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Robert T. Mccluskey - One of the best experts on this subject based on the ideXlab platform.
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preferential megalin mediated transcytosis of low hormonogenic thyroglobulin a control mechanism for Thyroid Hormone Release
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Simonetta Lisi, Francesca Menconi, L. Grasso, Aldo Pinchera, Thomas E Willnow, Robert T. Mccluskey, Paolo Vitti, Claudio Marcocci, Samuel Refetoff, F LuchettiAbstract:Hormone secretion by thyrocytes occurs by fluid phase uptake and lysosomal degradation of the proHormone thyroglobulin (Tg). However, some Tg internalized by megalin bypasses lysosomes and is transcytosed across cells and Released into the bloodstream. Because the Hormone content of Tg is variable, we investigated whether this affects transcytosis. We found that rat Tg with a low Hormone content [low-hormonogenic rat Tg (low-horm-rTg)] is transcytosed by megalin across Thyroid FRTL-5 cells to a greater extent than rat Tg with a high Hormone content [hormonogenic rat Tg (horm-rTg)]. In immunoprecipitation experiments, the Tg sequence Arg-2489-Lys-2503 (required for binding to megalin and heparan sulfate proteoglycans) was found to be more exposed in low-horm-rTg, which accounted for its preferential transcytosis. Thus, removal of surface heparan sulfate proteoglycans from FRTL-5 cells or blocking of 2489-2503 reduced transcytosis of low-horm-rTg to a greater extent than that of horm-rTg. Preferential transcytosis of low-horm-rTg affected Hormone Release. Thus, the increase in Hormone Release from horm-rTg in FRTL-5 cells determined by megalin blocking (due to reduced transcytosis and enhanced Tg degradation) was rescued by low-horm-rTg, suggesting that megalin is required for effective Hormone Release. This finding was confirmed in a small number of megalin-deficient mice, which had serological features resembling mild hypoThyroidism. Reduced Hormone formation within Tg in vivo, due to treatment of rats with aminotriazole or of patients with Graves' disease with methimazole, resulted in increased Tg transcytosis via megalin, in confirmation of results with FRTL-5 cells. Our study points to a major role of megalin in Thyroid homeostasis with possible implications in Thyroid diseases.
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Role of thyroglobulin endocytic pathways in the control of Thyroid Hormone Release.
American Journal of Physiology-Cell Physiology, 2000Co-Authors: Michele Marino, Robert T. MccluskeyAbstract:Thyroglobulin (Tg), the Thyroid Hormone precursor, is synthesized by thyrocytes and secreted into the colloid. Hormone Release requires uptake of Tg by thyrocytes and degradation in lysosomes. This...
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role of megalin gp330 in transcytosis of thyroglobulin by Thyroid cells a novel function in the control of Thyroid Hormone Release
Journal of Biological Chemistry, 2000Co-Authors: Michele Marino, Aldo Pinchera, Luca Chiovato, David Andrews, Dennis Brown, Gang Zheng, Robert T. MccluskeyAbstract:Abstract When thyroglobulin (Tg) is endocytosed by thyrocytes and transported to lysosomes, Thyroid Hormones (T4 and T3) are Released. However, some internalized Tg is transcytosed intact into the bloodstream, thereby avoiding proteolytic cleavage. Here we show that megalin (gp330), a Tg receptor on Thyroid cells, plays a role in Tg transcytosis. Following incubation with exogenous rat Tg at 37 °C, Fisher rat Thyroid (FRTL-5) cells, a differentiated Thyroid cell line, Released T3 into the medium. However, when cells were incubated with Tg plus either of two megalin competitors, T3 Release was increased, suggesting that Tg internalized by megalin bypassed the lysosomal pathway, possibly with Release of undegraded Tg from cells. To assess this possibility, we performed experiments in which FRTL-5 cells were incubated with either unlabeled or 125I-labeled Tg at 37 °C to allow internalization, treated with heparin to remove cell surface-bound Tg, and further incubated at 37 °C to allow Tg Release. Intact 330-kDa Tg was Released into the medium, and the amount Released was markedly reduced by megalin competitors. To investigate whether Tg Release resulted from transcytosis, we studied FRTL-5 cells cultured as polarized layers with tight junctions on permeable filters in the upper chamber of dual chambered devices. Following the addition of Tg to the upper chamber and incubation at 37 °C, intact 330-kDa Tg was found in fluids collected from the lower chamber. The amount recovered was markedly reduced by megalin competitors, indicating that megalin mediates Tg transcytosis. We also studied Tg transcytosis in vivo, using a rat model of goiter induced by aminotriazole, in which increased Release of thyrotropin induces massive colloid endocytosis. This was associated with increased megalin expression on thyrocytes and increased serum Tg levels, with reduced serum T3 levels, supporting the conclusion that megalin mediates Tg transcytosis. Tg transcytosis is a novel function of megalin, which usually transports ligands to lysosomes. Megalin-mediated transcytosis may regulate the extent of Thyroid Hormone Release.
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Megalin-mediated transcytosis of thyroglobulin by Thyroid cells is a calmodulin-dependent process.
Thyroid, 2000Co-Authors: Michele Marino, Robert T. MccluskeyAbstract:Megalin, a multiligand receptor expressed on the apical surface of Thyroid cells, mediates transepithelial transport (transcytosis) of thyroglobulin (Tg) across thyrocytes, resulting in diversion of Tg from the lysosomal pathway and reduction of the extent of Thyroid Hormone Release from internalized Tg molecules. The calcium regulatory protein calmodulin facilitates some forms of transcytosis. Here we investigated the role of calmodulin in megalin-mediated transcytosis of Tg by Thyroid cells. For this purpose, we studied the effect of calmodulin antagonists on Tg transcytosis by Fisher rat Thyroid cells (FRTL-5), an established, differentiated Thyroid cell line. FRTL-5 cells were cultured on permeable filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper surface. Unlabeled Tg was added to the upper chamber at 37°C, and transcytosed Tg was detected by enzyme-linked immunosorbent assay (ELISA) in fluids collected 1 hour later from the lower chamber. To stu...
Michele Marino - One of the best experts on this subject based on the ideXlab platform.
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Role of thyroglobulin endocytic pathways in the control of Thyroid Hormone Release.
American Journal of Physiology-Cell Physiology, 2000Co-Authors: Michele Marino, Robert T. MccluskeyAbstract:Thyroglobulin (Tg), the Thyroid Hormone precursor, is synthesized by thyrocytes and secreted into the colloid. Hormone Release requires uptake of Tg by thyrocytes and degradation in lysosomes. This...
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role of megalin gp330 in transcytosis of thyroglobulin by Thyroid cells a novel function in the control of Thyroid Hormone Release
Journal of Biological Chemistry, 2000Co-Authors: Michele Marino, Aldo Pinchera, Luca Chiovato, David Andrews, Dennis Brown, Gang Zheng, Robert T. MccluskeyAbstract:Abstract When thyroglobulin (Tg) is endocytosed by thyrocytes and transported to lysosomes, Thyroid Hormones (T4 and T3) are Released. However, some internalized Tg is transcytosed intact into the bloodstream, thereby avoiding proteolytic cleavage. Here we show that megalin (gp330), a Tg receptor on Thyroid cells, plays a role in Tg transcytosis. Following incubation with exogenous rat Tg at 37 °C, Fisher rat Thyroid (FRTL-5) cells, a differentiated Thyroid cell line, Released T3 into the medium. However, when cells were incubated with Tg plus either of two megalin competitors, T3 Release was increased, suggesting that Tg internalized by megalin bypassed the lysosomal pathway, possibly with Release of undegraded Tg from cells. To assess this possibility, we performed experiments in which FRTL-5 cells were incubated with either unlabeled or 125I-labeled Tg at 37 °C to allow internalization, treated with heparin to remove cell surface-bound Tg, and further incubated at 37 °C to allow Tg Release. Intact 330-kDa Tg was Released into the medium, and the amount Released was markedly reduced by megalin competitors. To investigate whether Tg Release resulted from transcytosis, we studied FRTL-5 cells cultured as polarized layers with tight junctions on permeable filters in the upper chamber of dual chambered devices. Following the addition of Tg to the upper chamber and incubation at 37 °C, intact 330-kDa Tg was found in fluids collected from the lower chamber. The amount recovered was markedly reduced by megalin competitors, indicating that megalin mediates Tg transcytosis. We also studied Tg transcytosis in vivo, using a rat model of goiter induced by aminotriazole, in which increased Release of thyrotropin induces massive colloid endocytosis. This was associated with increased megalin expression on thyrocytes and increased serum Tg levels, with reduced serum T3 levels, supporting the conclusion that megalin mediates Tg transcytosis. Tg transcytosis is a novel function of megalin, which usually transports ligands to lysosomes. Megalin-mediated transcytosis may regulate the extent of Thyroid Hormone Release.
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Megalin-mediated transcytosis of thyroglobulin by Thyroid cells is a calmodulin-dependent process.
Thyroid, 2000Co-Authors: Michele Marino, Robert T. MccluskeyAbstract:Megalin, a multiligand receptor expressed on the apical surface of Thyroid cells, mediates transepithelial transport (transcytosis) of thyroglobulin (Tg) across thyrocytes, resulting in diversion of Tg from the lysosomal pathway and reduction of the extent of Thyroid Hormone Release from internalized Tg molecules. The calcium regulatory protein calmodulin facilitates some forms of transcytosis. Here we investigated the role of calmodulin in megalin-mediated transcytosis of Tg by Thyroid cells. For this purpose, we studied the effect of calmodulin antagonists on Tg transcytosis by Fisher rat Thyroid cells (FRTL-5), an established, differentiated Thyroid cell line. FRTL-5 cells were cultured on permeable filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper surface. Unlabeled Tg was added to the upper chamber at 37°C, and transcytosed Tg was detected by enzyme-linked immunosorbent assay (ELISA) in fluids collected 1 hour later from the lower chamber. To stu...
Kanji Sato - One of the best experts on this subject based on the ideXlab platform.
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Genes regulated by thyrotropin and iodide in cultured human Thyroid follicles: analysis by cDNA microarray.
Thyroid, 2003Co-Authors: Kazuko Yamazaki, Emiko Yamada, Yoshio Kanaji, Tetsuo Yanagisawa, Yoshiyuki Kato, Kazue Takano, Takao Obara, Kanji SatoAbstract:Thyrotropin (TSH) regulates a number of genes in thyrocytes, leading to iodide uptake, de novo synthesis and Release of Thyroid Hormones, and cell proliferation, accompanied by increased blood flow. At higher doses of iodide, however, the TSH-induced increases in Thyroid Hormone Release and blood flow are downregulated, and high iodide intake occasionally worsens autoimmune Thyroiditis. To elucidate the genes involved in such effects, we cultured human thyrocytes and examined genes modulated by TSH and iodide, using a cDNA microarray study, which can analyze 2400 genes in each run. When Thyroid follicles were cultured with TSH for 2 days, more than 100 genes were upregulated. These genes included those for enzymes involved in carbohydrate and lipid metabolism, adenylate and guanylate cyclases, and enzyme involved in cell proliferation. When Thyroid follicles were cultured with high iodide concentrations (10-5 M) for 24 hours, more than 100 genes were upregulated. Interesting genes were interleukin-8, IFP5...
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potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125i incorporation and de novo synthesized Thyroid Hormone Release in human Thyroid follicles
The Journal of Clinical Endocrinology and Metabolism, 1995Co-Authors: Kazuko Yamazaki, Kazuo Shizume, Takao Obara, Kanji Sato, Y Kanaji, Yukio Ito, T Nakagawa, T Koizumi, R NishimuraAbstract:Using a highly sensitive bioassay for TSH, in which human Thyroid follicles incorporate 125I and Release de novo synthesized Thyroid Hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in Thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than purified hCG. Asialo-hCG purified from a patient with choriocarcinoma had very potent TSH-like activity (468 microU hTSH/mg). Submaximal doses of bTSH and hCG variants produced additive stimulation of Thyroid function. Furthermore, the thyrotropic effect of hCG was inhibited by anti-TSH receptor antibody obtained from patients with myxedema. These in vitro findings suggest that although hCG is reported to exert potent cAMP-stimulating activity on rat Thyroid-like cells (FRTL-5) and Chinese hamster ovary cells transfected with hTSH receptor complementary DNA (0.092-0.72 microU hTSH/U hCG), the thyrotropic activity induced by authentic hCG in human Thyroid follicles is too weak to cause hyperThyroidism in normal pregnancy. However, hCG produced by some trophoblastic tumors, particularly asialo-hCG, has potent thyrotropic activity sufficient to cause clinically overt hyperThyroidism when produced excessively.
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Potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125I incorporation and de novo synthesized Thyroid Hormone Release in human Thyroid follicles.
The Journal of Clinical Endocrinology & Metabolism, 1995Co-Authors: Kazuko Yamazaki, Kazuo Shizume, Takao Obara, Kanji Sato, Y Kanaji, Yukio Ito, T Nakagawa, T Koizumi, R NishimuraAbstract:Using a highly sensitive bioassay for TSH, in which human Thyroid follicles incorporate 125I and Release de novo synthesized Thyroid Hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in Thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than pur...
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Reversible inhibition by interferons alpha and beta of 125I incorporation and Thyroid Hormone Release by human Thyroid follicles in vitro.
The Journal of Clinical Endocrinology & Metabolism, 1993Co-Authors: Kazuko Yamazaki, Kazuo Shizume, Takao Obara, Y Kanaji, Y. Yamakawa, Hiroshi Demura, Kanji SatoAbstract:When interferons (IFN-alpha-2a, IFN-alpha-2b, natural IFN-alpha and IFN-beta) were cultured with human Thyroid follicles, each IFN inhibited TSH-induced Thyroid function (125I incorporation and Release of 125I-T4) in a concentration-dependent manner. The minimal inhibitory effect was exerted at 1-10 U/ml. However, the inhibitory effect was reversible, and no inhibitory effect was detected when IFNs were removed from the medium within 12 h. These in vitro findings suggest that each IFN directly inhibits human Thyroid function at clinically attainable levels. However, since the inhibitory effect was reversible, IFN therapy would provoke only subtle Thyroid dysfunction in a majority of patients without preexisting Thyroid autoimmunity.
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Inhibition of 125I Organification and Thyroid Hormone Release by Interleukin-1, Tumor Necrosis Factor-α, and Interferon-γ in Human Thyrocytes in Suspension Culture*
The Journal of Clinical Endocrinology & Metabolism, 1990Co-Authors: Kanji Sato, Kazuo Shizume, Y Kanaji, Hiroshi Demura, T. Satoh, Ozawa M, D C Han, Imamura H, Toshio Tsushima, Yukio ItoAbstract:To elucidate the mechanism of decreased 131I uptake by the Thyroid gland in patients with subacute Thyroiditis and painless Thyroiditis, human Thyroid follicles were cultured with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF alpha), and/or interferon-gamma (IFN gamma), and the effects of these cytokines on Thyroid function were studied in vitro. When human thyrocytes were cultured in RPMI-1640 medium containing 0.5% fetal calf serum and TSH for 5-8 days, the cells incorporated 125I, synthesized de novo [125I]iodotyrosines and [125I]iodothyronines, and secreted [125I]T4 and [125I]T3 into the medium. IL-1 alpha and IL-1 beta inhibited 125I incorporation and [125I]iodothyronine Release in a concentration-dependent manner. The minimal inhibitory effect was detected at 10 pg/ml. Electron microscopic examination revealed a marked decrease in lysosome formation in IL-1-treated thyrocytes. TNF alpha and IFN gamma also inhibited Thyroid function in a concentration-dependent manner. Furthermore, when thyrocytes were cultured with IL-1, TNF alpha and IFN gamma, these cytokines more than additively inhibited Thyroid function. Although the main mechanism of 131I uptake suppression in the Thyroid gland in subacute Thyroiditis is due to cellular damage and suppression of TSH Release, our present findings suggest that IL-1, TNF alpha, and IFN gamma produced in the inflammatory process within the Thyroid gland further inhibit iodine incorporation and at least partly account for the decreased 131I uptake by the Thyroid gland in destruction-induced hyperThyroidism.
Aldo Pinchera - One of the best experts on this subject based on the ideXlab platform.
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Binding, uptake, and degradation of internalized thyroglobulin in cultured Thyroid and non-Thyroid cells
Journal of Endocrinological Investigation, 2011Co-Authors: R. Botta, Aldo Pinchera, S. Lisi, A. R. Taddei, A. M. Fausto, F. Giorgi, M. MarinòAbstract:Thyroid Hormone Release requires degradation of thyroglobulin (Tg) by Thyroid epithelial cells, which occurs mainly in the lysosomal pathway following Tg endocytosis. Non-specific fluid-phase endocytosis is thought to be the main route of Tg uptake leading to degradation, whereas receptor-mediated endocytosis is believed to lead to post-endocytic pathways other than degradation. To gain more insights into these issues, we investigated handling of Tg by various cell types. Tg bound similarly to Thyroid (FRTL-5, FRT) and non-Thyroid (COS-7, IRPT) cells, indicating the presence of membrane-binding sites, presumably receptors, in both cell types. Tg was internalized and degraded by all cells and degradation paralleled uptake, with the exception of FRTL-5 cells, in which a lower proportion of Tg was degraded, suggesting that in FRTL-5 cells mechanisms that target Tg to the various post-endocytic pathways (either receptors or post-receptorial factors) are differently represented. Immunoelectronmicroscopy showed a common path of endocytosis in FRTL-5, COS-7, and IRPT cells, namely the formation of pseudopods engulfing Tg, followed by internalization and accumulation of Tg in cytoplasmic vesicles and lysosomes. The fastest rate was observed in COS-7 cells, probably reflecting a lower impact of endocytic receptors. Our findings suggest that Tg uptake and degradation are not Thyroid-specific, that Tg binding sites exist in different cell types, and that uptake and/or degradation are differently regulated in differentiated Thyroid cells, presumably because of a different impact of endocytic receptors or post-endocytic mechanisms, which are probably responsible for the regulation of Hormone Release.
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preferential megalin mediated transcytosis of low hormonogenic thyroglobulin a control mechanism for Thyroid Hormone Release
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Simonetta Lisi, Francesca Menconi, L. Grasso, Aldo Pinchera, Thomas E Willnow, Robert T. Mccluskey, Paolo Vitti, Claudio Marcocci, Samuel Refetoff, F LuchettiAbstract:Hormone secretion by thyrocytes occurs by fluid phase uptake and lysosomal degradation of the proHormone thyroglobulin (Tg). However, some Tg internalized by megalin bypasses lysosomes and is transcytosed across cells and Released into the bloodstream. Because the Hormone content of Tg is variable, we investigated whether this affects transcytosis. We found that rat Tg with a low Hormone content [low-hormonogenic rat Tg (low-horm-rTg)] is transcytosed by megalin across Thyroid FRTL-5 cells to a greater extent than rat Tg with a high Hormone content [hormonogenic rat Tg (horm-rTg)]. In immunoprecipitation experiments, the Tg sequence Arg-2489-Lys-2503 (required for binding to megalin and heparan sulfate proteoglycans) was found to be more exposed in low-horm-rTg, which accounted for its preferential transcytosis. Thus, removal of surface heparan sulfate proteoglycans from FRTL-5 cells or blocking of 2489-2503 reduced transcytosis of low-horm-rTg to a greater extent than that of horm-rTg. Preferential transcytosis of low-horm-rTg affected Hormone Release. Thus, the increase in Hormone Release from horm-rTg in FRTL-5 cells determined by megalin blocking (due to reduced transcytosis and enhanced Tg degradation) was rescued by low-horm-rTg, suggesting that megalin is required for effective Hormone Release. This finding was confirmed in a small number of megalin-deficient mice, which had serological features resembling mild hypoThyroidism. Reduced Hormone formation within Tg in vivo, due to treatment of rats with aminotriazole or of patients with Graves' disease with methimazole, resulted in increased Tg transcytosis via megalin, in confirmation of results with FRTL-5 cells. Our study points to a major role of megalin in Thyroid homeostasis with possible implications in Thyroid diseases.
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role of megalin gp330 in transcytosis of thyroglobulin by Thyroid cells a novel function in the control of Thyroid Hormone Release
Journal of Biological Chemistry, 2000Co-Authors: Michele Marino, Aldo Pinchera, Luca Chiovato, David Andrews, Dennis Brown, Gang Zheng, Robert T. MccluskeyAbstract:Abstract When thyroglobulin (Tg) is endocytosed by thyrocytes and transported to lysosomes, Thyroid Hormones (T4 and T3) are Released. However, some internalized Tg is transcytosed intact into the bloodstream, thereby avoiding proteolytic cleavage. Here we show that megalin (gp330), a Tg receptor on Thyroid cells, plays a role in Tg transcytosis. Following incubation with exogenous rat Tg at 37 °C, Fisher rat Thyroid (FRTL-5) cells, a differentiated Thyroid cell line, Released T3 into the medium. However, when cells were incubated with Tg plus either of two megalin competitors, T3 Release was increased, suggesting that Tg internalized by megalin bypassed the lysosomal pathway, possibly with Release of undegraded Tg from cells. To assess this possibility, we performed experiments in which FRTL-5 cells were incubated with either unlabeled or 125I-labeled Tg at 37 °C to allow internalization, treated with heparin to remove cell surface-bound Tg, and further incubated at 37 °C to allow Tg Release. Intact 330-kDa Tg was Released into the medium, and the amount Released was markedly reduced by megalin competitors. To investigate whether Tg Release resulted from transcytosis, we studied FRTL-5 cells cultured as polarized layers with tight junctions on permeable filters in the upper chamber of dual chambered devices. Following the addition of Tg to the upper chamber and incubation at 37 °C, intact 330-kDa Tg was found in fluids collected from the lower chamber. The amount recovered was markedly reduced by megalin competitors, indicating that megalin mediates Tg transcytosis. We also studied Tg transcytosis in vivo, using a rat model of goiter induced by aminotriazole, in which increased Release of thyrotropin induces massive colloid endocytosis. This was associated with increased megalin expression on thyrocytes and increased serum Tg levels, with reduced serum T3 levels, supporting the conclusion that megalin mediates Tg transcytosis. Tg transcytosis is a novel function of megalin, which usually transports ligands to lysosomes. Megalin-mediated transcytosis may regulate the extent of Thyroid Hormone Release.
Kazuko Yamazaki - One of the best experts on this subject based on the ideXlab platform.
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Genes regulated by thyrotropin and iodide in cultured human Thyroid follicles: analysis by cDNA microarray.
Thyroid, 2003Co-Authors: Kazuko Yamazaki, Emiko Yamada, Yoshio Kanaji, Tetsuo Yanagisawa, Yoshiyuki Kato, Kazue Takano, Takao Obara, Kanji SatoAbstract:Thyrotropin (TSH) regulates a number of genes in thyrocytes, leading to iodide uptake, de novo synthesis and Release of Thyroid Hormones, and cell proliferation, accompanied by increased blood flow. At higher doses of iodide, however, the TSH-induced increases in Thyroid Hormone Release and blood flow are downregulated, and high iodide intake occasionally worsens autoimmune Thyroiditis. To elucidate the genes involved in such effects, we cultured human thyrocytes and examined genes modulated by TSH and iodide, using a cDNA microarray study, which can analyze 2400 genes in each run. When Thyroid follicles were cultured with TSH for 2 days, more than 100 genes were upregulated. These genes included those for enzymes involved in carbohydrate and lipid metabolism, adenylate and guanylate cyclases, and enzyme involved in cell proliferation. When Thyroid follicles were cultured with high iodide concentrations (10-5 M) for 24 hours, more than 100 genes were upregulated. Interesting genes were interleukin-8, IFP5...
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potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125i incorporation and de novo synthesized Thyroid Hormone Release in human Thyroid follicles
The Journal of Clinical Endocrinology and Metabolism, 1995Co-Authors: Kazuko Yamazaki, Kazuo Shizume, Takao Obara, Kanji Sato, Y Kanaji, Yukio Ito, T Nakagawa, T Koizumi, R NishimuraAbstract:Using a highly sensitive bioassay for TSH, in which human Thyroid follicles incorporate 125I and Release de novo synthesized Thyroid Hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in Thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than purified hCG. Asialo-hCG purified from a patient with choriocarcinoma had very potent TSH-like activity (468 microU hTSH/mg). Submaximal doses of bTSH and hCG variants produced additive stimulation of Thyroid function. Furthermore, the thyrotropic effect of hCG was inhibited by anti-TSH receptor antibody obtained from patients with myxedema. These in vitro findings suggest that although hCG is reported to exert potent cAMP-stimulating activity on rat Thyroid-like cells (FRTL-5) and Chinese hamster ovary cells transfected with hTSH receptor complementary DNA (0.092-0.72 microU hTSH/U hCG), the thyrotropic activity induced by authentic hCG in human Thyroid follicles is too weak to cause hyperThyroidism in normal pregnancy. However, hCG produced by some trophoblastic tumors, particularly asialo-hCG, has potent thyrotropic activity sufficient to cause clinically overt hyperThyroidism when produced excessively.
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Potent thyrotropic activity of human chorionic gonadotropin variants in terms of 125I incorporation and de novo synthesized Thyroid Hormone Release in human Thyroid follicles.
The Journal of Clinical Endocrinology & Metabolism, 1995Co-Authors: Kazuko Yamazaki, Kazuo Shizume, Takao Obara, Kanji Sato, Y Kanaji, Yukio Ito, T Nakagawa, T Koizumi, R NishimuraAbstract:Using a highly sensitive bioassay for TSH, in which human Thyroid follicles incorporate 125I and Release de novo synthesized Thyroid Hormone into the culture medium, the thyrotropic activities of various hCG preparations were studied. Under the culture conditions employed, bovine TSH (bTSH) was approximately 6- to 9-fold more active than human TSH (hTSH). Highly purified hCG prepared from urine of normal pregnant women (CR 127) had only a trivial thyrotropic activity equipotent to 0.00022 microU bTSH/U hCG or 0.0013 microU hTSH/U hCG (19.7 microU hTSH/mg hCG). Hybrid hCG (AB1ER) also elicited low thyrotropic activity (14.0 microU hTSH/mg), whereas crude hCG had moderate thyrotropic activity (0.041 hTSH microU/U hCG or 127 microU/mg protein). Deglycosylated hCG, a very weak LH/hCG receptor agonist, was the most potent agonist in Thyroid follicles (588 microU hTSH/mg protein). hCGs purified from urine of patients with trophoblastic tumors had greater TSH-like activity (37-84 microU hTSH/mg protein) than pur...
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Reversible inhibition by interferons alpha and beta of 125I incorporation and Thyroid Hormone Release by human Thyroid follicles in vitro.
The Journal of Clinical Endocrinology & Metabolism, 1993Co-Authors: Kazuko Yamazaki, Kazuo Shizume, Takao Obara, Y Kanaji, Y. Yamakawa, Hiroshi Demura, Kanji SatoAbstract:When interferons (IFN-alpha-2a, IFN-alpha-2b, natural IFN-alpha and IFN-beta) were cultured with human Thyroid follicles, each IFN inhibited TSH-induced Thyroid function (125I incorporation and Release of 125I-T4) in a concentration-dependent manner. The minimal inhibitory effect was exerted at 1-10 U/ml. However, the inhibitory effect was reversible, and no inhibitory effect was detected when IFNs were removed from the medium within 12 h. These in vitro findings suggest that each IFN directly inhibits human Thyroid function at clinically attainable levels. However, since the inhibitory effect was reversible, IFN therapy would provoke only subtle Thyroid dysfunction in a majority of patients without preexisting Thyroid autoimmunity.