-change plots comparing Tregs/Tconvs expression profiles between the peripheral blood and the thyroid. Real-time PCR comparing (G) thyroglobulin (Tg), (H) IL-4, (I) IFN-γ, and (J) IL-10 mRNA expressions between the peripheral blood and thyroid Tregs. Mean ± s
Yun-Qing Zhu, Yun Hu, Ke He, Na Li, Peng Jiang, Yu-Qin Pan, Hong Zhou, and Xiao-Ming Mao
D Fabbro, L Pellizzari, F Mercuri, G Tell, and G Damante
Pax proteins are transcription factors that control differentiation of several cell types. In adult organisms Pax-8 is expressed in the follicular thyroid cell where it interacts with sequences of thyroglobulin and thyroperoxidase promoters. In this study, we provide evidence indicating that Pax-8 protein levels regulate thyroglobulin gene transcription. The most critical approach consisted in increasing Pax-8 protein levels by transfecting thyroid cells with a Pax-8 expression vector. In this situation the thyroglobulin promoter transcriptional activity was significantly increased with respect to untransfected cells. In contrast, the transfection of thyroid transcription factor-1 (TTF-1) expression vector causes a modest decrease of thyroglobulin promoter activity, rather than an increase. Northern blots of human papillary cancers reveal a significant correlation between Pax-8 and thyroglobulin mRNAs. Gel-retardation assays suggest that the mechanism by which the Pax-8 protein levels modulate thyroglobulin promoter activity may occur through competition with TTF-1 for a common binding site. Since we also demonstrate that Pax-8 expression is subjected to TSH control, our data strongly suggest that Pax-8 protein levels could represent an important determinant for the regulation of thyroid cells.
M T den Hartog, C C Sijmons, P M P Kristel, O Bakker, and J J M de Vijlder
The synthesis is described of an N-terminal thyroglobulin (Tg) polypeptide of 27 kDa, which is capable of hormonogenesis, in a baculovirus system. This polypeptide was made using a 657 bp Tg cDNA cloned from human thyroid RNA by a polymerase chain reaction method. The cDNA contained the information for the Tg signal peptide, the N-terminally located site for thyroid hormone formation and, at the 3′ end, a sequence coding for six histidine residues. The fragments produced were purified using a nickel—nitrilotriacetic acid column using these six histidine residues. The products were analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and showed two glycosylated fragments of 32 and 34 kDa, both of which started with asparagine. Iodination of the fragments with lactoperoxidase in vitro resulted in the formation of thyroxine (T4). The formation rate of T4 in the fragments was about five times lower than that of the mature Tg dimer of 660 kDa, but ten times more rapid than in bovine serum albumin under the same conditions.
M. Chambard, D. Depetris, D. Gruffat, S. Gonzalvez, J. Mauchamp, and O. Chabaud
Exocytosis, the ultimate step in thyroglobulin secretion, has been studied in porcine thyroid cells cultured in monolayers on the permeable bottom of culture chambers. We have previously demonstrated, using this culture system, that apical secretion accounts for 85–95% of total secretion of newly synthesized thyroglobulin. When cells were cultured for several days with bovine TSH (25 μU/ml) in the basal medium, the rate of glycoprotein accumulation in the upper compartment was three times higher than that in the absence of TSH. In contrast, the rate of thyroglobulin released into the basal medium (5–15% of total secreted thyroglobulin) appeared unmodified by chronic TSH stimulation. To investigate the effect of acute TSH stimulation on thyroglobulin exocytosis in the apical and basal compartments, pulse-chase experiments were carried out with the same culture system. The release of radiolabelled thyroglobulin (1·5-h pulse) into the apical medium was increased threefold during the 2-h chase period under TSH stimulation. The radiolabelled thyroglobulin released into the basal medium was increased only 1·5- to 2-fold, and stimulation disappeared after 1 h. The effect of TSH was maximal when the chase medium contained 50 μU TSH/ml. However, cells cultured for several days in the presence of 25 μU TSH/ml before the pulse-chase experiment, appeared desensitized to acute TSH stimulation. Similar responses were observed when the chase medium contained 8-chloro-cyclic AMP or cholera toxin. This study provides another example of the pleiotropic effect of TSH, mediated by cyclic AMP, on the sequential steps of thyroglobulin gene expression in cultured thyroid cells in which the polar character of the epithelial cells is well preserved.
CR Espinoza, TL Schmitt, and U Loos
Thyroglobulin (Tg) is an essential thyroid-specific protein, which serves as the matrix for thyroid hormone biosynthesis. To obtain new insights in the regulation of Tg gene expression, we investigated the interaction of the human Tg promoter with the thyroid-specific transcription factors TTF-1 and Pax8. A reporter gene, containing a 202 bp fragment from the human Tg 5'-flanking region including the promoter sequence and the transcriptional start site, and expression vectors containing the cDNAs for human TTF-1 and Pax8 were used in cotransfection experiments, in the non-thyroidal cell lines COS-7 and HeLa. Pax8 increased the specific transcriptional activity of the Tg promoter about threefold, whereas cotransfection with the homeodomain-containing protein TTF-1 stimulated promoter activity from six- to tenfold. The simultaneous expression of both factors stimulated the Tg promoter activity in a multiplicative manner up to 25-fold. TTF-1 binding sites could be localized precisely by lectron mobility shift assay. The two binding elements corresponded to sites A and C in the rat Tg promoter. Site-directed mutagenesis of three nucleotides in each binding element inhibited binding of TTF-1 to the two oligonucleotides. In cotransfection experiments, the mutant site C decreased TTF-1 transactivation to 26% of the wild-type, whereas an additional mutation in the site A reduced this value to almost zero, thus proving the physiological relevance of these sites. The present results demonstrate that the activity of the human Tg promoter is closely dependent on the function of TTF-1 and Pax8, opening the field for further investigations of pathological alterations of Tg gene expression.
AW Kung and KS Lau
The release of interferon-gamma (IFNgamma) has been demonstrated from the infiltrating T lymphocytes of the thyroid gland from patients with autoimmune thyroid disease (AITD). We have shown previously that IFNgamma inhibited thyroglobulin (Tg) gene transcription, and that its action was mediated by an increase in intracellular calcium and inositol phosphates. In the present study, we tried to determine the specific site of action of IFNgamma on the Tg gene. A 565 bp fragment (position -530 to +34) spanning the transcriptional start site of the human Tg promoter was ligated to the luciferase plasmid and transiently transfected into human thyrocytes. Stimulation with TSH (10 mIU/l) and IFNgamma (500 IU/l) resulted in a twofold increase and a 60% reduction in the luciferase activity respectively, similar to the effect observed with the endogenous Tg gene. Deletion studies revealed that the region with the strongest suppression by IFNgamma lay between 5' -388 to -258. Mobility gel shift experiments and DNA footprinting experiments demonstrated that the action of IFNgamma was mediated through a trans-acting protein which complexed to position -282 to -262 TTGAGCCTGTTCCCTC CAAA. Position -272 to -261 TTCCCTCCAA corresponded to the gamma-interferon activation site (GAS) consensus sequence TTNC(C)T NNNA. The turnover time of the nuclear protein lasted for only 4 h although the suppressive effect of IFNgamma on Tg gene transcription lasted for 48 h. The effect of IFNgamma was lost when the thyrocytes were co-treated with genistein, a specific tyrosine kinase inhibitor. The presence of the GAS in the promoter sequence of the Tg gene confirms the specific action of IFNgamma in thyroid hormone metabolism. In conclusion, apart from its regulatory role in T cell development and perpetuation of the immune response in AITD, IFNgamma may also play a role in altering cellular function of the thyrocytes by its action on the Tg gene promoter.
Ikuhiro Maeda, Toru Takano, Hiroshi Yoshida, Fumio Matsuzuka, Nobuyuki Amino, and Akira Miyauchi
Introduction Thyroid-specific genes, such as thyroid peroxidase (TPO), thyroglobulin (TG), Na + /I − symporter (NIS) and thyroid-stimulating hormone receptor (TSHR) play fundamental roles in the functions of thyroid follicular cells
Vaishnavi Venugopalan, Maren Rehders, Jonas Weber, Lisa Rodermund, Alaa Al-Hashimi, Tonia Bargmann, Janine Golchert, Vivien Reinecke, Georg Homuth, Uwe Völker, Francois Verrey, Janine Kirstein, Heike Heuer, Ulrich Schweizer, Doreen Braun, Eva K Wirth, and Klaudia Brix
do so, the thyroid gland synthesizes TH on the backbone of thyroglobulin (Tg), liberates these upon sequential Tg proteolysis, and releases them into the bloodstream ( Brix et al. 2019 , 2020 , Coscia & Taler-Verčič 2021 ). Tg solubilization and
I. D. Phillips, E. G. Black, M. C. Sheppard, and K. Docherty
In order to study the regulation of cathepsin B expression in the thyroid, cathepsin B mRNA concentrations were measured in rat thyroid cells (FRTL5) in culture. Northern blot analysis demonstrated that cathepsin B mRNA concentrations were increased in FRTL5 cells cultured for up to 6 days in TSH. The effect of TSH on cathepsin B mRNA concentrations was dose dependent over the range 25–150 μunits/ml. Cytoplasmic dot-blot analysis was used to characterize this effect further. The TSH-induced increase in cathepsin B mRNA concentrations (approximately fivefold over that in untreated cells) was partially mimicked by forskolin (approximately threefold) and ionomycin, while phorbol ester decreased cathepsin B mRNA concentrations. Similar changes were observed for thyroglobulin and actin mRNA concentrations. TSH had no effect on cathepsin B enzymatic activity or immunoreactive protein concentration. These results demonstrate (1) that cathepsin B expression in the thyroid is regulated in parallel with that of thyroglobulin and actin, and (2) that cyclic AMP- and Ca2+-dependent processes stimulate gene expression, while phorbol ester treatment inhibits gene expression in FRTL5 cells.
C. Massart, C. Le Tellier, C. Lucas, J. Gibassier, G. Leclech, and M. Nicol
Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anticancer drug which induces many sideeffects, but its action on the thyroid gland is still unknown. We have investigated the effects of this drug on human thyrocytes cultured in monolayers or in follicles and stimulated with 200 μU TSH/ml.
After 72h in culture, different concentrations of cisplatin (15, 30 and 75 μm) caused partial or total inhibition of cyclic AMP (cAMP), thyroglobulin (Tg) and tri-iodothyronine (T3) production, whereas thyroxine levels increased in the medium of thyrocytes cultured as follicles. Small doses of the drug did not affect thyrocyte production. Decreases in neutral-red uptake by thyroid cells and in intracellular lactate dehydrogenase, α-hydroxybutyryldehydrogenase and creatine phosphokinase activities were induced by 30 and 75 μm cisplatin.
These data show that high concentrations of cisplatin had a cytotoxic effect on thyrocytes. Cisplatin also induced inhibition of the production of cAMP, Tg and T3.