Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells

hCG stimulates thyroid function, but it has been suggested that it is impurities in commercial hCG preparations or a variant of hCG that are responsible for the thyrotropic activity. In this study, we tested the thyrotropic activity of purified and commercial hCG and compared its action with that of bovine TSH (bTSH) in cultured rat FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and synthesis of DNA. Iodide uptake was measured after incubation of the cells for 48-72 h with the test hormones, followed by a 40-min incubation with 0.1 microCi Na125I and 10 mumol/L carrier NaI; the 125I in the washed cells was counted. Adenylate cyclase was measured after incubation of the cells with the test stimulators for 3 h in hypotonic medium by RIA of cAMP in the medium. DNA synthesis was measured after incubation of the cells with the test substances for 24 h, followed by addition of [3H]thymidine for 3 h and then measuring the incorporation of [3H]thymidine into the cells. Both purified and commercial hCG produced a dose-related increase in iodide uptake. The relative potency of commercial hCG was 0.024 microU bTSH/U hCG and that of purified hCG was 0.042 microU bTSH/U hCG; compared with human TSH, the potency of purified hCG was 0.72 microU/U hCG. hCG caused a dose-related increment of adenylate cyclase and [3H]thymidine incorporation. The effect of hCG on iodide uptake and [3H]thymidine incorporation was additive with that of bTSH; hCG was not an antagonist of TSH in these cultured rat thyroid cells. We conclude that hCG has intrinsic thyrotropic activity in FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and stimulation of DNA synthesis.     

Effects of retinoids on iodine metabolism, thyroid peroxidase gene expression, and deoxyribonucleic acid synthesis in porcine thyroid cells in culture.

Effects of retinoids on DNA synthesis, iodine metabolism, and thyroid peroxidase messenger RNA levels were studied in cultured porcine thyroid cells. Retinol (10(-8)-10(-5) M) alone did not affect DNA synthesis but potentiated that induced by epidermal growth factor or insulin-like growth factor-I without changes in the number or affinity of receptors for the growth factors, suggesting that retinol stimulates postreceptor events responsible for DNA synthesis. Retinol was an inhibitor of TSH-stimulated iodine metabolism. Iodide uptake and release of organified iodine stimulated by TSH or forskolin were inhibited dose dependently by treatment with retinol. The inhibition was detected at 10(-8) M and was approximately 50% at 10(-6) M. The potency of retinoic acid was comparable to that of retinol. The inhibitory effect of retinol was detected after treatments of thyroid cells for 24 h, and the maximal effect occurred after 48 h incubation. The cAMP accumulation in cultures treated with TSH plus retinol was lower than that of control cultures treated with TSH alone. However, iodide uptake stimulated by 8-bromo-cAMP was also inhibited by retinoids. Retinol reduced TSH- or 8-bromo-cAMP-stimulated gene expression of thyroid peroxidase. Thus, the data suggest that retinoids inhibit TSH-stimulated iodine metabolism by reducing cAMP accumulation and also by acting on the steps subsequent to cAMP production.     

Organic iodine inhibits deoxyribonucleic acid synthesis and growth in FRTL-5 thyroid cells

FRTL-5 thyroid epithelial cells in culture were used to study the possible inhibitory effects of iodine on thyroid growth. NaI exerted a dose-dependent, thyroid epithelial cell-specific inhibitory effect on [methyl-3H]thymidine incorporation into DNA, reduced the DNA content in the cell layer, and limited the increase in cell number mediated by TSH. The inhibitory effects of sodium iodide applied to growth stimulated by TSH-, cAMP-, and non-cAMP-dependent mechanisms and were prevented by 1-methylimidazole-2-thiol (methimazole) and 2-ethylthioisonicotinamide (ethionamide). The latter findings indicate that the inhibitory effects of NaI are mediated by some iodine-containing organic compound. The inhibitory effects of organic iodine on growth subsided 24-48 h after removal of excess NaI from the culture medium. In contrast, NaI had no effect on normal rat kidney fibroblast or thyroid fibroblast [methyl-3H]thymidine incorporation stimulated by epidermal growth factor or serum. These data demonstrate a specific inhibitory effect of organic iodine on thyroid epithelial cell growth.     

Thyroid hormone synthesis in thyroglobulin secreted by porcine thyroid cells cultured on porous bottom chambers. Effect of iodide.

The long-term iodination of thyroglobulin secreted into the apical medium of thyroid cells cultured as monolayers on porous bottom chambers reached 5.87 +/- 1.66 atoms of iodine/mol thyroglobulin after 11 days incubation in the presence of TSH (0.1 mU/ml) and iodide (0.5 microM) in the basal medium. This iodinated thyroglobulin contained thyroid hormones (T3 + T4) which involved 22.7% of the thyroglobulin iodine content. The iodoamino acid content was, in residues per mole, 2.2 +/- 0.35 for monoiodotyrosine, 0.74 +/- 0.04 for diiodotyrosine, 0.23 +/- 0.04 for T4, and 0.098 +/- 0.02 for T3. Kinetic studies showed that a minimal level of iodination (2.05 +/- 0.26 atoms iodine/mol thyroglobulin) was necessary for hormonogenesis. A maximal level of iodination and hormonogenesis was obtained with 0.5 microM iodide added daily to the basal medium. In these conditions, hormonogenesis efficiency reached about 40% (a value close to this one observed in vivo). Above 0.5 microM iodide, both iodination and T4 synthesis were inhibited (28.3% and 73.9%, respectively, for 1 microM iodide). Our culture system makes it possible to demonstrate that this high iodide concentration in the basal medium did not increase apical iodide concentration above 10 microM but decreased apical thyroglobulin concentration. The inhibitory effect of iodide on hormonogenesis cannot be due to a competition with tyrosine residues of thyroglobulin for their binding to thyroperoxidase although it could be related, at least in part, to a decrease in protein synthesis.     

Regulation of deoxyribonucleic acid synthesis in cultured porcine granulosa cells by growth factors and hormones

To assess the potential role of hormones and growth factors in ovarian follicular growth, we have developed a simple method for the evaluation of DNA synthesis in cultured porcine granulosa cells. Optimal conditions were found to entail newly established cultures that were growth arrested by serum deprivation and then treated with growth-promoting agents. Such treatment resulted in an easily quantitated 3- to 30-fold increase in [3H]thymidine incorporation into trichloroacetic acid-precipitable macromolecules. The radioactivity incorporated was localized to the DNA band on cesium chloride gradients and showed excellent correlation with labeling indices. Insulin, multiplication-stimulating activity, epidermal growth factor (EGF), platelet-derived growth factor, and ovarian follicular fluid were potent stimulators of DNA synthesis in this assay. While EGF and insulin effects were additive, indicating discrete modes of action, the effects of insulin and multiplication-stimulating activity were not additive at maximally effective concentrations. In contrast to the effects of these stimulatory substances, BSA and porcine relaxin were devoid of mitogenic activity under these circumstances. The major trophic hormones implicated in follicular growth, FSH, LH, and estradiol, as well as the cAMP analog 8-bromo-cAMP were without mitogenic effects. Instead, treatment with LH, 8-bromo-cAMP, and the combination of estradiol and FSH resulted in a distinct decrease in DNA synthesis. These data confirm and extend previous evidence of the mitogenic action of insulin-like peptides and EGF, and suggest that the hormones generally believed to regulate granulosa cell replication in vivo lack direct mitogenic effects in vitro.     

Iodine suppression of iodide uptake in FRTL-5 thyroid cells

Exposure of FRTL-5 cells to iodide (I-) in excess of 3 microM suppresses the concentrative uptake of I-. The depression of I- uptake measured at the steady state is due to decrease in the rate of I- influx and not to an effect on I- efflux. Exposure to NaI is associated with decreased T4 secretion and also depressed Na+-dependent amino acid accumulation. The depression in I- and amino acid transports increases proportionately with the duration of exposure and concentration of I- used but is not associated with alterations in FRTL-5 cell cAMP levels. The I- suppression effect is blocked, however, when methimazole is present during the incubation with NaI. In agreement with studies in vivo, I- suppression in FRTL-5 cells appears to depend on an intermediate in the organification process and to be independent of a TSH-induced cAMP-mediated action.     

Hydrogen peroxide inhibits iodide uptake and iodine organification in cultured porcine thyroid follicles.

We investigated the effect of hydrogen peroxide on the process of thyroid hormone formation in a physiologic culture system of porcine thyroid follicles that we recently established. Porcine thyroid follicles cultured in medium containing 1 mU/mL TSH were exposed to 0 to 500 microM hydrogen peroxide in the presence of 0.1 microCi carrier-free Na125 and sodium iodide for 2 h. Iodide uptake and iodine organification were measured in this incubation system. The kinetics of iodide uptake were used to explain the action of hydrogen peroxide. In addition, cAMP content and Na+,K(+)-ATPase activity (an enzyme necessary for iodide uptake) were measured to investigate the mechanism of hydrogen peroxide action. Hydrogen peroxide at concentrations of 100, 200, and 500 microM inhibited iodide uptake in a dose-dependent manner. Iodide organification was inhibited only when the concentration of hydrogen peroxide was greater than 200 microM. The kinetics of iodide uptake indicated that hydrogen peroxide was a noncompetitive inhibitor with iodide. Inhibition of iodide uptake and iodine organification by hydrogen peroxide were not mediated by alteration of cAMP content of Na+,K(+)-ATPase activity, since exposure to even 500 microM hydrogen peroxide did not change these parameters in the follicle when compared with those of control samples. Our results suggest that the iodide transport system in the thyroid follicle is inhibited at 200 microM hydrogen peroxide or greater.     

Inhibition of N-glycan processing affects iodide organification in porcine thyroid cells

The N-glycan-processing inhibitors swainsonine (Sw) and deoxymannojirimycin (dMM) were used to study the influence of N-glycans on iodide organification in cultured porcine thyroid cells. Incubations with [125I]NaI were followed by determination of labeled trichloroacetic acid-insoluble material in culture media, follicular contents and cells. In controls, most of this material was in the follicular contents. With Sw and dMM, total acid-insoluble material was less than 10% of control. Iodide uptake was slightly inhibited and hydrogen peroxide release was not affected by inhibitors. Cell-surface thyroid peroxidase (TPO) activity, assayed by its ability to iodinate bovine serum albumin, was strongly inhibited. Pronase glycopeptide analysis indicated that with drugs the content in complex-type N-glycans was strongly decreased while that in hybrid or oligomannosidic type was increased. In conclusion, inhibition of N-glycan processing prevents iodide organification in cultured porcine thyroid cells by decreasing the recovery of cell-surface TPO activity.     

Effect of increased iodide intake on thyroid function in subjects on chronic lithium therapy

Thyroid function tests were obtained in 10 patients on chronic lithium therapy before and after the administration of potassium iodide 250 mg q.i.d. Mean serum TSH rose by 8.9 muU/ml and mean serum T3 rose from 70 to 101 ng/dl. Two patients became hypothyroid; a third showed a rise in TSH without any change in T3 or T4. A fourth patient developed hyperthyroidism probably secondary to the Jod-Basedow phenomenon. Pharmacologic doses of iodine should be administered with caution to patients on chronic lithium therapy.     

Suppression of iodide uptake and thyroid hormone synthesis with stimulation of the type I interferon system by double-stranded ribonucleic acid in cultured human thyroid follicles

Although viral infection is thought to be associated with subacute thyroiditis and probably with autoimmune thyroid disease, possible changes in thyroid function during the prodromal period of infection or subclinical infection remain largely unknown. Recently, it was shown that pathogen-associated molecular patterns stimulate Toll-like receptors (TLR) and activate innate immune responses by producing type I interferons (IFN). Using a human thyroid follicle culture system, in which de novo synthesized thyroid hormones are released into the culture medium under physiological concentrations of human TSH, we studied the effects of polyinosinic-polycytidylic acid [Poly(I:C)], a chemical analog of viral double-stranded RNA (dsRNA), on TSH-induced thyroid function. Thyrocytes expressed ligands for dsRNA (TLR 3, CD14, and retinoic-acid-inducible protein-1) comparable with the TSH receptor. DNA microarray and real-time PCR analyses revealed that dsRNA increased the expression of mRNA for TLR3, IFN-beta, IFN-regulating factors, proinflammatory cytokines, and class I major histocompatibility complex (MHC), whereas genes associated with thyroid hormonogenesis (sodium/iodide symporter, peroxidase, deiodinases) were suppressed. In accordance to these data, Poly(I:C) suppressed TSH-induced 125I uptake and hormone synthesis dose dependently, accompanied by a decrease in the ratio of 125I-T3/125I-T4 released into the culture medium, whereas peptidoglycan, lipopolysaccharides, or unmethylated CpG DNA, ligands for TLR2, TLR4, and TLR9, respectively, had no significant effect. These inhibitory effects of Poly(I:C) were not blocked by a neutralizing antibody against TLR3 and an anti-IFN alpha/beta receptor antibody. These in vitro findings suggest that when thyrocytes are infected with certain viruses, dsRNA formed intracellularly in thyrocytes may be a cause for thyroid dysfunction, leading to development of autoimmune thyroiditis.     

Carbamazepine (Tegretol) inhibits in vivo iodide uptake and hormone synthesis in rat thyroid glands

Decreased serum concentrations of T3 and T4 occur in patients treated with the anticonvulsant drug carbamazepine (CBZ), but with rare exception, these patients remain euthyroid. The mechanism that accounts for diminished hormone levels is unknown, and our objective was to study the direct effect of CBZ on iodide uptake and hormone synthesis in thyroid glands of CBZ-treated and pair-fed control rats. Chronic ingestion (per os) of CBZ in male rats reduced the four hour thyroid 131I-iodide uptake by approximately 60%. This inhibition occurred after the animals had received sufficient CBZ to attain plasma CBZ concentrations of 0.8 microgram/ml. Continued treatment with CBZ ranging from 560 to 800 mg/kg/day for 14 days did not result in further inhibition of iodide uptake even though the plasma CBZ concentrations had increased 6-20 fold. No inhibition of iodide uptake was apparent when the animals initially received CBZ ranging from 40 to 152 mg/kg body weight for 22 days when there were no detectable levels of plasma CBZ. Overall growth rates of CBZ-treated rats were slightly (6-10%) less than the pair-fed control animals. Plasma T4 concentrations were reduced by 18% (p less than 0.05) in the CBZ-fed animals, while T3 concentrations were diminished by 53% (p less than 0.01). CBZ appeared to alter thyroidal iodide transport because the thyroid:plasma iodide ratios were decreased by 26% in the drug-treated rats. The distribution of radioiodine in thyroidal iodoamino acids was essentially the same in both groups of rats but the absolute quantities of radioiodine were more than 2.5 times greater in the control rats. CBZ failed to inhibit peroxidase-catalyzed iodide and guaiacol oxidation in vitro. No differences in thyroid gland morphology were noted in the two groups of animals, but weights of the glands from the CBZ-fed animals increased by 25% after 42 days. It is concluded that CBZ, or its metabolites, has a direct inhibitory effect on iodide utilization and hormone synthesis by the thyroid gland.     

Inhibition of cyclic AMP formation by iodide in suspension cultures of porcine thyroid follicle cells

In the present study porcine thyroid cells in suspension cultures were employed to investigate the suppressive effect of iodide on adenylate cyclase under basal conditions and following incubation with TSH, PGE1, cholera toxin and forskolin. Within 30 min of incubation with iodide (half-maximal effect 10(-5) M), inhibition was established and remained unchanged up to 40 h of culture. The inhibitory action was abolished by methimazole. TSH, PGE1, cholera toxin and forskolin stimulated cAMP accumulation 10-, 3-, 24- and 22-fold, respectively. Iodide pretreatment reduced basal cAMP levels and also made the cells less sensitive to stimulation by the various agents. High concentrations of TSH or PGE1 could not overcome the suppressive influence of iodide, whereas with high concentrations of cholera toxin and forskolin the reduction in cAMP levels in iodide-treated cultures was less pronounced. Membranes isolated from iodide-treated cultures produced significantly lower amounts of cAMP compared to control membranes. Furthermore, iodide did not inhibit basal or forskolin-stimulated cAMP production in human fibroblasts. The results demonstrate that iodide via an iodination-dependent mechanism influences cAMP generation in thyroid cells. It is suggested that the inhibitory activity, which has a long half-life, involves stable modification of the membrane-localized catalytic unit of adenylate cyclase such that its activation by the regulatory unit is rendered less efficient.     

Variations in the culture medium for FRTL5 cells: effects on growth and iodide uptake

We evaluated the composition of the medium in which FRTL5 cells were incubated for effects on basal and stimulated growth and differentiated function. Variables included the concentration of calf serum (CS) from 0% (0.2% BSA) to 5% and the addition of insulin or insulin-like growth factor-I (IGF-I); stimulation was by TSH, with some experiments using thyroid-stimulating antibody-immunoglobulin G. The basal rate of incorporation of [3H]thymidine and the content of DNA per well were enhanced progressively by the increasing concentration of CS, whether in a pretreatment period of 3 days or over a subsequent 2 days of incubation. Concomitantly, I- uptake was progressively inhibited. Stimulation of growth by TSH required serum and was greatest with 5% CS; TSH effect on I- uptake was progressively decreased by increments of CS. Insulin and IGF-I had effects comparable to each other, but much smaller concentrations of IGF-I were required; they both augmented growth and enhanced growth stimulation by TSH. These actions were progressively annulled by increasing concentrations of CS. I- uptake (basal and stimulated) was inhibited by insulin and IGF-I, but this inhibitory effect was abolished by CS, particularly at 5% concentrations. Overall, the influences of varying the concentrations of CS and the effects of insulin or IGF-I were consistent; if there was enhancement of growth stimulation, there was inhibition of the TSH effect on I- uptake. Underlying mechanisms for these observations have yet to be elucidated.     

维甲酸对甲状腺癌细胞钠/碘转运体基因的调节作用

以RTPCR法检测维甲酸对甲状腺癌细胞钠/碘转运体(NIS)基因表达的影响,发现在5～50μmol/L的剂量区间,维甲酸能促进NIS基因表达,提示维甲酸在甲状腺癌及其转移灶的治疗中可能起到重要作用。     

Ultrastructural morphology and evidence of iodide uptake and protein synthesis in normal human thyroid transplants in nude mice

Normal human thyroid tissue was xenotransplanted to athymic mice. Well preserved structural properties of the transplants were observed at both light and electron microscopical level. Analysis of the soluble proteins in the transplants by polyacrylamide gel electrophoresis showed that the major fractions were the 19S thyroglobulin, the 27S iodoprotein and a larger protein fraction. Protein synthesizing capacity of the transplants, as measured by in vivo 3H-leucine incorporation into the soluble transplants proteins was demonstrated. The observed 3H-leucine incorporation was in the same magnitude as that found in the original donor tissue at the time of transplantation. Four weeks after transplantation a significant 125I-iodide uptake (2.0%) was observed in the transplants. Corresponding value for the host thyroids was 6.6%. The biological half-time of the accumulated 125I-iodide in the transplants was reduced from 97 days to 38 days between 2 and 4 weeks after transplantation. The biological half-time in the host thyroid increased from 27 days to 32 days during the same period. This investigation shows that normal human thyroid tissue can be transplanted to nude mice with preserved tissue organisation and maintained cellular function.