PCR, using single-stranded cDNA reverse transcribed from Fischer rat thyroid glands as the template. Oligonucleotide primers were chosen in regions corresponding to the specific 5'-end of the short DUOX2 mRNA and to the 3'-UTR identical in both mRNA species, respectively. These oligonucleotides could be expected to prime the amplification of an 1880-bp DNA fragment corresponding to the short DUOX2 cDNA. RT-PCR analysis showed that no amplification of the thyroid gland DUOX2 cDNA was obtained (Fig. 3, lane 1) under these conditions, although the 1880-bp fragment with cDNAs from the FRTL-5 cells used as control was amplified (Fig. 3, lane 3). Amplification of a 5467-bp fragment of the rat DUOX2 cDNA could be obtained only using an oligonucleotide primer corresponding to the sequence of the 5'-UTR of the normal rat DUOX2 mRNA with cDNAs from rat thyroid gland (Fig. 3, lane 2) and from FRTL-5 cells (Fig. 3, lane 4). Consequently, FRTL-5 cells expressed both the short and the normal DUOX2 mRNAs, whereas only the normal DUOX2 transcript was expressed in normal rat thyroid tissue. These PCR experiments also confirmed that the nucleotide sequence of the 5'-end of the short DUOX2 transcript was not an exon of the normal transcript.
fig.ommitteedk, http://www.100md.com
Figure 3. Identification of DUOX2 mRNA transcripts in Fischer rat thyroid tissue and in FRTL-5 cells by RT-PCR. PCR was performed using as the templates single-stranded cDNA generated by reverse transcription of RNAs from Fischer rat thyroids and from FRTL-5 cells. Lane 1, cDNA template from Fischer rat thyroid tissue amplified by PCR with sense and antisense oligonucleotide primers designed from the specific 5'-end sequence of the short DUOX2 cDNA and from the 3'-UTR sequence of DUOX2, respectively. Lane 2, cDNA template from Fischer rat thyroid tissue amplified by PCR with sense and antisense oligonucleotide primers designed from the 5'-UTR and the 3'-UTR sequences of the normal DUOX2 cDNA, respectively. Lane 3, cDNA template from FRTL-5 cells amplified with the same primers as used in lane 1. Lane 4, cDNA template from FRTL-5 cells amplified with the same primers as used in lane 2. The amplified products were analyzed by agarose gel electrophoresis and ethidium bromide staining. Size markers are shown on the left.k, http://www.100md.com
Expression of the short DUOX2 transcript in PC Cl3 cells
Interestingly, both forms of DUOX2 mRNA were also expressed in another Fischer rat thyroid cell line: the PC Cl3 cells. As previously shown in FRTL-5 cells, the short form of DUOX2 mRNA was more highly expressed than the normal form, but its level was much lower than in FRTL-5 cells (Fig. 4A). To confirm that the same species of short DUOX2 mRNA was present in these cells, we performed PCR with specific oligonucleotides previously used in PCR experiments with thyroid tissue and FRTL-5 cDNA (Fig. 3). An 1880-bp DNA fragment corresponding to the short DUOX2 cDNA was also amplified with PC Cl3 cDNAs (Fig. 4B).y[v!, 百拇医药
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Figure 4. Identification of truncated DUOX2 mRNA in PC Cl3 cells by Northern blot (A) and RT-PCR (B) analysis. A, Twenty micrograms of total RNA from FRTL-5 cells (lane 1) and from PC Cl3 cells (lane 2) were analyzed by Northern blot with the 3'-UTR probe, as described in Materials and Methods. The bottom panel shows the 28S and 18S ribosomal RNA bands detected by methylene blue staining. B, Total RNA from PC Cl3 cells was reverse-transcribed, as described in Materials and Methods, and amplified by PCR with sense and antisense oligonucleotide primers designed from the specific 5'-end sequence of the short DUOX2 cDNA and from the 3'-UTR sequence of the normal DUOX2 cDNA, respectively. Size markers are shown on the left.
Translation efficiency of the short DUOX2 mRNA(te), http://www.100md.com
In the absence of an antibody against the C-Ter domain of Duox2 protein, nothing is yet known about the translation of the short DUOX2 mRNA. H2O2 generation activity has been measured in FRTL-5 cells (18, 19) and in PC Cl3 cells (13) and has been shown to be enhanced by adding the Ca2+ ionophore A23187, indicating that H2O2 generation could be ascribed to the Duox system. We had previously observed that Ca2+ modulates the Km of pig thyroid NADPH oxidase for NADPH (20), suggesting that the first intracellular loop of Duox2 containing the Ca2+-binding sites could interact with the C-Ter domain containing the NADPH binding site. The putative expression of a truncated Duox2 protein corresponding to the C-Ter domain in FRTL-5 cells and PC Cl3 cells could therefore interfere with the formation or activation of a functional Ca2+-dependent H2O2 generating system at the plasma membrane. Recent data (14) have shown that a heterozygous inactivating mutation resulting in the possible translation of the N-Ter domain comprising the first transmembrane domain of human Duox2 was responsible for a case of transient congenital hypothyroidism. These data might also reflect the dominant negative properties of the mutant protein resulting from aberrant interactions with other components likely to be required for the activity (10) and/or the targeting of the oxidase system to the plasma membrane (13).
We tested the ability of short DUOX2 mRNA to generate protein in vitro. Transcripts encoding proteins were synthetized in vitro by bacteriophage T7 RNA polymerase using PCR-generated DNA fragments as template (see Materials and Methods). Three PCR-generated DNA fragments were used (Fig. 5, A and B): the first corresponds to the 5'-region of the normal DUOX2 containing 192 additional nucleotides upstream of ATG, in addition to 1305 nucleotides coding for 435 amino acids; the second corresponds to the ORF sequence of the short DUOX2 mRNA, beginning at the Met1063 and not at Tyr1032 (Fig. 2); and the third corresponds to the full-length sequence of the short DUOX2. Transcription-coupled translation of the full-length cDNA of truncated DUOX2 followed by SDS-PAGE and autoradiography revealed no radiolabeled proteins (Fig. 5C, lane 3), indicating that synthetic truncated DUOX2 RNA failed to support protein synthesis in a translation system in vitro. On the other hand, radiolabeled protein of the expected size (48 kDa) was synthetized from the 5'-UTR cDNA sequence of the normal DUOX2, confirming that this mRNA contained the translation initiation site (Fig. 5C, lane 1). The absence of the 5'-UTR and the addition of a Kozak consensus sequence upstream of the first ATG of the short DUOX2 coding sequence allowed efficient translation with production of a protein with an apparent molecular mass of 46 kDa. The predicted molecular mass of the truncated Duox2 protein was 52 kDa. It is known that binding of a greater amount of sodium dodecyl sulfate to hydrophobic proteins accelerates their relative rate of migration during electrophoresis, leading to an underestimation of their molecular mass. Because truncated Duox2 has five hydrophobic stretches, a mechanism of this type may be responsible for its low apparent molecular mass in our experiments. Alternatively, degradation of the protein involving the release of a 6-kDa peptide cannot be excluded. These findings showed that, unlike normal DUOX2 mRNA, the 5'-UTR of the short DUOX2 species could not support translation, indicating that this mRNA was not functional. Suppression of the 3'-UTR region did not modify the translation rate (data not shown). Even if the short DUOX2 mRNA is not translated, its abundance could modify the translation rate of the normal DUOX2 mRNA by recruiting large amounts of the translation factors that interact with the 3'-UTR common to both species. Moreover, although FRTL-5 and PC Cl3 cells constitute a good model for investigating a number of thyroid cell functions, this work highlights the importance of a careful choice of oligonucleotides and cDNA probes used to study DUOX2 mRNA.
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Figure 5. In vitro translation. A, Diagram of the various PCR-generated DNA templates used for coupled transcription/translation reactions (see Materials and Methods). B, Ethidium bromide-stained 1% agarose gel of PCR-generated DNA templates. Lane M, DNA size markers; lane 1, PCR DNA containing the 5'-UTR and the N-terminal coding region of the normal DUOX2 (1497 bp); lane 2, PCR DNA corresponding to the ORF of the truncated DUOX2 (1371 bp); lane 3, PCR DNA corresponding to the full length of the truncated DUOX2 (2672 bp). C, Autoradiogram of [35S]methionine-labeled in vitro translation products. Lane 1, Translation product from transcription-coupled translation of the 5'-end of the normal DUOX2; lane 2, translation product from transcription-coupled translation of the short DUOX2 ORF; lane 3, translation product from transcription-coupled translation of the full-length short DUOX2; and lane 4, control reaction containing no added DNA.{;j(d, http://www.100md.com
Effect of serum on the regulation of the DUOX2 mRNA level by forskolin{;j(d, http://www.100md.com
We evaluated the effect of the concentration of serum on the expression of the two species of DUOX2 mRNA in FRTL-5 cells (Fig. 6). The cells were maintained for 6 d without TSH (i.e. in 5H medium) in the presence of 5% or 0.2% newborn calf serum. As expected (15), Northern blots showed that DUOX2 mRNA was enhanced after 24 h of stimulation with forskolin (10 µM) in cells cultured in 5% serum. When FRTL-5 cells were cultured in 5H medium with a lower concentration (0.2%) of serum, the level of DUOX2 mRNA was high in untreated cells, indicating that serum contains some factor(s) that repress DUOX2 gene expression. At low concentrations of these factors (0.2% serum), forskolin itself markedly repressed DUOX2 mRNA expression. Interestingly, we found that the short and the normal species of DUOX2 mRNA displayed the same regulation. The same negative effect was also found at a low serum concentration with 1 mU/ml TSH instead of forskolin (data not shown). This confirmed that some factors strongly modulate the up-regulation of DUOX2 mRNA expression by cAMP-dependent cascades. This had previously been observed in vivo (15) in the hyperstimulated thyroid gland from rats chronically treated with antithyroid drug, where DUOX2 mRNA expression was lower than in tissue from untreated animals, whereas the expression of other thyroid cAMP-regulated genes, such as TPO and NIS, was markedly increased. Although a posttranslational effect of cAMP is likely, the substantial decrease in Duox immunostaining seen in Graves’ thyroid tissues and toxic adenoma (21) and the decrease of H2O2 generation observed in toxic adenoma (22) could also be attributable, to some extent, to a down-regulation of DUOX2 mRNA expression by cAMP. These mechanisms may protect hyperstimulated thyrocytes from the oxidative stress resulting from overexpression of the NADPH oxidase.
fig.ommitteedw/, http://www.100md.com
Figure 6. Effect of serum concentration and forskolin (FK) stimulation on the levels of DUOX2 mRNAs in FRTL-5. FRTL-5 cells were maintained for 6 d in 5H (TSH-free) medium containing either 0.2% or 5% newborn calf serum, then for 24 h in the same medium, with (+) or without (-) forskolin. Total RNA was analyzed by Northern blotting with a cDNA probe corresponding to either the 5' region in the ORF (N-ter probe) or the 3'-UTR (3'-UTR probe) of the normal DUOX2 cDNA, as described in Materials and Methods.w/, http://www.100md.com
Effect of insulin on short and normal DUOX2 mRNA expression in FRTL-5 cellsw/, http://www.100md.com
Because DUOX2 gene expression in 5H medium was high in 0.2% serum (Fig. 6), we checked the effect of three hormones (insulin, somatostatin, and Gly-His-Lys-acetate) on the basal level (without TSH) and on the 24-h forskolin-stimulated expression of DUOX2 mRNA. The cells were maintained for 6 d in 5H or 4H medium, i.e. medium lacking TSH and one of the other three hormones as indicated, and DUOX2 mRNA expression was analyzed by Northern blot (Fig. 7). In the absence of forskolin, the level of DUOX2 mRNA was high under all conditions, except when the medium contained no insulin, indicating that, in these conditions, this hormone was implicated in the regulation of the DUOX2 gene expression. Moreover, the inhibiting effect of forskolin on the level of DUOX2 mRNA in 0.2% serum was obtained only when insulin was present in the culture medium. Insulin is involved in the regulation of thyroid-specific genes, but to date nothing had been known about its effect on DUOX2 gene expression. We evaluated the time course of the effect of insulin on the expression of the two species of DUOX2 mRNA in FRTL-5 cells (Fig. 8). Northern blotting showed that the expression of both DUOX2 mRNA species peaked after 6–12 h of stimulation. As expected (23), insulin alone had no effect on NIS mRNA level under these conditions (Fig. 8). Consequently, like the thyroglobulin (Tg) and TPO genes (24, 25), insulin also positively regulates DUOX2 gene expression in FRTL-5 cells, but this effect is counteracted in 5% serum (Fig. 6), suggesting that serum contains some repressive factor(s) that interact with the insulin pathway.
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Figure 7. Effect of hormone withdrawal on the amount of short DUOX2 mRNA using NIS mRNA as reference. FRTL-5 cells were maintained for 6 d in 5H or 4H medium lacking insulin or somatostatin or Gly-His-Lys-acetate as indicated and containing 0.2% serum. Forskolin (FK) was then added (+) or not (-) for 24 h. Total RNA was extracted and analyzed by Northern blotting. The bottom panel shows the 18S ribosomal RNA band detected by methylene blue staining.#w, http://www.100md.com
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Figure 8. Time course of the effect of insulin on DUOX2 and NIS mRNA levels in FRTL-5. FRTL-5 cells were maintained for 6 d in 4H medium containing 0.2% serum and then incubated with 10 µg/ml insulin for the periods indicated. A, Total RNA was analyzed by Northern blotting for both DUOX2 and NIS mRNA levels. 18S ribosomal RNA bands were detected by methylene blue staining. B, The ratio of the short DUOX2 mRNA, quantified using an INSTANTIMAGER (Packard Instrument SA), to the corresponding 18 S ribosomal RNA density, quantified by densitometry using the NIH Image program, was calculated. Values calculated from cells incubated 6 d in 4H medium were chosen as a reference and arbitrarily taken to be 1.0. Data are the mean ± SEM from three separate experiments (n = 3) using different batches of cells.
Effect of insulin on the regulation by forskolin of DUOX2 mRNA expression in FRTL-58fod|], 百拇医药
As shown in Fig. 7, the inhibitory effect of forskolin on the level of DUOX2 mRNA in 0.2% serum was obtained only when insulin was present in the culture medium. We determined the time course of the forskolin effect on the concentration of short and normal DUOX2 mRNA in FRTL-5 cells cultured in 0.2% serum, without (Fig. 9A) and with (Fig. 9B insulin. Northern blots showed that the expression of both DUOX2 mRNA species was markedly increased by forskolin in the absence of insulin (Fig. 9, A and C), peaking after 6 h of stimulation at a level that could be up to three times the initial level, and then declined. As expected (26), the expression of the NIS gene under these conditions became apparent after 6 h and was strongly enhanced after 14 h, when the levels of both species of DUOX2 mRNA had already decreased.8fod|], 百拇医药
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Figure 9. Time course of the effects of forskolin on DUOX2 and NIS mRNA levels in FRTL-5 cultured in the absence (A) or presence (B) of insulin. FRTL-5 cells were maintained in 4H medium (A) or 5H medium (B) containing 0.2% serum for 6 d, and then incubated with 10 µM forskolin for the periods indicated. Total RNA was then extracted and analyzed by Northern blotting as described in Fig. 8.
Interestingly, in the presence of insulin, forskolin had no stimulating effect on DUOX2 mRNA expression (Fig. 9, B and C), but still produced a definite inhibitory effect (Fig. 9, B and C). Under the same conditions, NIS mRNA level was positively regulated by forskolin (Fig. 9B), but the concentration of NIS mRNA was much lower than in the absence of insulin, confirming that insulin down-regulated its forskolin-induced expression, as previously reported (23).u, 百拇医药
Insulin is another important factor affecting thyroid function that collaborates with TSH in regulating thyroid proliferation and differentiation. Insulin and IGF-I both stimulate Tg and TPO mRNA expression (24, 25). Both hormones increase the transcription of the Tg gene, and their effects are additive to those of TSH/cAMP (24). In contrast, our data show that at low serum concentrations, the positive effects of insulin and cAMP on DUOX2 gene expression were not additive, but antagonistic. Similarly, and in accordance with previous reports showing that insulin/IGF-I inhibited iodide uptake stimulated by TSH in FRTL-5 cells (27), recent data have demonstrated that IGF-I inhibits the TSH stimulation of NIS gene expression. This effect was also obtained when cells were maintained under low serum conditions, and was phosphatidylinositide-3-kinase mediated (23). It was not attributable to an inhibitory effect on TSH-induced cAMP generation, but rather to cAMP signal action (27). Our results also demonstrate that insulin interferes with the effect of cAMP on DUOX2 mRNA expression and could therefore be an important regulator of the DUOX2 gene expression in hyperfunctioning thyroid tissues.
Effect of insulin and forskolin on the expression of DUOX2 mRNA in pig thyroid follicles!!., 百拇医药
We compared the effect of insulin on DUOX2 gene expression in FRTL-5 cells to that in thyrocytes in primary culture (Fig. 10A). Pig thyroid follicles were cultured in 4H medium in the presence of 0.2% newborn calf serum. Northern blots showed that unlike rat thyroid cell lines, only one DUOX2 mRNA species was detected, corresponding to an approximately 6-kb-long mRNA. The DUOX2 mRNA level was markedly increased after 24 h of insulin stimulation and reached a peak after 48 h. As a control, the NIS mRNA level was also determined under these conditions. Using an NIS-specific 3'-UTR probe, we detected two mRNA species, 3.5 kb and 3 kb in length, respectively, probably corresponding to the pig NIS transcripts published in GenBank by Dr. S. Selmi-Rubi (Lyon, France; accession no. AJ277989 and AJ487855). It is interesting to note that, in contrast to what was observed in FRTL-5 cells under these conditions, the NIS mRNA level was still markedly increased after stimulating for 48 h.
fig.ommitteedl#uc\?h, 百拇医药
Figure 10. Time course of the effects of insulin alone (A) or forskolin plus insulin (B) on DUOX2 and NIS mRNA levels in pig thyroid follicles. A, Pig thyroid follicles were maintained for 3 d in 4H medium containing 0.2% serum and then incubated with 10 µg/ml insulin for the periods indicated. Total RNA was analyzed by Northern blotting. 18 S ribosomal RNA bands were revealed by methylene blue staining. DUOX2 and NIS mRNA levels were quantified using specific porcine probes and an INSTANTIMAGER as described in Fig. 8. B, Pig thyroid follicles were maintained in 4H or 5H medium containing 0.2% serum for 3 d, and then incubated with 10 µM forskolin for the periods indicated. Total RNA was isolated and analyzed by Northern blotting as in A.l#uc\?h, 百拇医药
We also evaluated the time course of the forskolin effect on DUOX2 mRNA accumulation (Fig. 10B) in pig thyroid follicles cultured with (5H) or without (4H) insulin. In the absence of insulin, DUOX2 mRNA expression peaked after 14 h of stimulation and declined after 24 h. As observed in the FRTL-5 cells, the highest expression of the NIS gene under the same conditions was reached later (24 h) than that of DUOX2.
In 5H medium, DUOX2 mRNA was strongly expressed without forskolin because of the presence of insulin. Forskolin did not inhibit DUOX2 mRNA expression, in contrast to the effect in FRTL-5 cells. On the contrary, the expression of DUOX2 mRNA was still high 48 h after adding forskolin, suggesting that the positive effects of the cAMP and of insulin on DUOX2 gene expression are additive in porcine thyrocytes. This discrepancy could be ascribed to species-related differences in the signaling pathways in rat and pig thyroid, or it could reflect differences between the rat thyroid gland and the rat thyroid cell line FRTL-5 with regard to the regulation of DUOX2 gene.7+, http://www.100md.com
In conclusion, we have identified a truncated form of DUOX2 mRNA in two rat thyroid cell lines widely used in studies of thyroid cell function. It is more abundantly expressed than the normal DUOX2 mRNA, but fails to support protein synthesis in an in vitro translation system.7+, http://www.100md.com
This work highlights the importance of a careful choice of oligonucleotides and cDNA probes to study DUOX2 mRNA in both cell lines. This report also provides the first evidence that insulin up-regulates the expression of DUOX2 gene at the mRNA level, an effect that is antagonized by cAMP in FRTL-5 cells.
Acknowledgments3es0, 百拇医药
We are indebted to Etablissements HARANG (Houdan) for allowing us to collect pig thyroid glands from their slaughterhouse.3es0, 百拇医药
Accepted for publication November 6, 2002.3es0, 百拇医药
References3es0, 百拇医药
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The Duox2 flavoprotein is strongly expressed in the thyroid gland, where it plays a critical role in the synthesis of thyroid hormones likely by providing thyroperoxidase with H2O2. A truncated DUOX2 mRNA was isolated from the rat thyroid cell line FRTL-5. The cDNA sequence predicted an open reading frame of 1458 bp, encoding a polypeptide of 486 amino acids corresponding to the carboxyl fragment of the Duox2 flavoprotein. The truncated form of DUOX2 mRNA, expressed in another rat thyroid cell line, the PC Cl3 cell line, was absent from Fischer rat thyroid glands. Although it was expressed in both cell lines to a greater extent than normal mRNA, it failed to support protein synthesis in an in vitro translation system. Insulin increased the levels of both normal and truncated DUOX2 mRNA in FRTL-5 cells grown in TSH-free medium containing a low concentration of serum. The stimulating effect of insulin on DUOX2 mRNA expression was reproduced in pig thyroid follicles in primary culture. The presence of insulin in the culture medium converted forskolin from a stimulator to an inhibitor in FRTL-5 cells maintained in low serum conditions, but not in porcine thyrocytes in primary culture.
Introductionpa., 百拇医药
THE SYNTHESIS OF thyroid hormone is catalyzed by thyroperoxidase (Tpo) in the presence of H2O2 (1) on the apical membrane of the thyroid follicular cells (2). H2O2 is produced on the apical plasma membrane of rat and pig open follicles (3, 4) by a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in both pig (5, 6, 7) and human thyroid (8), as proposed by Björkman et al. (3). Thyroid NADPH oxidase requires micromolar concentrations of calcium to acquire a functional conformation and to generate H2O2 (7, 8, 9). A functional NADPH oxidase, generating H2O2 in a Ca2+-dependent manner, was solubilized from pig thyroid (10), and a flavoprotein with an apparent molecular mass of about 180 kDa was purified from it (11). Microsequences were used to clone its porcine and human partial cDNAs, making it possible to localize its gene on chromosome 15q15 (11). The full-length cDNA, encoding a 1548- amino-acid protein known as thyroid oxidase gene (Thox)2, has subsequently been cloned from human thyroid (12). Its sequence is 83% similar to that of the Thox1, also expressed in the thyroid gland (12). Thox1,2 proteins and THOX1,2 genes are also known as Duox1,2 and DUOX1,2 genes, according to the recommendations of the Human Gene Nomenclature Committee. Although a functional Duox-based H2O2 generating system has not yet been reconstituted (13), the essential role of Duox2 in thyroid hormone synthesis has been confirmed by the recent observation of permanent and severe congenital hypothyroidism in a patient with a biallelic inactivating mutation in the DUOX2 gene (14).
Little is known about the regulation of DUOX2 expression. In vitro studies have shown that DUOX2 mRNA is up-regulated by cAMP agonists in human (12), porcine (11), and dog thyroid cells in primary culture (12), as well as in the FRTL-5 rat thyroid cell line (15). However, the level of DUOX2 mRNA is reduced in the hyperfunctioning thyroid tissues of rats treated with an antithyroid drug (methimazole; Ref. 15). These findings indicate that the up-regulation of the expression of DUOX2 mRNA by cAMP detected in cultured thyrocytes is strongly modulated in vivo by factors that repress its expression in hyperfunctioning tissues, in contrast to Na+/I- symporter (NIS) and TPO mRNAs, two other markers of thyroid differentiation. Because FRTL-5 cells are often used as a thyrocyte model to investigate thyroid gene expression, we also studied the regulation of DUOX2 expression in these cells and compared it to that observed in thyrocytes in primary culture.n, http://www.100md.com
In the present report, we show that rat FRTL-5 and PC Cl3 thyroid cells, unlike rat thyroid tissue, express relatively high levels of a truncated DUOX2 mRNA compared with the normal DUOX2 transcript. In vitro translation studies show that the synthetic truncated DUOX2 mRNA fails to support protein synthesis. Insulin increased the levels of both species of DUOX2 mRNA in FRTL-5 cells grown in a TSH-free medium containing 0.2% serum. Insulin had a similar effect on the expression of normal DUOX2 mRNA in similarly treated primary cultures of pig thyroid cells. However, in FRTL-5 cells, but not in pig thyrocytes in primary culture, the presence of insulin in the medium converted forskolin from a stimulator to an inhibitor of DUOX2 expression in cells maintained in low serum conditions. Our findings make it clear that caution is required when using FRTL-5 cells to study DUOX gene expression.
Materials and Methods9, 百拇医药
cDNA cloning9, 百拇医药
The amplification of the short DUOX2 cDNA from FRTL-5 cells was achieved by RACE using the SMART RACE cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA) and applying the manufacturer’s protocol with 1 µg total mRNA. PCR products were cloned in pCR-XL-TOPO (Invitrogen, Cergy Pontoise, France) for sequencing. Sequencing was performed three times in the 5' to 3' direction and three times in the reverse direction.9, 百拇医药
Cell culture9, 百拇医药
FRTL-5 cells (CRL no. 8305, American Type Culture Collection, Rockville, MD), a rat thyroid cell line, were a generous gift from Dr. M. Eggo (University of Birmingham, Birmingham, UK). They were grown to 80–90% confluence in Coon’s modified Ham’s F12 medium (Eurobio, Les Ulis, France) supplemented with 5% newborn calf serum, 0.5 mU/ml bovine TSH (Sigma), 10 µg/ml insulin, 5 µg/ml transferrin, 10 ng/ml somatostatin, 10 ng/ml Gly-His-Lys-acetate, and 10 nM hydrocortisone (6H medium). The cells were transferred to TSH-free 5-hormone medium (5H), or in TSH- and insulin-free 4-hormone medium (4H), containing 0.2% newborn calf serum for 6 d; forskolin or insulin was then added to the medium as required.
PC Cl3 cells, developed as previously described (16), were a generous gift from Dr. R. Di Lauro (Stazione Zoologica Anton Dohrn, Napoli, Italy). They were cultured in Coon’s modified Ham’s F12 medium supplemented with 10% fetal calf serum, 10 µU/ml bovine TSH (Sigma), 1 µg/ml insulin, 5 µg/ml transferrin, and 10 nM hydrocortisone.g@y, http://www.100md.com
Isolation of pig thyroid folliclesg@y, http://www.100md.com
Fresh porcine thyroid glands were obtained from a slaughterhouse and immediately transported to the laboratory in cold PBS solution. After the connective tissue was removed, the glands (90 g) were washed briefly in 70% ethanol and rinsed twice with PBS containing antibiotics and fungizone. The glands were minced, and follicles were isolated by collagenase digestion (360 U/ml) for 75 min at 37 C in 200 ml of PBS containing 1 mM calcium, antibiotics, and fungizone. The digest was filtered through a mesh (250 µm) and washed twice by centrifuging (100 x g, 3 min) with 20 ml cold PBS containing antibiotics and fungizone. The final pellet was resuspended in 20 ml of the same solution and sedimented for 10 min on ice. The preparation was suspended in 250 ml DMEM supplemented with antibiotics and fungizone, 5% fetal calf serum, and 1 mU/ml bovine TSH, and cultured in suspension for 3 d in a 95% air/5% CO2 incubator at 37 C. Follicles were then transferred into TSH-free 5H medium or TSH and insulin-free 4H medium, containing 0.2% newborn calf serum for 3 d; forskolin or insulin was then added to the medium as required.
RNA isolation and Northern blot analysis0{9du'{, http://www.100md.com
RNA was extracted from all of the cells and from Fischer rat thyroids by the method of Chomczynski and Sacchi (17). Northern blot analysis was performed as previously described (11). Final washes were carried out at 60 C in 0.1x SSC, 0.1% sodium dodecyl sulfate (1x SSC = 0.15 M NaCl, 15 mM sodium citrate). The rat cDNA probes used were the N-Ter cDNA corresponding to the rat 3'-RACE cDNA EcoRI insert obtained as previously described (15), the rat 3'-untranslated region (UTR) and NIS cDNAs prepared by RT-PCR, using total RNA from Fischer rat thyroids. The sense and antisense 3'-UTR PCR primers (MWG Biotech, Ebersberg, Germany) were 5'-GGGCAGGTAGACAAGGTCAG-3' and 5'-ACAGCTCAGCTTTTTAGTAA-3', respectively. The sense and antisense NIS PCR primers were 5'-CCTTCTGGACTTTCATAGTGC-3' and 5'-TGGGACCAGTAAGGTAGCTGA-3', respectively. The porcine cDNA probes used were DUOX2 cDNA and NIS cDNA prepared by RT-PCR, using total RNA from pig thyroids. The sense and antisense DUOX2 primers, designed on the 3'-UTR of the DUOX2 cDNA, were 5'-CACTTCAGGCCTTAGCTGGA-3' and 5'-GACCAAACGAATCTAGAGCA-3', respectively. The sense and antisense NIS primers were 5'-GGACAGACATCACACATGCTCT-3' and 5'-GCAAGTTTATTCTTTGCAGGCT-3', respectively.
The cDNA probes were {alpha} -32P-labeled by random priming extension using a kit (Amersham Pharmacia Biotech, Saclay, France). Membranes were analyzed by electronic autoradiography using INSTANTIMAGER (Packard Instrument SA, Rungis, France).+4, 百拇医药
RT-PCR experiment+4, 百拇医药
Total RNA (2 µg) was treated with 15 U ThermoScript reverse transcriptase (Invitrogen, Inc.) in 20 µl PCR buffer for 90 min at 60 C according to the manufacturer’s protocol. The control was run without reverse transcriptase. The rat DUOX2 cDNA and truncated DUOX2 cDNA were amplified by 30 temperature cycles (95 C, 5 sec; 62 C, 10 sec; 72 C, 8 min) in a GeneAmp 2400 temperature cycler (Applied Biosystems, Courtaboeuf, France) in 50 µl of prewarmed PCR buffer containing 1 µl Advantage 2 polymerase Mix (CLONTECH Laboratories, Inc.), 250 nM of each sense and antisense oligonucleotide primer, and 200 µM of each deoxynucleoside triphosphate. Glyceraldehyde 3-phosphate dehydrogenase cDNA was amplified in parallel. The sense and antisense PCR primers for rat DUOX2 were designed from the rat DUOX2 cDNA sequence and were 5'-TGAAAGTGCATCCAGGAAGGTGAAGATT-3' and 5'-TACAGCTCAGCTTTTTAGTA-3', respectively. The expected size of the amplified product was 5467 bp. The sense and antisense PCR primers for truncated DUOX2 cDNA were 5'-ATGGCAGAGCTCAGGCACTCTGACTCA-3' and 5'-TACAGCTCAGCTTTTTAGTA-3', respectively; and the expected size of the amplified product was 1880 bp. PCR products were directly sequenced using sense and antisense oligonucleotides.
Translation in vitro5?, 百拇医药
mRNAs used in this study were synthetized using the TNT T7 Quick for PCR DNA kit (Promega, Lyon, France) and PCR DNA as a template, according to the manufacturer’s instructions. PCR-generated DNA for coupled transcription/translation reactions was prepared as follows. First, for PCR DNA containing the 5'-UTR and the N-terminal coding region of the normal rat thyroid gland DUOX2, the 5'-primer that installs the T7 promoter with six additional nucleotides upstream of the T7 promoter was 5'-GGATCCTAATACGACTCACTATAGGAAAGTGGTAACAACGCAGAGTACGAG-3' (the consensus T7 promoter is underlined), the 3'-primer was 5'-TCAATCTCGGCCACTCTGGATACTGCT-3', and the template was the normal rat thyroid gland 5'-RACE cDNA DUOX2 insert (1.8 kb) of pCR4-TOPO previously sequenced (15). Second, for PCR DNA corresponding to the full length of the truncated DUOX2, the 5'-primer was 5'-GGATCCTAATACGACTCACTATAGGAAGCGGGGCCTCAGTGTCTGGATG-3', the 3'-primer was 5'-ACAATCAGCCAAGCCCAGAAAC-3', and the template was the FRTL-5 short DUOX2 cDNA insert (2.7 kb) of pCR-XL-TOPO. Thirdly, for PCR DNA corresponding to the ORF of the truncated DUOX2, the 5'-primer was 5'-GGATCCTAATACGACTCACTATAGGAACAGACCACCATGTTCTCCTACATCCTG-3' (the Kozak consensus sequence and the start codon are in bold type), the 3'-primer was 5'-TCAGAAGTTCTCATAATGATGCACAAA-3', and the template was the FRTL-5 short DUOX2 cDNA insert (2.7 kb) of pCR-XL-TOPO. All of the PCRs were performed with 30 temperature cycles (95 C, 30 sec; 55 C, 30 sec; 72 C, 3 min) in a GeneAmp 2400 temperature cycler (Applied Biosystems) in 50 µl of prewarmed PCR buffer containing 2.5 U of PfuTurbo hotstart DNA polymerase (Stratagene, Amsterdam, The Netherlands), 250 nM of each sense and antisense oligonucleotide primer, and 200 µM of each deoxynucleoside triphosphate. Five microliters from the amplification reaction and 20 µCi [35S]methionine (Amersham Pharmacia Biotech) were mixed with 40 µl of TNT T7 Quick Master Mix and incubated at 30 C for 90 min after adjusting the final volume to 50 µl with water. After 90 min, 2 µl of the reaction mixture was subjected to SDS-PAGE analysis. In the case of full-length short DUOX2, 5 µl and 10 µl of the reaction mixture were also subjected to SDS-PAGE analysis. Gels were analyzed by electronic autoradiography using INSTANTIMAGER (Packard Instrument SA).
Results and Discussion$(rjs, http://www.100md.com
Expression of a truncated DUOX2 mRNA in FRTL-5 cells$(rjs, http://www.100md.com
Total RNA was extracted from the Fischer rat thyroids and FRTL-5 cells and subjected to Northern blot analysis. As previously shown (15), the use of a probe interacting with the 5' region of the DUOX2 mRNA revealed in both the rat thyroid tissue and the rat thyroid cell line an mRNA species approximately 6 kb long corresponding to the full-length DUOX2 mRNA (Fig. 1). Interestingly, by using a specific 3'-UTR probe, we detected a second mRNA species of about 3 kb, which was expressed in FRTL-5 cells to a greater extent than the normal DUOX2 mRNA. Northern blot analysis did not detect this short transcript in the rat thyroid gland (Fig. 1). Because the open reading frame (ORF) probe failed to detect this new mRNA species, it could correspond to a truncated form of DUOX2 mRNA. A 2698-bp cDNA was isolated from FRTL-5 cells. Its sequence was identical to the 3'-end of the normal DUOX2 cDNA. It predicted an ORF of 1458 bp encoding a polypeptide with a theoretical molecular mass of 56 kDa, corresponding to the last 486 amino acids of the C-terminal part of the rat Duox2 (Tyr1032 to Phe1517), which comprises the last five membrane spanning domains (Fig. 2). The 3'-UTR of the normal and short transcripts were identical, whereas the first 102 nucleotides of the truncated transcript were missing from the normal DUOX2 mRNA. There is no evidence that this mRNA can be translated, but if it is, the translation will probably start at Met1063 and not at Tyr1032. Under these conditions, the protein produced will contain neither a signal peptide nor a glycosylation site, and consequently will probably not be adequately targeted to the plasma membrane. We repeated the experiment with RNA prepared from FRTL-5 cells obtained from another source (kindly provided by Dr. G. Vassart, Brussels, Belgium). The same truncated DUOX2 mRNA species was also detected in this cell line (data not shown).
fig.ommitteedz$, 百拇医药
Figure 1. Northern blot analysis of mRNA from Fischer rat thyroid tissue and from FRTL-5 cells. Twenty micrograms of total RNA were analyzed by Northern blot with a cDNA probe corresponding to either the 5'-region of the ORF (N-ter probe) or the 3'-UTR (3'-UTR probe) of the rat DUOX2 mRNA, as described in Materials and Methods.z$, 百拇医药
fig.ommitteedz$, 百拇医药
Figure 2. Primary structure of the putative truncated Duox2 protein. A, cDNA and deduced amino acid sequences of the truncated Duox2 (GenBank accession no. AF547268). B, Presumed topology and functional domains of the normal and truncated (dark) rat Duox2 proteins. FAD, Flavine adenine dinucleotide.z$, 百拇医药
Absence of the short DUOX2 transcript in rat thyroid tissuez$, 百拇医药
We wanted to find out whether small amounts of the short DUOX2 mRNA are produced in the rat thyroid gland. For this purpose, we used the PCR, using single-stranded cDNA reverse transcribed from Fischer rat thyroid glands as the template. Oligonucleotide primers were chosen in regions corresponding to the specific 5'-end of the short DUOX2 mRNA and to the 3'-UTR identical in both mRNA species, respectively. These oligonucleotides could be expected to prime the amplification of an 1880-bp DNA fragment corresponding to the short DUOX2 cDNA. RT-PCR analysis showed that no amplification of the thyroid gland DUOX2 cDNA was obtained (Fig. 3, lane 1) under these conditions, although the 1880-bp fragment with cDNAs from the FRTL-5 cells used as control was amplified (Fig. 3, lane 3). Amplification of a 5467-bp fragment of the rat DUOX2 cDNA could be obtained only using an oligonucleotide primer corresponding to the sequence of the 5'-UTR of the normal rat DUOX2 mRNA with cDNAs from rat thyroid gland (Fig. 3, lane 2) and from FRTL-5 cells (Fig. 3, lane 4). Consequently, FRTL-5 cells expressed both the short and the normal DUOX2 mRNAs, whereas only the normal DUOX2 transcript was expressed in normal rat thyroid tissue. These PCR experiments also confirmed that the nucleotide sequence of the 5'-end of the short DUOX2 transcript was not an exon of the normal transcript.
fig.ommitteedk, http://www.100md.com
Figure 3. Identification of DUOX2 mRNA transcripts in Fischer rat thyroid tissue and in FRTL-5 cells by RT-PCR. PCR was performed using as the templates single-stranded cDNA generated by reverse transcription of RNAs from Fischer rat thyroids and from FRTL-5 cells. Lane 1, cDNA template from Fischer rat thyroid tissue amplified by PCR with sense and antisense oligonucleotide primers designed from the specific 5'-end sequence of the short DUOX2 cDNA and from the 3'-UTR sequence of DUOX2, respectively. Lane 2, cDNA template from Fischer rat thyroid tissue amplified by PCR with sense and antisense oligonucleotide primers designed from the 5'-UTR and the 3'-UTR sequences of the normal DUOX2 cDNA, respectively. Lane 3, cDNA template from FRTL-5 cells amplified with the same primers as used in lane 1. Lane 4, cDNA template from FRTL-5 cells amplified with the same primers as used in lane 2. The amplified products were analyzed by agarose gel electrophoresis and ethidium bromide staining. Size markers are shown on the left.k, http://www.100md.com
Expression of the short DUOX2 transcript in PC Cl3 cells
Interestingly, both forms of DUOX2 mRNA were also expressed in another Fischer rat thyroid cell line: the PC Cl3 cells. As previously shown in FRTL-5 cells, the short form of DUOX2 mRNA was more highly expressed than the normal form, but its level was much lower than in FRTL-5 cells (Fig. 4A). To confirm that the same species of short DUOX2 mRNA was present in these cells, we performed PCR with specific oligonucleotides previously used in PCR experiments with thyroid tissue and FRTL-5 cDNA (Fig. 3). An 1880-bp DNA fragment corresponding to the short DUOX2 cDNA was also amplified with PC Cl3 cDNAs (Fig. 4B).y[v!, 百拇医药
fig.ommitteedy[v!, 百拇医药
Figure 4. Identification of truncated DUOX2 mRNA in PC Cl3 cells by Northern blot (A) and RT-PCR (B) analysis. A, Twenty micrograms of total RNA from FRTL-5 cells (lane 1) and from PC Cl3 cells (lane 2) were analyzed by Northern blot with the 3'-UTR probe, as described in Materials and Methods. The bottom panel shows the 28S and 18S ribosomal RNA bands detected by methylene blue staining. B, Total RNA from PC Cl3 cells was reverse-transcribed, as described in Materials and Methods, and amplified by PCR with sense and antisense oligonucleotide primers designed from the specific 5'-end sequence of the short DUOX2 cDNA and from the 3'-UTR sequence of the normal DUOX2 cDNA, respectively. Size markers are shown on the left.
Translation efficiency of the short DUOX2 mRNA(te), http://www.100md.com
In the absence of an antibody against the C-Ter domain of Duox2 protein, nothing is yet known about the translation of the short DUOX2 mRNA. H2O2 generation activity has been measured in FRTL-5 cells (18, 19) and in PC Cl3 cells (13) and has been shown to be enhanced by adding the Ca2+ ionophore A23187, indicating that H2O2 generation could be ascribed to the Duox system. We had previously observed that Ca2+ modulates the Km of pig thyroid NADPH oxidase for NADPH (20), suggesting that the first intracellular loop of Duox2 containing the Ca2+-binding sites could interact with the C-Ter domain containing the NADPH binding site. The putative expression of a truncated Duox2 protein corresponding to the C-Ter domain in FRTL-5 cells and PC Cl3 cells could therefore interfere with the formation or activation of a functional Ca2+-dependent H2O2 generating system at the plasma membrane. Recent data (14) have shown that a heterozygous inactivating mutation resulting in the possible translation of the N-Ter domain comprising the first transmembrane domain of human Duox2 was responsible for a case of transient congenital hypothyroidism. These data might also reflect the dominant negative properties of the mutant protein resulting from aberrant interactions with other components likely to be required for the activity (10) and/or the targeting of the oxidase system to the plasma membrane (13).
We tested the ability of short DUOX2 mRNA to generate protein in vitro. Transcripts encoding proteins were synthetized in vitro by bacteriophage T7 RNA polymerase using PCR-generated DNA fragments as template (see Materials and Methods). Three PCR-generated DNA fragments were used (Fig. 5, A and B): the first corresponds to the 5'-region of the normal DUOX2 containing 192 additional nucleotides upstream of ATG, in addition to 1305 nucleotides coding for 435 amino acids; the second corresponds to the ORF sequence of the short DUOX2 mRNA, beginning at the Met1063 and not at Tyr1032 (Fig. 2); and the third corresponds to the full-length sequence of the short DUOX2. Transcription-coupled translation of the full-length cDNA of truncated DUOX2 followed by SDS-PAGE and autoradiography revealed no radiolabeled proteins (Fig. 5C, lane 3), indicating that synthetic truncated DUOX2 RNA failed to support protein synthesis in a translation system in vitro. On the other hand, radiolabeled protein of the expected size (48 kDa) was synthetized from the 5'-UTR cDNA sequence of the normal DUOX2, confirming that this mRNA contained the translation initiation site (Fig. 5C, lane 1). The absence of the 5'-UTR and the addition of a Kozak consensus sequence upstream of the first ATG of the short DUOX2 coding sequence allowed efficient translation with production of a protein with an apparent molecular mass of 46 kDa. The predicted molecular mass of the truncated Duox2 protein was 52 kDa. It is known that binding of a greater amount of sodium dodecyl sulfate to hydrophobic proteins accelerates their relative rate of migration during electrophoresis, leading to an underestimation of their molecular mass. Because truncated Duox2 has five hydrophobic stretches, a mechanism of this type may be responsible for its low apparent molecular mass in our experiments. Alternatively, degradation of the protein involving the release of a 6-kDa peptide cannot be excluded. These findings showed that, unlike normal DUOX2 mRNA, the 5'-UTR of the short DUOX2 species could not support translation, indicating that this mRNA was not functional. Suppression of the 3'-UTR region did not modify the translation rate (data not shown). Even if the short DUOX2 mRNA is not translated, its abundance could modify the translation rate of the normal DUOX2 mRNA by recruiting large amounts of the translation factors that interact with the 3'-UTR common to both species. Moreover, although FRTL-5 and PC Cl3 cells constitute a good model for investigating a number of thyroid cell functions, this work highlights the importance of a careful choice of oligonucleotides and cDNA probes used to study DUOX2 mRNA.
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Figure 5. In vitro translation. A, Diagram of the various PCR-generated DNA templates used for coupled transcription/translation reactions (see Materials and Methods). B, Ethidium bromide-stained 1% agarose gel of PCR-generated DNA templates. Lane M, DNA size markers; lane 1, PCR DNA containing the 5'-UTR and the N-terminal coding region of the normal DUOX2 (1497 bp); lane 2, PCR DNA corresponding to the ORF of the truncated DUOX2 (1371 bp); lane 3, PCR DNA corresponding to the full length of the truncated DUOX2 (2672 bp). C, Autoradiogram of [35S]methionine-labeled in vitro translation products. Lane 1, Translation product from transcription-coupled translation of the 5'-end of the normal DUOX2; lane 2, translation product from transcription-coupled translation of the short DUOX2 ORF; lane 3, translation product from transcription-coupled translation of the full-length short DUOX2; and lane 4, control reaction containing no added DNA.{;j(d, http://www.100md.com
Effect of serum on the regulation of the DUOX2 mRNA level by forskolin{;j(d, http://www.100md.com
We evaluated the effect of the concentration of serum on the expression of the two species of DUOX2 mRNA in FRTL-5 cells (Fig. 6). The cells were maintained for 6 d without TSH (i.e. in 5H medium) in the presence of 5% or 0.2% newborn calf serum. As expected (15), Northern blots showed that DUOX2 mRNA was enhanced after 24 h of stimulation with forskolin (10 µM) in cells cultured in 5% serum. When FRTL-5 cells were cultured in 5H medium with a lower concentration (0.2%) of serum, the level of DUOX2 mRNA was high in untreated cells, indicating that serum contains some factor(s) that repress DUOX2 gene expression. At low concentrations of these factors (0.2% serum), forskolin itself markedly repressed DUOX2 mRNA expression. Interestingly, we found that the short and the normal species of DUOX2 mRNA displayed the same regulation. The same negative effect was also found at a low serum concentration with 1 mU/ml TSH instead of forskolin (data not shown). This confirmed that some factors strongly modulate the up-regulation of DUOX2 mRNA expression by cAMP-dependent cascades. This had previously been observed in vivo (15) in the hyperstimulated thyroid gland from rats chronically treated with antithyroid drug, where DUOX2 mRNA expression was lower than in tissue from untreated animals, whereas the expression of other thyroid cAMP-regulated genes, such as TPO and NIS, was markedly increased. Although a posttranslational effect of cAMP is likely, the substantial decrease in Duox immunostaining seen in Graves’ thyroid tissues and toxic adenoma (21) and the decrease of H2O2 generation observed in toxic adenoma (22) could also be attributable, to some extent, to a down-regulation of DUOX2 mRNA expression by cAMP. These mechanisms may protect hyperstimulated thyrocytes from the oxidative stress resulting from overexpression of the NADPH oxidase.
fig.ommitteedw/, http://www.100md.com
Figure 6. Effect of serum concentration and forskolin (FK) stimulation on the levels of DUOX2 mRNAs in FRTL-5. FRTL-5 cells were maintained for 6 d in 5H (TSH-free) medium containing either 0.2% or 5% newborn calf serum, then for 24 h in the same medium, with (+) or without (-) forskolin. Total RNA was analyzed by Northern blotting with a cDNA probe corresponding to either the 5' region in the ORF (N-ter probe) or the 3'-UTR (3'-UTR probe) of the normal DUOX2 cDNA, as described in Materials and Methods.w/, http://www.100md.com
Effect of insulin on short and normal DUOX2 mRNA expression in FRTL-5 cellsw/, http://www.100md.com
Because DUOX2 gene expression in 5H medium was high in 0.2% serum (Fig. 6), we checked the effect of three hormones (insulin, somatostatin, and Gly-His-Lys-acetate) on the basal level (without TSH) and on the 24-h forskolin-stimulated expression of DUOX2 mRNA. The cells were maintained for 6 d in 5H or 4H medium, i.e. medium lacking TSH and one of the other three hormones as indicated, and DUOX2 mRNA expression was analyzed by Northern blot (Fig. 7). In the absence of forskolin, the level of DUOX2 mRNA was high under all conditions, except when the medium contained no insulin, indicating that, in these conditions, this hormone was implicated in the regulation of the DUOX2 gene expression. Moreover, the inhibiting effect of forskolin on the level of DUOX2 mRNA in 0.2% serum was obtained only when insulin was present in the culture medium. Insulin is involved in the regulation of thyroid-specific genes, but to date nothing had been known about its effect on DUOX2 gene expression. We evaluated the time course of the effect of insulin on the expression of the two species of DUOX2 mRNA in FRTL-5 cells (Fig. 8). Northern blotting showed that the expression of both DUOX2 mRNA species peaked after 6–12 h of stimulation. As expected (23), insulin alone had no effect on NIS mRNA level under these conditions (Fig. 8). Consequently, like the thyroglobulin (Tg) and TPO genes (24, 25), insulin also positively regulates DUOX2 gene expression in FRTL-5 cells, but this effect is counteracted in 5% serum (Fig. 6), suggesting that serum contains some repressive factor(s) that interact with the insulin pathway.
fig.ommitteed#w, http://www.100md.com
Figure 7. Effect of hormone withdrawal on the amount of short DUOX2 mRNA using NIS mRNA as reference. FRTL-5 cells were maintained for 6 d in 5H or 4H medium lacking insulin or somatostatin or Gly-His-Lys-acetate as indicated and containing 0.2% serum. Forskolin (FK) was then added (+) or not (-) for 24 h. Total RNA was extracted and analyzed by Northern blotting. The bottom panel shows the 18S ribosomal RNA band detected by methylene blue staining.#w, http://www.100md.com
fig.ommitteed#w, http://www.100md.com
Figure 8. Time course of the effect of insulin on DUOX2 and NIS mRNA levels in FRTL-5. FRTL-5 cells were maintained for 6 d in 4H medium containing 0.2% serum and then incubated with 10 µg/ml insulin for the periods indicated. A, Total RNA was analyzed by Northern blotting for both DUOX2 and NIS mRNA levels. 18S ribosomal RNA bands were detected by methylene blue staining. B, The ratio of the short DUOX2 mRNA, quantified using an INSTANTIMAGER (Packard Instrument SA), to the corresponding 18 S ribosomal RNA density, quantified by densitometry using the NIH Image program, was calculated. Values calculated from cells incubated 6 d in 4H medium were chosen as a reference and arbitrarily taken to be 1.0. Data are the mean ± SEM from three separate experiments (n = 3) using different batches of cells.
Effect of insulin on the regulation by forskolin of DUOX2 mRNA expression in FRTL-58fod|], 百拇医药
As shown in Fig. 7, the inhibitory effect of forskolin on the level of DUOX2 mRNA in 0.2% serum was obtained only when insulin was present in the culture medium. We determined the time course of the forskolin effect on the concentration of short and normal DUOX2 mRNA in FRTL-5 cells cultured in 0.2% serum, without (Fig. 9A) and with (Fig. 9B insulin. Northern blots showed that the expression of both DUOX2 mRNA species was markedly increased by forskolin in the absence of insulin (Fig. 9, A and C), peaking after 6 h of stimulation at a level that could be up to three times the initial level, and then declined. As expected (26), the expression of the NIS gene under these conditions became apparent after 6 h and was strongly enhanced after 14 h, when the levels of both species of DUOX2 mRNA had already decreased.8fod|], 百拇医药
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Figure 9. Time course of the effects of forskolin on DUOX2 and NIS mRNA levels in FRTL-5 cultured in the absence (A) or presence (B) of insulin. FRTL-5 cells were maintained in 4H medium (A) or 5H medium (B) containing 0.2% serum for 6 d, and then incubated with 10 µM forskolin for the periods indicated. Total RNA was then extracted and analyzed by Northern blotting as described in Fig. 8.
Interestingly, in the presence of insulin, forskolin had no stimulating effect on DUOX2 mRNA expression (Fig. 9, B and C), but still produced a definite inhibitory effect (Fig. 9, B and C). Under the same conditions, NIS mRNA level was positively regulated by forskolin (Fig. 9B), but the concentration of NIS mRNA was much lower than in the absence of insulin, confirming that insulin down-regulated its forskolin-induced expression, as previously reported (23).u, 百拇医药
Insulin is another important factor affecting thyroid function that collaborates with TSH in regulating thyroid proliferation and differentiation. Insulin and IGF-I both stimulate Tg and TPO mRNA expression (24, 25). Both hormones increase the transcription of the Tg gene, and their effects are additive to those of TSH/cAMP (24). In contrast, our data show that at low serum concentrations, the positive effects of insulin and cAMP on DUOX2 gene expression were not additive, but antagonistic. Similarly, and in accordance with previous reports showing that insulin/IGF-I inhibited iodide uptake stimulated by TSH in FRTL-5 cells (27), recent data have demonstrated that IGF-I inhibits the TSH stimulation of NIS gene expression. This effect was also obtained when cells were maintained under low serum conditions, and was phosphatidylinositide-3-kinase mediated (23). It was not attributable to an inhibitory effect on TSH-induced cAMP generation, but rather to cAMP signal action (27). Our results also demonstrate that insulin interferes with the effect of cAMP on DUOX2 mRNA expression and could therefore be an important regulator of the DUOX2 gene expression in hyperfunctioning thyroid tissues.
Effect of insulin and forskolin on the expression of DUOX2 mRNA in pig thyroid follicles!!., 百拇医药
We compared the effect of insulin on DUOX2 gene expression in FRTL-5 cells to that in thyrocytes in primary culture (Fig. 10A). Pig thyroid follicles were cultured in 4H medium in the presence of 0.2% newborn calf serum. Northern blots showed that unlike rat thyroid cell lines, only one DUOX2 mRNA species was detected, corresponding to an approximately 6-kb-long mRNA. The DUOX2 mRNA level was markedly increased after 24 h of insulin stimulation and reached a peak after 48 h. As a control, the NIS mRNA level was also determined under these conditions. Using an NIS-specific 3'-UTR probe, we detected two mRNA species, 3.5 kb and 3 kb in length, respectively, probably corresponding to the pig NIS transcripts published in GenBank by Dr. S. Selmi-Rubi (Lyon, France; accession no. AJ277989 and AJ487855). It is interesting to note that, in contrast to what was observed in FRTL-5 cells under these conditions, the NIS mRNA level was still markedly increased after stimulating for 48 h.
fig.ommitteedl#uc\?h, 百拇医药
Figure 10. Time course of the effects of insulin alone (A) or forskolin plus insulin (B) on DUOX2 and NIS mRNA levels in pig thyroid follicles. A, Pig thyroid follicles were maintained for 3 d in 4H medium containing 0.2% serum and then incubated with 10 µg/ml insulin for the periods indicated. Total RNA was analyzed by Northern blotting. 18 S ribosomal RNA bands were revealed by methylene blue staining. DUOX2 and NIS mRNA levels were quantified using specific porcine probes and an INSTANTIMAGER as described in Fig. 8. B, Pig thyroid follicles were maintained in 4H or 5H medium containing 0.2% serum for 3 d, and then incubated with 10 µM forskolin for the periods indicated. Total RNA was isolated and analyzed by Northern blotting as in A.l#uc\?h, 百拇医药
We also evaluated the time course of the forskolin effect on DUOX2 mRNA accumulation (Fig. 10B) in pig thyroid follicles cultured with (5H) or without (4H) insulin. In the absence of insulin, DUOX2 mRNA expression peaked after 14 h of stimulation and declined after 24 h. As observed in the FRTL-5 cells, the highest expression of the NIS gene under the same conditions was reached later (24 h) than that of DUOX2.
In 5H medium, DUOX2 mRNA was strongly expressed without forskolin because of the presence of insulin. Forskolin did not inhibit DUOX2 mRNA expression, in contrast to the effect in FRTL-5 cells. On the contrary, the expression of DUOX2 mRNA was still high 48 h after adding forskolin, suggesting that the positive effects of the cAMP and of insulin on DUOX2 gene expression are additive in porcine thyrocytes. This discrepancy could be ascribed to species-related differences in the signaling pathways in rat and pig thyroid, or it could reflect differences between the rat thyroid gland and the rat thyroid cell line FRTL-5 with regard to the regulation of DUOX2 gene.7+, http://www.100md.com
In conclusion, we have identified a truncated form of DUOX2 mRNA in two rat thyroid cell lines widely used in studies of thyroid cell function. It is more abundantly expressed than the normal DUOX2 mRNA, but fails to support protein synthesis in an in vitro translation system.7+, http://www.100md.com
This work highlights the importance of a careful choice of oligonucleotides and cDNA probes to study DUOX2 mRNA in both cell lines. This report also provides the first evidence that insulin up-regulates the expression of DUOX2 gene at the mRNA level, an effect that is antagonized by cAMP in FRTL-5 cells.
Acknowledgments3es0, 百拇医药
We are indebted to Etablissements HARANG (Houdan) for allowing us to collect pig thyroid glands from their slaughterhouse.3es0, 百拇医药
Accepted for publication November 6, 2002.3es0, 百拇医药
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