- Email: [email protected]
iodide symporter
Molecular and Cellular Endocrinology 213 (2003) 109–113
Graves’ IgG activate upstream enhancer of the sodium/iodide symporter Ekaterina Breous, Achim Wenzel, Ulrich Loos∗ Abteilung Innere Medizin I, Universität Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany
Abstract Graves’ thyroid tissue has been shown to express elevated levels of human sodium/iodide symporter (hNIS) mRNA and protein. In the present work, we demonstrate for the first time that hNIS gene expression in Graves’ disease (GD) is up-regulated by Graves’ IgG. Here, in transient transfection experiments using FRTL-5 cells, hNIS promoter and enhancer/luciferase construct showed an up to six-fold increase in transcriptional activity after incubation with purified Graves’ IgG. Mutation of a CRE site in hNIS enhancer inhibited Graves’ IgG response. In addition, mutation of a novel putative regulatory region in hNIS promoter reduced the stimulation three-fold. This discovered putative regulatory sequence might play a role in hNIS up-regulation by Graves’ IgG and TSH. The data presented here complement our current knowledge of the pathogenesis of GD and will contribute to a better understanding of mechanisms regulating the thyroid iodide concentrating activity. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Graves’ disease; TSH receptor autoantibodies; Sodium/iodide symporter; Regulatory elements
1. Introduction Graves’ disease (GD) is an autoimmune disease classically characterised by the presence of thyroid-stimulating antibodies. These antibodies mimic TSH by binding to the TSH receptor (TSHR), thus resulting in the activation of adenylyl cyclase cascade (Kohn et al., 1983; Rees Smith et al., 1988). Consequently, this leads to the uncontrolled increase in iodine uptake, stimulation of protein synthesis and thyroid growth (Rapoport et al., 1998; Weetman and McGregor, 1994). The thyroid is able to accumulate I− via the sodium/iodide symporter (NIS) (Carrasco, 1993). NIS is an intrinsic plasma membrane symporter protein that couples the inward “downhill” translocation of Na+ to the inward “uphill” translocation of I− (Taurog, 1996). Graves’ thyroid tissue has been shown to express elevated levels of NIS mRNA and protein (Ajjan et al., 1998; Saito et al., 1997). In addition, it is known that I− uptake activity is increased in patients with GD (Cavalieri, 1986). Furthermore, Shoda et al. (1993) reported that Graves’ IgG could stimulate iodide efflux from FRTL-5 rat thyroid cells. Regulatory elements in hNIS promoter and upstream enhancer (NUE) have been characterised by our group and oth∗ Corresponding author. Tel.: +49-731-5002-4306; fax: +49-731-5002-4745. E-mail address: [email protected] (U. Loos).
ers (Behr et al., 1998; Schmitt et al., 2002; Taki et al., 2002). TSH was shown to activate NUE via the cAMP response element (CRE), while the CRE was not found in hNIS promoter region. Here, for the first time we demonstrate that human NIS (hNIS) gene expression in GD is up-regulated by Graves’ IgG. The present study establishes that, like TSH, Graves’ IgG stimulate hNUE and not hNIS promoter. In addition, we describe a novel putative regulatory region in the hNIS promoter, which is involved in hNIS up-regulation by TSH and Graves’ IgG. This regulatory sequence is highly homologous to rat NIS (rNIS) TSH-responsive element (rTRE) (Ohmori et al., 1998), hence we named it hTRE.
2. Materials and methods 2.1. Cell culture FRTL-5 rat thyroid cells were grown in a 1:1 mixture of Click’s medium and RPMI (Gibco, Karlsruhe, Germany) containing 5% calf serum (Gibco) and a mixture of hormones and growth factors (Sigma, Taufkirchen, Germany), including glycyl-l-histidyl-l-lysine acetate (10 ng/ml), hydrocortisone (3.6 ng/ml), somatostatin (10 ng/ml), insulin (10 g/ml), transferrin (5 g/ml) and 1 U/l bovine thyrotropin (TSH).
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2.2. NIS-luciferase constructs The construction of the hNIS reporter constructs in the luciferase expression vector pGL3-Basic (Promega, Manheim, Germany)—promoter (N3), promoter and enhancer (N3 + NUE) and promoter and CRE-mutated enhancer (N3 + mtNUE), was performed by our group (Behr et al., 1998; Schmitt et al., 2002). hTRE-mutated promoter and enhancer (mtN3 + NUE) construct was generated by using ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, CA). Oligonucleotides used were hTREFor: 5 -TTAAAGCAGGCTGTGCAGGCTTGGA-3 and hTRERev: 5 -CACTCAAAGCCGTATTGTGCTTGAAACCTT3 . The accuracy of plasmid DNA was determined by sequencing. 2.3. IgG purification Sera were obtained from 29 patients with active GD and from seven normal subjects. TSHR-binding autoantibodies were measured in a commercial TRAK (TSH Rezeptor Antikoerper Konzentration) assay (BRAHMS Diagnostics, Berlin, Germany). IgG were purified using HiTrap protein GHP affinity columns (Amersham Biosciences, Uppsala, Sweden) according to the instructions of manufacturer. Centricon YM-100 concentrators (Millipore, Bedford, MA) were used for IgG desalting and dialysis against PBS. 2.4. Transfection and luciferase assays FRTL-5 cells were seeded into 12-well plates and transiently transfected with N3, N3 + NUE, mtN3 + NUE and N3 + mtNUE using the Effectene transfection reagent Qiagen (Hilden, Germany). As a control, pGL3-Basic empty vector (Promega) was also transfected into these cells. Following transfection, FRTL-5 cells were incubated for 48 h in TSH-free medium and then treated with 100 g/ml IgG, 0.0008 IU/l thyroid-stimulating antibody (TSA) standard (TSA) (National Institute for Biological Standards and Control, Hertfordshire, UK) or 1 mU/ml TSH for 24 h. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (IBMX, 0.5 mmol/l) (Sigma) was added to the medium containing IgG, TSA or TSH (Kraiem et al., 1988; Nguyen et al., 2002). Negative control consisted of medium supplemented with 0.5 mmol/l IBMX. Luciferase activity of cell extracts was measured in a luminometer of Berthold (Lumat LB9501, Wildbad, Germany). Luciferase assay materials were purchased from Promega (Manheim, Germany). For normalisation of the luciferase activities, the protein content was estimated by the BioRad protein assay (BioRad, Munich, Germany). 2.5. Statistical analysis Experiments were performed in triplicate wells and repeated three times. For statistical analysis of the
data, Student’s t-test was used. Values are the mean ± S.D.
3. Results 3.1. Graves’ IgG activate hNUE We assayed 29 IgG from patients with active GD and seven normal subjects for their ability to stimulate hNIS upstream regulatory elements—promoter and enhancer. FRTL-5 cells, transiently transfected with hNIS promoter (N3) and hNIS promoter and enhancer (N3 + NUE) luciferase-fused constructs, were treated with purified Graves’ IgG, IgG from normal subjects, thyroid-stimulating antibody standard (TSA) or TSH. In cells transfected with N3, no activation was observed after the treatment with Graves’ IgG at 100 g/ml (Fig. 1). In contrast, luciferase activity was significantly increased when N3 + NUE-transfected cells were treated with Graves’ IgG. N3 + NUE stimulation was even higher when cells were treated with TSA or TSH, whereas N3 was not stimulated by either. Furthermore, N3 + NUE stimulation by IgG from normal subjects was not significant, while N3 was not activated. Graves’ IgG-induced activation of CRE mutation-containing construct N3 + mtNUE was reduced approximately 30-fold compared to the stimulation of the wild-type CRE. The mean individual N3 + NUE luciferase activities and TRAK values of 29 Graves’ IgG are presented in Table 1. A linear relationship between NUE activation and the TRAK values could be demonstrated for the majority of the 29 samples. However, in a minority of instances there was a divergence from this relationship. 3.2. A novel putative regulatory region is involved in hNIS up-regulation by Graves’ IgG and TSH It has been reported by Ohmori et al. (1998) that TSH/cAMP-induced up-regulation of the rat NIS (rNIS) gene expression requires a novel thyroid transcription factor interacting with TSH-responsive element (rTRE) in the promoter region. Kogai et al. (2001) detected two possible hTRE sites with a consensus sequence GNNCGGANG in hNIS promoter (one and two base mismatch). We identified a third putative hTRE sequence located at −699 to −690 (one base mismatch) of hNIS promoter, which positioning is very similar to that of rTRE (Fig. 2). To determine the functional relevance of this novel hTRE, we performed its site-directed mutagenesis in N3 + NUE construct. The mutant construct was called mtN3 + NUE. When two nucleotides in hTRE were mutated, mtN3 + NUE was still activated by Graves’ IgG, TSA or TSH but the activation was reduced approximately three-fold compared to that of N3 + NUE with wild-type hTRE (Fig. 1). No significant stimulation was found when mtN3 + NUE-transfected cells were treated with IgG from normal subjects.
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Fig. 1. Stimulation of FRTL-5 cells, transiently transfected with pGL3-Basic, pGL-N3, pGL-N3 + NUE, pGL-mtN3 + NUE or pGL-N3 + mtNUE, by 1 mU/ml TSH (2), 100 g/ml IgG from normal subjects (n = 7) (3), 100 g/ml Graves’ IgG (n = 29) (4), or 0.0008 IU/l TSA (5). IgG from 29 GD patients or seven normal subjects have been tested individually and the results have then been pooled. 0.5 mmol/l IBMX was added to the medium containing IgG, TSH or TSA. Negative control consisted of medium supplemented with 0.5 mmol/l IBMX (1). Values are the mean ± S.D.
Fig. 2. Sequence alignment between promoter regions of the rat (upper) and human (lower) NIS gene. In rat promoter, TRE motif is in bold and underlined, GC-box is in italics and TATA-box is boxed. In human promoter, novel rTRE-like motif is in bold and underlined, the mutated nucleotides are in bold and boxed, two previously found TRE-like motifs are in bold (Kogai et al., 2001), GC-box is in italics and TATA-box is boxed. Numbering is relative to the ATG start codon in rat and human NIS genes, respectively.
4. Discussion IgG produced by B-lymphocytes infiltrating the thyroid play a major role in the development of GD (Kendall-Taylor et al., 1984; McLachlan et al., 1986). IgG bind the TSH receptor and stimulate cAMP production in several thyroid systems, including human thyroid slices and membranes (Rees Smith et al., 1988; Zakarija and McKenzie, 1987). Activation of the cAMP pathway is related to increased iodide uptake and thyroid growth in GD (Jin et al., 1986; Tramontano et al., 1986; Zakarija et al., 1988). In the present study for the first time we present direct evidence of Graves’ IgG stimulation of hNIS gene. In addition, our results help to elucidate the molecular mechanisms involved in hNIS gene activation by Graves’ IgG and TSH. By testing IgG from 29 patients with GD we
found that, similar to TSH, Graves’ IgG stimulate hNIS gene upstream enhancer (NUE). The stimulation of NUE containing a mutation in cAMP response element (CRE) (Schmitt et al., 2002) was reduced approximately 30-fold, thus suggesting that Graves’ IgG activate NUE via this regulatory sequence. We did not detect any significant stimulation of hNIS promoter-only construct by Graves’ IgG or TSH. This observation is in accordance with the previous data on TSH stimulation of hNIS cis-acting elements (Schmitt et al., 2002). Furthermore, we compared N3 + NUE-stimulating luciferase activities of 29 Graves’ IgG with their TRAK values, which represent a pool of TSHR stimulating and blocking autoantibodies. For most patients, we found a distinct correlation between the two values, however, in a few patients such correlation was not observed. This is likely due to the heterogeneity of
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Table 1 N3 + NUE-stimulating Graves’ IgG luciferase activities in FRTL-5 cells GD patient no.
N3+NUE (mean RLU/OD ± SD)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
121300 120200 122700 119600 81840 86750 82790 88260 92090 75310 77880 76420 61670 65090 65830 69620 66420 64370 68830 62420 58120 54510 60510 44390 48830 43700 33410 38300 35740
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1993 6796 1806 5329 1633 1382 5642 3423 1856 5499 4491 2541 4078 2462 4191 4558 1870 2297 3103 3371 6854 2566 5621 4142 5531 2684 4598 1653 2450
TRAK >40 >40 >40 >40 13,7 26,8 26,2 >40 >40 16,3 10,7 16,3 13,4 16,5 18,5 14,7 16,9 16,2 14,7 12,5 >40 8,2 8,3 8,9 >40 5,5 6,9 4,7 4,2
For each GD patient the experiments were performed in triplicate wells and repeated three times. TRAK values represent TSHR-binding autoantibodies.
Graves TSHR autoantibodies, in particular the presence of blocking TSHR-autoantibodies in IgG fraction of these patients. Based on the sequence homology of hNIS and rNIS promoter regions, we found a novel putative regulatory region in hNIS promoter. It is located 232 bp upstream of the GC-box, which is a very similar positioning to known rNIS TSH-responsive element (rTRE). Interestingly, the mutation of two nucleotides of this novel region, which we termed hTRE, reduced the activation of mtN3 + NUE construct approximately three-fold but did not abolish it completely. This may indicate that additional nucleotides participate in the interaction of the discovered regulatory region and its nuclear factor. To summarise, hTRE might play a role in hNIS up-regulation by Graves’ IgG and TSH, however, further studies are required to examine the nuclear factor interacting with this novel putative regulatory element. The obtained results complement our current knowledge of the pathogenesis of GD. The discovery of a novel putative regulatory element in hNIS gene should allow a further detailed investigation of transcriptional regulation of human NIS gene expression. The data presented here will contribute to a better understanding of the complex mechanisms regulating the thyroid iodide concentrating activity.
References Ajjan, R.A., Kamaruddin, N.A., Crisp, M., Watson, P.F., Ludgate, M., Weetman, A.P., 1998. Regulation and tissue distribution of the human sodium iodide symporter gene. Clin. Endocrinol. (Oxford) 49, 49,517– 49,523. Behr, M., Schmitt, T.L., Espinoza, C.R., Loos, U., 1998. Cloning of a functional promoter of the human sodium/iodide-symporter gene. Biochem. J. 331, 359–363. Carrasco, N., 1993. Iodide transport in the thyroid gland. Biochim. Biophys. Acta 1154, 65–82. Cavalieri, R.R., 1986. Quantitive in vivo tests. In: Ingbar, S.H., Braverman, L.E. (Eds.), Werner’s: the Thyroid. Lippincott, Philadelphia, pp. 445–458. Jin, S., Hornicek, F.J., Neylan, D., Zakarija, M., McKenzie, J.M., 1986. Evidence that adenosine 3 ,5 -monophosphate mediates stimulation of thyroid growth in FRTL5 cells. Endocrinology 119, 802–810. Kendall-Taylor, P., Knox, A.J., Steel, N.R., Atkinson, S., 1984. Evidence that thyroid-stimulating antibody is produced in the thyroid gland. Lancet 1, 654–656. Kogai, T., Hershman, J.M., Motomura, K., Endo, T., Onaya, T., Brent, G.A., 2001. Differential regulation of the human sodium/iodide symporter gene promoter in papillary thyroid carcinoma cell lines and normal thyroid cells. Endocrinology 142, 3369–3379. Kohn, L.D., Valente, W.A., Laccetti, P., Cohen, J.L., Aloj, S.M., Grollman, E.F., 1983. Multicomponent structure of the thyrotropin receptor: relationship to Graves’ disease. Life Sci. 32, 15–30. Kraiem, Z., Alkobi, R., Sadeh, O., 1988. Sensitization and desensitization of human thyroid cells in culture: effects of thyrotrophin and thyroid-stimulating immunoglobulin. J. Endocrinol. 119, 341–349. McLachlan, S.M., Pegg, C.A., Atherton, M.C., Middleton, S.L., Clark, F., Rees Smith, B., 1986. TSH receptor antibody synthesis by thyroid lymphocytes. Clin. Endocrinol. (Oxford) 24, 24,223–24,230. Nguyen, L.Q., Arseven, O.K., Gerber, H., Stein, B.S., Jameson, J.L., Kopp, P., 2002. Cloning of the cat TSH receptor and evidence against an autoimmune etiology of feline hyperthyroidism. Endocrinology 143, 395–402. Ohmori, M., Endo, T., Harii, N., Onaya, T., 1998. A novel thyroid transcription factor is essential for thyrotropin-induced up-regulation of Na+ /I− symporter gene expression. Mol. Endocrinol. 12, 727–736. Rapoport, B., Chazenbalk, G.D., Jaume, J.C., McLachlan, S.M., 1998. The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr. Rev. 20, 100. Rees Smith, B., McLachlan, S.M., Furmaniak, J., 1988. Autoantibodies to the thyrotropin receptor. Endocr. Rev. 9, 106–121. Saito, T., Endo, T., Kawaguchi, A., Ikeda, M., Nakazato, M., Kogai, T., Onaya, T., 1997. Increased expression of the Na+ /I− symporter in cultured human thyroid cells exposed to thyrotropin and in Graves’ thyroid tissue. J. Clin. Endocrinol. Metab. 82, 3331–3336. Schmitt, T.L., Espinoza, C.R., Loos, U., 2002. Characterization of a thyroid-specific and cyclic adenosine monophosphate-responsive enhancer far upstream from the human sodium iodide symporter gene. Thyroid 12, 273–279. Shoda, Y., Kondo, Y., Kobayashi, I., 1993. Graves’ immunoglobulin G stimulates iodide efflux in FRTL-5 thyroid cells. J. Clin. Endocrinol. Metab. 77, 94–97. Taki, K., Kogai, T., Kanamoto, Y., Hershman, J.M., Brent, G.A., 2002. A thyroid-specific far-upstream enhancer in the human sodium/iodide symporter gene requires Pax-8 binding and cyclic adenosine 3 ,5 -monophosphate response element-like sequence binding proteins for full activity and is differentially regulated in normal and thyroid cancer cells. Mol. Endocrinol. 16, 2266–2282. Taurog, A., 1996. Hormone synthesis: thyroid iodine metabolism. In: Braverman, L.E., Utiger, R.D. (Eds.), The Thyroid: A Fundamental and Clinical Text, seventh ed. Lippincott/Raven, Philadelphia, New York, pp. 47–81.
E. Breous et al. / Molecular and Cellular Endocrinology 213 (2003) 109–113 Tramontano, D., Cushing, G.W., Moses, A.C., Ingbar, S.H., 1986. Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves’-IgG. Endocrinology 119, 940–942. Weetman, A.P., McGregor, A.M., 1994. Autoimmune thyroid disease: further developments in our understanding. Endocr. Rev. 15, 788– 830.
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Zakarija, M., McKenzie, J.M., 1987. The spectrum and significance of autoantibodies reacting with the thyrotropin receptor. Endocrinol. Metab. Clin. North Am. 16, 343–363. Zakarija, M., Jin, S., McKenzie, J.M., 1988. Evidence supporting the identity in Graves’ disease of thyroid-stimulating antibody and thyroid growth-promoting immunoglobulin G as assayed in FRTL5 cells. J. Clin. Invest. 81, 879–884.
Graves’ IgG activate upstream enhancer of the sodium/iodide symporter Ekaterina Breous, Achim Wenzel, Ulrich Loos∗ Abteilung Innere Medizin I, Universität Ulm, Robert-Koch-Strasse 8, D-89081 Ulm, Germany
Abstract Graves’ thyroid tissue has been shown to express elevated levels of human sodium/iodide symporter (hNIS) mRNA and protein. In the present work, we demonstrate for the first time that hNIS gene expression in Graves’ disease (GD) is up-regulated by Graves’ IgG. Here, in transient transfection experiments using FRTL-5 cells, hNIS promoter and enhancer/luciferase construct showed an up to six-fold increase in transcriptional activity after incubation with purified Graves’ IgG. Mutation of a CRE site in hNIS enhancer inhibited Graves’ IgG response. In addition, mutation of a novel putative regulatory region in hNIS promoter reduced the stimulation three-fold. This discovered putative regulatory sequence might play a role in hNIS up-regulation by Graves’ IgG and TSH. The data presented here complement our current knowledge of the pathogenesis of GD and will contribute to a better understanding of mechanisms regulating the thyroid iodide concentrating activity. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Graves’ disease; TSH receptor autoantibodies; Sodium/iodide symporter; Regulatory elements
1. Introduction Graves’ disease (GD) is an autoimmune disease classically characterised by the presence of thyroid-stimulating antibodies. These antibodies mimic TSH by binding to the TSH receptor (TSHR), thus resulting in the activation of adenylyl cyclase cascade (Kohn et al., 1983; Rees Smith et al., 1988). Consequently, this leads to the uncontrolled increase in iodine uptake, stimulation of protein synthesis and thyroid growth (Rapoport et al., 1998; Weetman and McGregor, 1994). The thyroid is able to accumulate I− via the sodium/iodide symporter (NIS) (Carrasco, 1993). NIS is an intrinsic plasma membrane symporter protein that couples the inward “downhill” translocation of Na+ to the inward “uphill” translocation of I− (Taurog, 1996). Graves’ thyroid tissue has been shown to express elevated levels of NIS mRNA and protein (Ajjan et al., 1998; Saito et al., 1997). In addition, it is known that I− uptake activity is increased in patients with GD (Cavalieri, 1986). Furthermore, Shoda et al. (1993) reported that Graves’ IgG could stimulate iodide efflux from FRTL-5 rat thyroid cells. Regulatory elements in hNIS promoter and upstream enhancer (NUE) have been characterised by our group and oth∗ Corresponding author. Tel.: +49-731-5002-4306; fax: +49-731-5002-4745. E-mail address: [email protected] (U. Loos).
ers (Behr et al., 1998; Schmitt et al., 2002; Taki et al., 2002). TSH was shown to activate NUE via the cAMP response element (CRE), while the CRE was not found in hNIS promoter region. Here, for the first time we demonstrate that human NIS (hNIS) gene expression in GD is up-regulated by Graves’ IgG. The present study establishes that, like TSH, Graves’ IgG stimulate hNUE and not hNIS promoter. In addition, we describe a novel putative regulatory region in the hNIS promoter, which is involved in hNIS up-regulation by TSH and Graves’ IgG. This regulatory sequence is highly homologous to rat NIS (rNIS) TSH-responsive element (rTRE) (Ohmori et al., 1998), hence we named it hTRE.
2. Materials and methods 2.1. Cell culture FRTL-5 rat thyroid cells were grown in a 1:1 mixture of Click’s medium and RPMI (Gibco, Karlsruhe, Germany) containing 5% calf serum (Gibco) and a mixture of hormones and growth factors (Sigma, Taufkirchen, Germany), including glycyl-l-histidyl-l-lysine acetate (10 ng/ml), hydrocortisone (3.6 ng/ml), somatostatin (10 ng/ml), insulin (10 g/ml), transferrin (5 g/ml) and 1 U/l bovine thyrotropin (TSH).
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2.2. NIS-luciferase constructs The construction of the hNIS reporter constructs in the luciferase expression vector pGL3-Basic (Promega, Manheim, Germany)—promoter (N3), promoter and enhancer (N3 + NUE) and promoter and CRE-mutated enhancer (N3 + mtNUE), was performed by our group (Behr et al., 1998; Schmitt et al., 2002). hTRE-mutated promoter and enhancer (mtN3 + NUE) construct was generated by using ExSite PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, CA). Oligonucleotides used were hTREFor: 5 -TTAAAGCAGGCTGTGCAGGCTTGGA-3 and hTRERev: 5 -CACTCAAAGCCGTATTGTGCTTGAAACCTT3 . The accuracy of plasmid DNA was determined by sequencing. 2.3. IgG purification Sera were obtained from 29 patients with active GD and from seven normal subjects. TSHR-binding autoantibodies were measured in a commercial TRAK (TSH Rezeptor Antikoerper Konzentration) assay (BRAHMS Diagnostics, Berlin, Germany). IgG were purified using HiTrap protein GHP affinity columns (Amersham Biosciences, Uppsala, Sweden) according to the instructions of manufacturer. Centricon YM-100 concentrators (Millipore, Bedford, MA) were used for IgG desalting and dialysis against PBS. 2.4. Transfection and luciferase assays FRTL-5 cells were seeded into 12-well plates and transiently transfected with N3, N3 + NUE, mtN3 + NUE and N3 + mtNUE using the Effectene transfection reagent Qiagen (Hilden, Germany). As a control, pGL3-Basic empty vector (Promega) was also transfected into these cells. Following transfection, FRTL-5 cells were incubated for 48 h in TSH-free medium and then treated with 100 g/ml IgG, 0.0008 IU/l thyroid-stimulating antibody (TSA) standard (TSA) (National Institute for Biological Standards and Control, Hertfordshire, UK) or 1 mU/ml TSH for 24 h. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (IBMX, 0.5 mmol/l) (Sigma) was added to the medium containing IgG, TSA or TSH (Kraiem et al., 1988; Nguyen et al., 2002). Negative control consisted of medium supplemented with 0.5 mmol/l IBMX. Luciferase activity of cell extracts was measured in a luminometer of Berthold (Lumat LB9501, Wildbad, Germany). Luciferase assay materials were purchased from Promega (Manheim, Germany). For normalisation of the luciferase activities, the protein content was estimated by the BioRad protein assay (BioRad, Munich, Germany). 2.5. Statistical analysis Experiments were performed in triplicate wells and repeated three times. For statistical analysis of the
data, Student’s t-test was used. Values are the mean ± S.D.
3. Results 3.1. Graves’ IgG activate hNUE We assayed 29 IgG from patients with active GD and seven normal subjects for their ability to stimulate hNIS upstream regulatory elements—promoter and enhancer. FRTL-5 cells, transiently transfected with hNIS promoter (N3) and hNIS promoter and enhancer (N3 + NUE) luciferase-fused constructs, were treated with purified Graves’ IgG, IgG from normal subjects, thyroid-stimulating antibody standard (TSA) or TSH. In cells transfected with N3, no activation was observed after the treatment with Graves’ IgG at 100 g/ml (Fig. 1). In contrast, luciferase activity was significantly increased when N3 + NUE-transfected cells were treated with Graves’ IgG. N3 + NUE stimulation was even higher when cells were treated with TSA or TSH, whereas N3 was not stimulated by either. Furthermore, N3 + NUE stimulation by IgG from normal subjects was not significant, while N3 was not activated. Graves’ IgG-induced activation of CRE mutation-containing construct N3 + mtNUE was reduced approximately 30-fold compared to the stimulation of the wild-type CRE. The mean individual N3 + NUE luciferase activities and TRAK values of 29 Graves’ IgG are presented in Table 1. A linear relationship between NUE activation and the TRAK values could be demonstrated for the majority of the 29 samples. However, in a minority of instances there was a divergence from this relationship. 3.2. A novel putative regulatory region is involved in hNIS up-regulation by Graves’ IgG and TSH It has been reported by Ohmori et al. (1998) that TSH/cAMP-induced up-regulation of the rat NIS (rNIS) gene expression requires a novel thyroid transcription factor interacting with TSH-responsive element (rTRE) in the promoter region. Kogai et al. (2001) detected two possible hTRE sites with a consensus sequence GNNCGGANG in hNIS promoter (one and two base mismatch). We identified a third putative hTRE sequence located at −699 to −690 (one base mismatch) of hNIS promoter, which positioning is very similar to that of rTRE (Fig. 2). To determine the functional relevance of this novel hTRE, we performed its site-directed mutagenesis in N3 + NUE construct. The mutant construct was called mtN3 + NUE. When two nucleotides in hTRE were mutated, mtN3 + NUE was still activated by Graves’ IgG, TSA or TSH but the activation was reduced approximately three-fold compared to that of N3 + NUE with wild-type hTRE (Fig. 1). No significant stimulation was found when mtN3 + NUE-transfected cells were treated with IgG from normal subjects.
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Fig. 1. Stimulation of FRTL-5 cells, transiently transfected with pGL3-Basic, pGL-N3, pGL-N3 + NUE, pGL-mtN3 + NUE or pGL-N3 + mtNUE, by 1 mU/ml TSH (2), 100 g/ml IgG from normal subjects (n = 7) (3), 100 g/ml Graves’ IgG (n = 29) (4), or 0.0008 IU/l TSA (5). IgG from 29 GD patients or seven normal subjects have been tested individually and the results have then been pooled. 0.5 mmol/l IBMX was added to the medium containing IgG, TSH or TSA. Negative control consisted of medium supplemented with 0.5 mmol/l IBMX (1). Values are the mean ± S.D.
Fig. 2. Sequence alignment between promoter regions of the rat (upper) and human (lower) NIS gene. In rat promoter, TRE motif is in bold and underlined, GC-box is in italics and TATA-box is boxed. In human promoter, novel rTRE-like motif is in bold and underlined, the mutated nucleotides are in bold and boxed, two previously found TRE-like motifs are in bold (Kogai et al., 2001), GC-box is in italics and TATA-box is boxed. Numbering is relative to the ATG start codon in rat and human NIS genes, respectively.
4. Discussion IgG produced by B-lymphocytes infiltrating the thyroid play a major role in the development of GD (Kendall-Taylor et al., 1984; McLachlan et al., 1986). IgG bind the TSH receptor and stimulate cAMP production in several thyroid systems, including human thyroid slices and membranes (Rees Smith et al., 1988; Zakarija and McKenzie, 1987). Activation of the cAMP pathway is related to increased iodide uptake and thyroid growth in GD (Jin et al., 1986; Tramontano et al., 1986; Zakarija et al., 1988). In the present study for the first time we present direct evidence of Graves’ IgG stimulation of hNIS gene. In addition, our results help to elucidate the molecular mechanisms involved in hNIS gene activation by Graves’ IgG and TSH. By testing IgG from 29 patients with GD we
found that, similar to TSH, Graves’ IgG stimulate hNIS gene upstream enhancer (NUE). The stimulation of NUE containing a mutation in cAMP response element (CRE) (Schmitt et al., 2002) was reduced approximately 30-fold, thus suggesting that Graves’ IgG activate NUE via this regulatory sequence. We did not detect any significant stimulation of hNIS promoter-only construct by Graves’ IgG or TSH. This observation is in accordance with the previous data on TSH stimulation of hNIS cis-acting elements (Schmitt et al., 2002). Furthermore, we compared N3 + NUE-stimulating luciferase activities of 29 Graves’ IgG with their TRAK values, which represent a pool of TSHR stimulating and blocking autoantibodies. For most patients, we found a distinct correlation between the two values, however, in a few patients such correlation was not observed. This is likely due to the heterogeneity of
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Table 1 N3 + NUE-stimulating Graves’ IgG luciferase activities in FRTL-5 cells GD patient no.
N3+NUE (mean RLU/OD ± SD)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
121300 120200 122700 119600 81840 86750 82790 88260 92090 75310 77880 76420 61670 65090 65830 69620 66420 64370 68830 62420 58120 54510 60510 44390 48830 43700 33410 38300 35740
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1993 6796 1806 5329 1633 1382 5642 3423 1856 5499 4491 2541 4078 2462 4191 4558 1870 2297 3103 3371 6854 2566 5621 4142 5531 2684 4598 1653 2450
TRAK >40 >40 >40 >40 13,7 26,8 26,2 >40 >40 16,3 10,7 16,3 13,4 16,5 18,5 14,7 16,9 16,2 14,7 12,5 >40 8,2 8,3 8,9 >40 5,5 6,9 4,7 4,2
For each GD patient the experiments were performed in triplicate wells and repeated three times. TRAK values represent TSHR-binding autoantibodies.
Graves TSHR autoantibodies, in particular the presence of blocking TSHR-autoantibodies in IgG fraction of these patients. Based on the sequence homology of hNIS and rNIS promoter regions, we found a novel putative regulatory region in hNIS promoter. It is located 232 bp upstream of the GC-box, which is a very similar positioning to known rNIS TSH-responsive element (rTRE). Interestingly, the mutation of two nucleotides of this novel region, which we termed hTRE, reduced the activation of mtN3 + NUE construct approximately three-fold but did not abolish it completely. This may indicate that additional nucleotides participate in the interaction of the discovered regulatory region and its nuclear factor. To summarise, hTRE might play a role in hNIS up-regulation by Graves’ IgG and TSH, however, further studies are required to examine the nuclear factor interacting with this novel putative regulatory element. The obtained results complement our current knowledge of the pathogenesis of GD. The discovery of a novel putative regulatory element in hNIS gene should allow a further detailed investigation of transcriptional regulation of human NIS gene expression. The data presented here will contribute to a better understanding of the complex mechanisms regulating the thyroid iodide concentrating activity.
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