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Endo
Extracellular adenosine increases Na+/I- symporter gene expression in rat thyroid FRTL-5 cells.Harii N, Endo T, Ohmori M, Onaya T. Mol Cell Endocrinol. 1999 Nov 25;157(1-2):31-9. [abstract only]
"We studied the effect of extracellular adenosine on iodide (I-) transport in FRTL-5 thyroid cells. I- accumulation increases after a 48 h exposure to adenosine in a concentration-dependent manner, reaching a maximum of 7.9-fold basal levels at 72 h after the addition of 300 microM adenosine. Neither I- efflux nor intracellular cyclic adenosine monophosphate accumulation is affected by the exposure to adenosine. The stimulation of I- transport by adenosine is partly as a result of an increase in Na+/I- symporter (NIS) mRNA and protein levels. Northern blot analysis revealed that adenosine increases NIS mRNA levels at 24 h, reaching a maximum at 36 h. Western blot analysis demonstrated that adenosine increases NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of adenosine-induced I- transport. Adenosine increased the promoter activity of a full-length NIS promoter-luciferase chimera, suggesting that the effect of adenosine on NIS mRNA levels is transcriptional. The stimulatory effect of adenosine on NIS mRNA levels, is mimicked by N6-(L-2-phenylisopropyl) adenosine (PIA), an A1 adenosine receptor agonist, and inhibited by 1,3-dipropyl-8-cyclopentylxanthine, an A1 adenosine receptor antagonist, suggesting that the effect is mediated via the A1 adenosine receptor stimulation in FRTL-5 cells. Incubating cells with islet-activating protein inhibited the adenosine-induced NIS mRNA levels. In sum, extracellular adenosine increases NIS gene expression and stimulates I- transport via the A1 adenosine receptor-Gi/Go protein signal transduction pathway."
A novel thyroid transcription factor is essential for thyrotropin-induced up-regulation of Na+/I- symporter gene expression.Ohmori M, Endo T, Harii N, Onaya T. Mol Endocrinol. 1998 May;12(5):727-36.
"The stimulation of iodide (I-) transport by TSH in FRTL-5 thyroid cells is partly due to an increase in Na+/I- symporter (NIS) gene expression. The identification of a TSH-responsive element (TRE) in the NIS promoter and its relationship to the action of thyroid transcription factor-1 (TTF-1) on the promoter are the subjects of this report. By transfecting NIS promoter-luciferase chimeric plasmids into FRTL-5 cells in the presence or absence of TSH, we identify a TRE between -420 and -370 bp of the NIS 5'-flanking region. Nuclear extracts from FRTL-5 cells cultured in the absence of TSH form two groups of protein-DNA complexes, A and B, in gel mobility shift assays using an oligonucleotide having the sequence from -420 to -385 bp. Only the A complex is increased by exposure of FRTL-5 cells to TSH or forskolin. The addition of TSH to FRTL-5 cells can increase the A complex at 3-6 h, reaching a maximum at 12 h. FRTL-5, but not nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cell nuclear extracts, form the A complex. The TSH-increased nuclear factor in FRTL-5 cells interacting with the NIS TRE is distinct from TTF-1, thyroid transcription factor-2, or Pax-8, as evidenced by the absence of competition using oligonucleotides specific for these factors in gel shift assays. Neither is it the nuclear protein interacting with cAMP response element. The TRE is in the upstream of a TTF-1-binding site, -245 to -230 bp. Mutation of the TRE causing a loss of TSH responsiveness also decreases TTF-1-induced promoter activity in a transfection experiment. The formation of the A complex between FRTL-5 nuclear extracts and the NIS TRE is redox-regulated. In sum, TSH/cAMP-induced up-regulation of the NIS requires a novel thyroid transcription factor, which also appears to be involved in TTF-1-mediated thyroid-specific NIS gene expression."
Increased expression of the sodium/iodide symporter in papillary thyroid carcinomas.Saito T, Endo T, Kawaguchi A, Ikeda M, Katoh R, Kawaoi A, Muramatsu A, Onaya T. J Clin Invest. 1998 Apr 1;101(7):1296-300.
"Iodide is concentrated to a much lesser extent by papillary thyroid carcinoma as compared with the normal gland. The Na+/I- symporter (NIS) is primarily responsible for the uptake of iodide into thyroid cells. Our objective was to compare NIS mRNA and protein expression in papillary carcinomas with those in specimens with normal thyroid. Northern blot analysis revealed a 2.8-fold increase in the level of NIS mRNA in specimens with papillary carcinoma versus specimens with normal thyroid. Immunoblot analysis using anti-human NIS antibody that was produced with a glutathione S-transferase fusion protein containing NIS protein (amino acids 466-522) showed the NIS protein at 77 kD. The NIS protein level was elevated in 7 of 17 cases of papillary carcinoma but was not elevated in the normal thyroid. Immunohistochemical staining revealed abundant NIS in 8 of 12 carcinomas, whereas NIS protein was barely detected in specimens with normal thyroid. Although considerable patient-to-patient variation was observed, our results indicate that NIS mRNA is elevated, and its protein tends to be more abundant, in a subset of papillary thyroid carcinomas than in normal thyroid tissue."
Transforming growth factor-beta1 suppresses thyrotropin-induced Na+/I- symporter messenger RNA and protein levels in FRTL-5 rat thyroid cells.Kawaguchi A, Ikeda M, Endo T, Kogai T, Miyazaki A, Onaya T. Thyroid. 1997 Oct;7(5):789-94. [abstract only]
"Iodide transport into the thyroid catalyzed by the Na+/I- symporter (NIS), is the first and main rate-limiting step in thyroid hormone synthesis. Recently, we have demonstrated that thyrotropin (TSH) increases NIS messenger RNA (mRNA) and protein levels, as well as iodide uptake activity. Although transforming growth factor-beta1 (TGFbeta1) is known to affect thyroid cell function, it is still unclear how TGFbeta1 regulates TSH-stimulated iodide accumulation. Therefore, the effects of TGFbeta1 on TSH-stimulated NIS mRNA and protein levels were examined in FRTL-5 rat thyroid cells by Northern and Western blot analyses, and iodide uptake was assessed. Northern blot analysis revealed that TGFbeta1 suppressed TSH-stimulated NIS mRNA levels in a dose- and time-dependent manner. Western blot analysis demonstrated that TGFbeta1 suppressed TSH-stimulated NIS protein levels. TGFbeta1 also suppressed (Bu)2 cyclic adenosine monophosphate (cAMP)- and forskolin-stimulated NIS mRNA and protein levels, indicating a role for TGFbeta1 downstream of cAMP production. As predicted, TGFbeta1 inhibited TSH-stimulated iodide uptake activity. These results suggest that the inhibitory effect of TGFbeta1 on TSH-stimulated iodide uptake is at least in part due to a suppression of NIS specific transcription. Therefore, TGFbeta1 may act as an autocrine or paracrine local modulator of thyroid hormone synthesis by influencing NIS mRNA levels in the thyroid."
Increased expression of the Na+/I- symporter in cultured human thyroid cells exposed to thyrotropin and in Graves' thyroid tissue.Saito T, Endo T, Kawaguchi A, Ikeda M, Nakazato M, Kogai T, Onaya T. J Clin Endocrinol Metab. 1997 Oct;82(10):3331-6.
"The Na+/I- symporter (NIS) is important in hormone synthesis in the thyroid gland. NIS activity, as reflected by I- uptake, was increased by TSH (1 mU/mL) or forskolin (10 mumol/L) in primary cultured human thyroid cells. Northern blot analysis revealed that incubation of these cells with TSH or forskolin for 24 h increased the abundance of NIS messenger ribonucleic acid (mRNA) 2.3- and 2.5-fold, respectively. Immunoblot analysis revealed 2.7- and 2.4-fold increases, respectively, in the amount of NIS protein after 48 h, suggesting that elevated levels of intracellular cAMP induced the expression of NIS in human thyrocytes. We then studied the levels of NIS mRNA and protein in Graves' thyroid tissue and found that the amount of NIS mRNA in thyroid tissue from individuals with Graves' disease (n = 5) was 3.8 times that in normal thyroid tissue (n = 5). The abundance of NIS mRNA was significantly correlated with that of thyroid peroxidase or thyroglobulin mRNAs, but not with that of TSH receptor mRNA, in the Graves' and normal thyroid tissue specimens. The amount of NIS protein was also increased 3.1-fold in Graves' thyroid tissue compared with that in normal thyroid tissue. The increased expression of NIS may thus contribute to the development of Graves' disease."
Iodide uptake and experimental 131I therapy in transplanted undifferentiated thyroid cancer cells expressing the Na+/I- symporter gene.Shimura H, Haraguchi K, Miyazaki A, Endo T, Onaya T. Endocrinology. 1997 Oct;138(10):4493-6.
"131I therapy is a widely accepted treatment for differentiated thyroid cancers which can accumulate iodide. We evaluated the efficiency of 131I therapy against tumors which are transfected with the Na+/I- symporter (NIS) gene. We transfected the rat NIS cDNA expression vector into malignantly transformed rat thyroid cells (FRTL-Tc) which do not concentrate iodide. The resultant cell line (Tc-rNIS) accumulated 125I 60-fold in vitro. The FRTL-Tc cells formed solid tumors after injection of cells into subcutaneous tissues of Fischer 344 rats. Tumors formed with Tc-rNIS cells accumulated up to 27.3% of total 125I administered, and concentrated 125I 11 to 27-fold in the tumors. Extracorporeal measurement of radioactivity in the tumors revealed that 125I accumulation peaked at 90 min, and decreased to half levels 6 h after the injections. To investigate the effect of 131I administration on the tumor growth, we injected Na131I 2 and 3 weeks after the transplantation of the cells. The Na131I did not change the tumor volume significantly in either the FRTL-Tc or the Tc-rNIS-induced tumors. The short (6 h) effective half life of 131I in the tumors diminished the radiation dose to the tumor cells. However, this approach may prove beneficial in the treatment of radiosensitive cancers, and could be employed diagnostically."
Autoantibody against thyroid iodide transporter in the sera from patients with Hashimoto's thyroiditis possesses iodide transport inhibitory activity.Endo T, Kaneshige M, Nakazato M, Kogai T, Saito T, Onaya T. Biochem Biophys Res Commun. 1996 Nov 1;228(1):199-202. [abstract only]
"Recently we have newly identified an autoantibody against thyroid iodide transporter (TIT) in the sera from patients with autoimmune thyroid disease. In order to study the function of these autoantibodies, we established CHO-KI cells stably expressing recombinant rat TIT (CHO-TIT cells), and the effect of IgGs from the patients with Hashimoto's thyroiditis on iodide uptake activity of CHO-TIT cells was investigated. We found that 4 out of 34 sera from patients with Hashimoto's thyroiditis strongly recognized TIT by Western blot analysis. These 4 IgGs, purified by protein A column chromatography, caused 14 to 62% inhibition of I- accumulation in CHO-TIT cells. Next, using synthetic peptides, we determined the recognition site of the autoantibody on the TIT molecule. The autoantibody reacted with the synthetic peptide corresponding to the 6th extracellular loop of the TIT molecule. These results suggest that autoantibody against TIT in the sera from patients with Hashimoto's thyroiditis binds to the 6th extracellular loop of TIT protein and inhibits I- transport into the thyrocytes. Anti-TIT autoantibody might participate in the pathogenesis of Hashimoto's thyroiditis and modulate thyroid function of patients with the disease."
Autoantibody against Na+/I- symporter in the sera of patients with autoimmune thyroid disease.Endo T, Kogai T, Nakazato M, Saito T, Kaneshige M, Onaya T. Biochem Biophys Res Commun. 1996 Jul 5;224(1):92-5. [abstract only]
"Using recombinant rat Na+/I- symporter (NaIS) protein, we have immunochemically searched for the autoantibody in the sera from patients with autoimmune thyroid disease. We found that 22 out of 26 sera (84%) from patients with Graves' disease and 3 out of 20 sera (15%) from patients with Hashimoto's thyroiditis recognized it. By Western blot, these IgGs reacted with 80 kDa protein in FRTL-5 cell membrane, which co-migrated with the band stained by rabbit antibody toward NaIS. These results indicate that autoantibody against NaIS, newly identified antibody, frequently exists in patients with autoimmune thyroid disease, especially in Graves' disease."
Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells.Kogai T, Endo T, Saito T, Miyazaki A, Kawaguchi A, Onaya T. Endocrinology. 1997 Jun;138(6):2227-32.
"To investigate the mechanism of I- transport stimulation by TSH, we studied the effects of TSH on Na+/I- symporter (NIS) messenger RNA (mRNA) and protein levels in FRTL-5 cells and correlated these with I- transport activity. When 1 mU/ml TSH was added to quiescent FRTL-5 cells, a 12-h latency was observed before the onset of increased I- transport activity, which reached a maximum [approximately 27 times basal (5H medium) levels] at 72 h. In contrast, Northern blot analysis, using rat NIS complementary DNA as a probe, revealed that addition of TSH to these cells significantly increased NIS mRNA at 3-6 h, reaching a maximum after 24 h (approximately 5.9 times basal levels). Forskolin and (Bu)2cAMP mimicked this stimulatory effect on both the I- transport activity and mRNA levels. D-ribofranosylbenzimidazole, a transcription inhibitor, almost completely blocked TSH-induced stimulation of I- transport and NIS mRNA levels. Western blot analysis demonstrated that TSH increased NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of TSH-induced I- transport activity. However, it also showed that the amount of NIS protein already present in FRTL-5 cell membranes before the addition of TSH was about one third of the maximum level induced by TSH. These results indicate that stimulation of I- transport activity by TSH in thyrocytes is partly due to a rapid increase in NIS gene expression, followed by a relatively slow NIS protein synthesis. However, the existence of an abundant amount of protein in quiescent FRTL-5 cells with very low I- transport activity also suggests that this activity is controlled by another TSH-regulated factor(s)."
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