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  • Author: Osamu Isozaki x
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Osamu Isozaki, Pilar Santisteban, John Chan, Evelyn Grollman and Leonard Kohn

Thyroglobulin (Tg) is the major protein synthesized by the thyroid cell and is the macromolecular precursor of thyroid hormones. Thyrotropin (TSH) has been shown to increase Tg synthesis approximately 2-fold both in vivo (Van Heuverswyn et al. 1984, 1985) and in rat FRTL-5 thyroid cells (Ambesi-Impiombato et al. 1982; Avvedimento et al. 1984) a continuous functioning line whose growth is TSH dependent but requires medium containing insulin and insulin-like growth factor-I (IGF-I) (Ambesi-Impiombato et al. 1982). In evaluating the role of insulin and IGF-I, we found that 1) they can induce the synthesis of Tg in the absence of TSH and cell growth, 2) their action was at the level of Tg mRNA transcription, and 3) their action on Tg synthesis was quantitatively greater than and additive to the effect of TSH.

The present report, which summarizes these observations, adds a new dimension to our understanding of the hormonal control

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John Chan, Pilar Santisteban, Michele De Luca, Osamu Isozaki, Evelyn Grollman and Leonard Kohn

Abstract. When solubilized, radiolabelled membrane preparations from FRTL-5 rat thyroid cells are applied to TSH affinity columns, two separate peaks of protein can be eluted by high salts/high pH and low pH buffers, respectively. Immunoprecipitation with monoclonal antibodies to the TSH receptor shows that both peaks contain proteins related to the TSH receptor. If extracts were from cells grown without TSH, one peak has a ~ 300 K and the other a ~ 70 K protein the 70 K protein can be derived from the purified 300 K protein in vitro. A 50 and 20 K protein can be derived from the 70 K protein. If extracts are from cells grown with TSH, the peaks contain a multiplicity of additional immunoprecipitable bands of ~ 200, 175, 130, 90, 50, 20 K etc. These bands are shown to result from the ability of TSH to increase the synthesis (3–4-fold) and degradation (2–3-fold) of the 300 and 70 K proteins. The 300/70 K protein fractions are reactive with monoclonal autoimmune thyroid stimulating antibodies and contain a specific disialo ganglioside. The ganglioside migrates near GM2, i.e., like a lower order ganglioside, and contains fucose. In translation experiments, the monoclonal antibodies to the TSH receptor identify a single mRNA component which produces a protein of ~ 220 K. This protein is not present in thyroid cells which have no functional TSH receptor and which cannot be surface labelled with monoclonal antibodies to the TSH receptor. The data thus indicate that the multiplicity of TSH binding proteins demonstrated in many labs may be breakdown products of a receptor which is synthesized by a single message but has both 330 and 70 K forms and is tightly complexed with a specific thyroid ganglioside. The 70 K form is composed of ~ 50 and ~ 20 K fragments seen in TSH cross-linking studies.

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Kanji Sato, Hisako Mimura, Shuichi Kato, Osamu Isozaki, Toshio Tsushima and Kazuo Shizume

Abstract. The serum levels of propylthiouracil (PTU) were determined by radioimmunoassay in 10 normal subjects and in 11 patients with Graves' disease after a single 100 or 200 mg oral dose of PTU. The serum half-life of PTU in the normal subjects and in hyperthyroid patients with uneventful clinical course was 75 ± 19 min (mean ± sd, n = 6) and 73 ± 13 min (n = 7), respectively. Maximum serum PTU concentrations were usually attained within 1 h after a single 200 mg oral dose and at 1 h were 5.3 ± 1.4 μg/ml (3.1 ± 0.82 × 10−5 m) in normal subjects (n=6) and 4.8 ± 2.4 μg/ml (2.8 ± 1.4 × 10−5 m) in hyperthyroid patients (n = 7). These betweengroup differences were not significant. Serum PTU concentrations were low in a pregnant hyperthyroid patient with a weak response to PTU treatment. In another patient, who appeared resistant to PTU therapy, the serum PTU level increased as expected at testing, and it was later confirmed that, during treatment, he had not taken the drug as prescribed. In a patient who developed agranulocytosis due to methimazole and subsequently fever due to PTU, the half-life of PTU was prolonged to about 130 min.

These findings suggest that monitoring the serum PTU levels in patients with Graves' disease can be of clinical value in patients who do not respond to treatment. Furthermore, it may provide some clues as to the mechanism by which toxic reaction develops.

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Osamu Isozaki, Toshio Tsushima, Kanji Sato, Motoyasu Saji, Yoshito Ohba, Naoya Emoto, Yuji Sato and Kauzo Shizume

Abstract. A 54 year old man with markedly elevated serum T3, but without an apparent thyroid disease, was found to have a specific antibody to T3. His serum thyroxine, TBG and TSH were in normal range, but T3-RSU was markedly low. Antibodies to thyroglobulin and microsome were negative. He was judged euthyroid because of a normal basal metabolic rate and a normal thyroidal 123I uptake which was suppressed by T3 adminnistration. When serum was extracted with ethanol prior to assay, serum T3 was found to be in the upper border of normal range. Several experiments revealed the presence of an antibody to T3 in his serum with an affinity constant of 3.3 × 109 m −1. The binding capacity of the antibody was 7.6 ng/mg of IgG. The binding of [125I]T3 was almost specific to T3, and potencies of T4 and fT3 in displacing [125I]T3 binding were only 1.0 and 0.3%, respectively, of that of T3. The antibody contained both kappa and lambda chains and was therefore polyclonal.

The T3 metabolic clearance rate, which was determined by disappearance of injected [125I]T3 from serum, was lower in this patient (7.44 I/day) than in normal. The T3-production rate was decreased to 14.9 μg/day, and serum free T3 concentration as well as urinary T3 excretion rate were also reduced. Since both serum total and free T4 concentrations were normal, the supply of T4 to peripheral tissues would be sufficient to keep this patient in a euthyroid state in spite of the anti-T3 antibody.

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Evelyn F. Grollman, Elisabeth Bone, John Chan, Daniella Corda, Osamu Isozaki, Claudio Marcocci, Pilar Santisteban and Leonard D. Kohn

The primary role of the thyroid cell is to synthesize and secrete the thyroid hormones, T3 and T4 (Dumont et al. 1971; Robbins et al. 1980). This must be accomplished in a regulated manner and despite a variable intake of iodine. This role must also be accomplished despite the fact that the synthesis and secretion of thyroid hormones involves numerous steps. These include 1) the uptake of iodide across the basal membrane of the cell in a scavanging and concentrative fashion; 2) efflux of iodide across the apical membrane into the follicular lumen of the thyroid follicle; 3) the synthesis of thyroglobulin and its vectorial transport to the site of iodide efflux; 4) iodination of thyroglobulin; 5) regulated storage of the iodinated thyroglobulin; 6) lysosomal targeting and biodegradation, of the iodinated thyroglobulin; and 7) ultimately, secretion of T3/T4 into the bloodstream, across the basal membrane

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Mariko Arai, Toshio Tsushima, Osamu Isozaki, Hiroshi Demura, Kazuo Shizume, Naoya Emoto, Megumi Miyakawa, Yasuko Nozoe, Hitomi Murakami and Eiji Ohmura

Arai M, Tsushima T, Isozaki 0, Demura H, Shizume K, Emoto N, Miyakawa M, Nozoe Y, Murakami H, Ohmura E. Effects of transforming growth factor α (TGF-α) on DNA synthesis and thyrotropin-induced iodine metabolism in cultured porcine thyroid cells. Eur J Endocrinol 1995;132:242–8. ISSN 0804–4643

Transforming growth factor α (TGF-α) is a potent mitogen that is similar structurally to epidermal growth factor (EGF). As EGF is a potent growth stimulator and an inhibitor of iodine metabolism in cultured thyroid cells of several species, we studied whether TGF-α has similar effects using porcine thyroid cells in culture. Recombinant human TGF-α dose-dependently stimulated DNA synthesis of thyroid cells, with maximal stimulation (eight- to ninefold above basal) occurring at 2 nmol/l. The potency was approximately 50% that of mouse EGF and correlated with the ability to compete with EGF for receptor binding, suggesting that the action of TGF-α is mediated by interaction with EGF receptors. When thyroid cells were cultured for 3 days with thyrotropin (TSH) in the presence of TGF-α, TSH-induced iodide uptake was inhibited in a dose-dependent manner. The potency of TGF-α again was approximately 50% that of EGF. Transforming growth factor α did not inhibit TSH-stimulated cAMP production. Moreover, iodide uptake stimulated by either forskolin or 8-bromo-cAMP also was inhibited by TGF-α. Thus, we conclude that TGF-α inhibits TSH-induced iodine metabolism largely by acting at the steps distal to cAMP production. Northern blot analysis revealed expression of TGF-α mRNA in porcine thyroid cells. These observations suggest that TGF-α acts as an autocrine modulator of growth and differentiated functions in porcine thyroid cells.

T Tsushima, Department of Medicine 2, Tokyo Women's Medical College, Kawadacho 8–1, Shinjukuku, Tokyo 162, Japan

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Motoyasu Saji, Osamu Isozaki, Toshio Tsushima, Mariko Arai, Megumi Miyakawa, Yoshito Ohba, Yumi Tsuchiya, Tomohide Sano and Kazuo Shizume

Abstract. The effect of iodide on growth of rat thyroid cells (FRTL-5) was studied. TSH-stimulated cell growth was inhibited by iodide in a concentration-dependent manner, and an effect of iodide was detected at 10−6 mol/l. KClO4 or 1-methylimidazole-2-thiol blocked the effect of iodide, suggesting that iodide uptake and its organification are required to produce the inhibitory effect of iodide on cell growth. Iodide not only decreased TSH-stimulated cAMP production in FRTL-5 cells but also cell growth induced by cAMP. These observations suggest that iodide inhibits TSH-stimulated growth of the cells by attenuating cAMP production and also by acting on the step(s) distal to cAMP generation. The inhibitory effect of iodide was also seen in growth stimulated by insulin, insulin-like growth factor-I or 12-O-tetradecanoyl phorbol 13-acetate, suggesting multiple sites of action of iodide in the process of growth of FRTL-5 cells.