Aspects ultrastructuraux et synthèse de la thyroglobouline dans les cultures de thyroides avant et après transplantation chez des rats hypophysectomisés

Two aspects concerning the study of thyroglobulin synthesis and of the ultrastructure of thyroid glands in organotypic cultures have been studied: (1) the chronology of ultrastructural alterations in thyroid cultures leading to the loss of thyroglobulin synthesis; and (2) the role of TSH in the re-establishment of the characteristic features in the cultured cells following transplantation.Alterations of the endoplasmic reticulum start on the very first day of culture. The ribosomes detach themselves from the membranes which then disappear. On the third and fourth day, significant alterations are observed on mitochondria and nuclei. The lysosomes disappear, while the Golgi apparatus is hardly affected.All the above modifications are reversible, since it is possible to re-establish the characteristic features of the endoplasmic reticulum and the synthesis of thyroglobulin after transplantation of the cultured glands into normal animals.The reappearance of phenotypic traits of the thyroid cells can also be obtained after transplantation into hypophysectomized rats. However, the level of thyroglobulin synthesis in these transplants, which is similar to that in the host gland, is lower than in grafts into normal animals.This results shows that TSH is not required for differentiation and for manifestation of the phenotypic traits of the thyroid gland. It seems therefore that the role of TSH is not to stimulate differentiation, but to maintain and regulate the level of specific processes in the already differentiated thyroid cell.     

Hormonal regulation of some steps of thyroglobulin synthesis and secretion in bicameral cell culture

Porcine thyroid cells were cultured for 15 days on porous bottom chambers with or without different mixtures of hormones added to serum-free basal medium. Assays with 10% serum were also performed for comparison with previously published results. The effects of the hormones, particularly insulin, TSH and hydrocortisone, were studied on total RNA content, thyroglobulin mRNA level, the amount of thyroglobulin secreted into the apical medium and on glycosylation. Insulin and TSH similarly increased the total RNA content, and their effects were additive. Thyroglobulin mRNA content was increased twofold by insulin and threefold by TSH. When they were added simultaneously, the maximal level of thyroglobulin mRNA was reached, showing that TSH and insulin effects on thyroglobulin gene expression were additive. Hydrocortisone alone did not modify total RNA or thyroglobulin mRNA content but the hormone amplified total RNA when insulin and TSH were present together. The basal level of thyroglobulin secreted into the apical medium was increased threefold by insulin and fourfold by TSH. The effects of these two hormones added together appeared to be additive. Hydrocortisone had no effect alone or even when combined with insulin or TSH. However, when the three hormones were added together, the hormonal response was amplified. TSH effect and insulin effect on the incorporation of ³H-mannose into thyroglobulin as well as on the anionic residue content of the molecule were additive. © 1994 Wiley-Liss, Inc.     

Mechanism of corticotropin action on adrenal protein synthesis. The effects of inhibitors of protein synthesis and calcium chelators

We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E, in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [14C]- or [3H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 micrograms) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 micrograms cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 micrograms per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 micrograms per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 micrograms per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.     

In vitro and in vivo regulation of thyrotropin receptor mRNA levels in dog and human thyroid cells.

Regulation of thyrotropin (TSH) receptor (TSHr) mRNA accumulation as compared with two other thyroid differentiation markers (thyroglobulin and thyroperoxidase (TPO] has been investigated by Northern blot. In dogs in vivo, chronic stimulation of the thyroid TSHr mRNA although it increased the levels of thyroglobulin and TPO mRNA. In dogs treated with thyroxin, the quiescent thyroids expressed normal levels of TSHr and TPO mRNA but depressed levels of thyroglobulin mRNA. In primary cultures of dog thyrocytes, dedifferentiation of the cells by treatment with epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate led to decreased TSHr mRNA levels and nearly abolished thyroglobulin and TPO gene expression. However, TSHr mRNA was always present, compatible with the fact that these cells, when treated by TSH, reexpress differentiation. Treatment of the cells with TSH or forskolin transiently increased the TSHr mRNA level after 20 h, an effect inhibited by cycloheximide. This up-regulation was confirmed at the protein level: forskolin-treated cells showed an enhanced cAMP response to TSH and an increased binding of labeled TSH to their membranes. Long term TSH treatment led to a slight down-regulation of TSHr mRNA in dog thyrocytes, but in human thyroid cells no marked down-regulation was observed.     

Morphological and functional changes in several endocrine glands induced by hypothyroidism in the rat

The effect of hypothyroidism upon the morphology and the function of several endocrine glands was studied in radiothyroidectomized male rats. It was found that T3, T4, insulin, prolactin and corticosterone levels were significantly lower in hypothyroid rats. TSH levels were significantly higher in these animals while no changes were depicted in testosterone levels. The administration of T4 drew back to normal range the above-mentioned altered serum hormone levels. The studies performed with light microscopy revealed alterations only in the TSH secretory cells of the adenohypophysis. Conversely, when using the electron microscope to study the different endocrine glands, clear alterations were depicted in the TSH and prolactin secretory cells of the adenohypophysis, as well as in the pancreatic B cells and the cells of the zona fasciculata of the adrenal cortex. No abnormal changes were demonstrable at the level of the seminiferous tubules of the testis. All the above morphological changes were corrected by the administration of T4 to hypothyroid rats. These results suggest that the hypothyroid state is a complex hormonal dysfunction rather than a single hormonal defect. The secretory alterations are accompanied by fine cellular alterations in the corresponding glands.     

Preferential response of thyroid glycosyltransferases to changes in thyrotropin stimulation

The level of thyrotropin stimulation of rat thyroid was modified to permit a study of the regulation of some of the enzymes of this tissue. This was accomplished by the administration of either propylthiouracil to increase the endogenous thyrotropin levels or thyroxine to suppress production of the trophic hormone. The enzymes measured included two glycosyltransferases involved in the synthesis of the main secretory protein of the gland (thyroglobulin), two lysosomal enzymes which may contribute to its catabolism, as well as two other enzymes of a more general nature. In the propylthiouracil-treated animals changes in the activities of succinic dehydrogenase, protease, and 5′-nucleotidase corresponded to the increase in the weight of the gland and appeared to be nonspecific in nature; these three enzymes, moreover, showed no changes after thyroxine treatment. The level of the N-acetylglucosaminidase was significantly decreased, per gram of tissue, in both groups of treated animals. The only enzymes which appeared to be specifically affected by the modulation of thyrotropin stimulation were the glycosyltransferases, with both mannosyl- and galactosyltransferases showing an increase in the thyroids of the propylthiouracil-treated animals and a decrease in those treated with thyroxine. This suggests that post-translational steps, such as carbohydration, may play an important role in regulating the turnover of thyroglobulin and therefore influence the overall rate of thyroid hormone formation. The distribution of each of the enzymes between the soluble and particulate fractions of the tissue was also measured and it was noted that the glycosyltransferases, which showed the most marked increase in total activity as a result of thyrotropin stimulation, also showed a statistically significant increase in the percentage present in the particle-bound form.     

Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells

Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors.     

Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum

Protein synthesis in the G1 period of the cell cycle has been investigated using two-dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone. The analysis of the proteins, pulse-labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr approximately equal to 81 000) was maximal 9-12 h after stimulation by the proliferative agents but began to decrease at 15-18 h and was still decreased at 29-32 h. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (proteins 2-9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6') while TSH specifically decreased the labeling of four polypeptides (proteins 1'-4') and increased the labeling of one polypeptide (protein 5') whose synthesis was decreased by EGF and serum. The specific effect of TSH on one protein labeling (protein 7; Mr approximately equal to 39 000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a correlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence. In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of iodide administration on hog thyroid gland and the composition of thyroglobulin and 27-S iodoprotein.

The effect of excess iodide on hog thyroid gland has been examined with regard to the change in the chemical composition of thyroglobulin and in the accumulation of 27-S iodoprotein by the in vivo treatment of hogs with iodide for various lengths of time. The iodine content of thyroglobulin was either unchanged by short term administration of excess iodide, or somewhat lowered. However, the iodine content as well as the total amount of thyroglobulin increased in the glands enlarged by prolonged treatment with iodide. The iodine highest reached 1.17% of the protein on an average. On the other hand, 27-S iodoprotein decreased and finally disappeared after the chronic treatment. Monoiodotyrosine and diiodotyrosine increased in parallel with the increase in the iodine content (0.15 to 1.17%) caused by the iodide treatment, while thyroxine increased but reached a plateau at the level of three residues per mole of thyroglobulin, and no change was observed even in the proteins with the higher iodine content than 0.75%. Proteolytic activity measured by amino acid release from the thyroid protein was depressed by the chronic treatment. On the other hand, the amount of iodocompound released by the autoproteolysis, which may reflect hormone secretion, increased, possibly because of the marked increase in the iodine content of thyroglobulin.     

Changes in the hypothalamo-pituitary-adrenocortical and hypothalamo-pituitary-thyroid axes in diabetic rats acclimated to moderate hyperthermic environment

We examined the effect of acclimation to moderate hyperthermic environment on the ACTH, TSH, T₃, T₄ and corticosterone level, as well as the relative weight of hypophysis, thyroid and adrenal glands in streptozotocin-diabetic rats. Increased activity of the hypothalamo-pituitary-adrenocortical (HPA) axis has been demonstrated in diabetic animals, whereas insulin treatment restores the changes. Heat acclimation reduces the level of ACTH and corticosterone in control animals and moderates the hormonal disturbances caused by diabetes. Simultaneously, our study revealed impairment in the activity of the hypothalamo-pituitary-thyroid (HPT) axis. Acclimation to 35±1 °C resulted in significantly lower T₃ and T₄ levels in control, diabetic and insulin-treated animals. Relative weight of the hypophysis, thyroid and adrenal glands is decreased in heat-acclimated rats. Our assumption is that there might be a cross tolerance between diabetes and heat acclimation on a hormonal level.     

Hormonogenesis in thyroid cells cultured on porous bottom chambers

Different processes implied in thyroid hormonogenesis (thyroglobulin, thyroperoxidase and hydrogen peroxide generating system expressions) and their regulation by TSH and iodide have been studied using porcine thyroid cells cultured in porous bottomed chambers. This system allowed to reproduce the functional bipolarity. Cells form a tight and polarized monolayer. Both apical and basolateral poles of epithelial cells were independently accessible and the cell layer separated two compartments which can contain different media. A major polarized secretion of thyroglobulin into the apical compartment was observed; it was increased in the presence of TSH as well as the thyroglobulin synthesis and mRNA level. These TSH effects were the consequence of adenylcyclase stimulation. Active transport of iodide, iodination of thyroglobulin and hormonosynthesis took place only in the presence of TSH. These steps occurred at the apical pole of cells. In the culture chamber system, thyroglobulin was weakly iodinated (6 atoms of iodide per mole of thyroglobulin; in vivo up to 40 atoms per mole) but hormonogenesis efficiency was close to this one observed in vivo (40%). Iodide concentrations higher than 0.5 µM daily added to the basal medium inhibited iodination of thyroglobulin and hormonosynthesis. Some components contained in culture media were inhibitors for iodination when they were present in the apical medium such as vitamins, amino acids and phenol red. The culture system appears to be interesting for pharmacological and toxicological studies.     

Differential expression of thrombospondin,collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[¹²⁵l] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450–480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [¹³¹l]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450–480 resolved to Mr 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin l. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from Mrs 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factor 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium. © 1994 Wiley-Liss, Inc.     

Effect of thyrotropin on the phosphorylation of thyroid chromosomal proteins.

Thyrotropin (TSH) stimulated the phosphorylation of histone H1 in calf thyroid slices but had no effect on other classes of histones. Phosphorylation of total phenol-soluble nonhistone chromosomal proteins was not affected by incubation with TSH. However, when these phenol-soluble nonhistone chromosomal proteins were analysed by two-dimensional gels involving isoelectrofocusing and dodecyl sulfate-polyacrylamide gel electrophoresis, TSH was shown to stimulate the phosphorylation of two specific groups of phosphoproteins with molecular weights between 35,000 and 45,000 and isoelectric points at pH values of 5.4-6.0. This increase in phosphorylation with TSH stimulation was confirmed by quantitative analysis of one-dimensional isoelectrofocusing gels.     

Changes in the synthesis of histone H1(0) and H1 in rat FRTL-5 thyroid cells exposed to thyrotropin

In absence of thyrotropin (TSH), FRTL-5 rat thyroid cells stop proliferating and lose the functional characteristics of thyroid tissue. FRTL-5 cells regain their differentiated state and their proliferation activity upon addition of TSH. In this study we investigated the synthesis of histone H1 variants and H19(0) in FRTL-5 cells exposed to 10(-8) M TSH, two days after TSH withdrawal. TSH induced the synthesis of some H1 variants and H1. This effect was already evident six hours after TSH addition, thus well before proliferation, DNA or thyroglobulin synthesis was induced. These data indicate that the induction of H1(0) and some H1 variants is an early event after TSH stimulation and may thus be related to the functional differentiation of FRTL-5 cells.     

TSH stimulation of iodine organification in early foetal rat thyroid in vitro

Thyroid glands from 15 day-old rat foetuses were incubated in Eagle's medium containing Na 125I and supplemented, or not, with TSH for 4 or 24 hours. Electron microscopic radioautographic study shows silver grains mainly in follicular cavities only in the thyroids submitted to TSH during 24 hr. A functional differentiation must therefore take place in thyroid cells under TSH stimulation.     

Multihormonal regulation of thyroglobulin production by the OVNIS 6H thyroid cell line

The hormonal regulation of thyroglobulin production has been studied using a clone of the ovine thyroid cell line: OVNIS 6H. 3 among the 6 hormones proposed for serum replacement are required for an optimal thyroglobulin production; insulin, hydrocortisone and thyrotropin. Insulin alone stimulates thyroglobulin production. The presence of insulin is also required to observe hydrocortisone and TSH stimulations. Newborn calf serum inhibits thyroglobulin production. The best conditions for optimal thyroglobulin expression and TSH responsiveness are obtained in serum-free medium supplemented with 5 micrograms/ml insulin, 100 nM hydrocortisone and 1 mU/ml TSH.     

Modulation of the carbohydrate moiety of thyroglobulin by thyrotropin and calcium in Fisher rat thyroid line-5 cells.

Thyroglobulin secreted in the medium by Fisher rat thyroid line-5 (FRTL-5) cells cultured in the presence of thyroid stimulating hormone (TSH) shows a slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a higher density position in a CsCl gradient than thyroglobulin secreted by FRTL-5 cells cultured in the absence of TSH for 5-7 days. Such a TSH effect is much less or not evident when secreted thyroglobulin is digested with peptide N-glycohydrolase F or when intracellular thyroglobulin is compared. Intracellular thyroglobulin migrates faster than thyroglobulin secreted either in the presence or in the absence of TSH. Evaluation of the mannose and galactose content of thyroglobulin demonstrates that intracellular thyroglobulin has more mannose and less galactose than extracellular thyroglobulin; it also shows that TSH decreases the mannose content of thyroglobulin while increasing its galactose content. Bio-Gel P6 chromatography shows that TSH increases the complex type carbohydrate chains while decreasing the high mannose chains in the secreted thyroglobulin. High mannose type oligosaccharides were characterized by fast atom bombardment-mass spectrometry analysis. Treatment with the calcium ionophore A23187 (5 microM) of FRTL-5 cells cultured with or without TSH causes the appearance of a "fast" migrating form of thyroglobulinin in the culture medium. Bio-Gel P6 chromatography shows that A23187 causes a dramatic decrease of the complex carbohydrate chains of the secreted thyroglobulin.     

Increased Phosphorylation of a Membrane Protein Consequent to Amygdaloid Kindling

The phosphorylation of both particulate and soluble proteins in the amygdala was examined in electrically kindled rats. In animals receiving electrical stimulation in the left amygdala for 5-6 days that displayed electrical after-discharges but no motor seizures, no changes were observed in the phosphorylation of either particulate or soluble proteins. In animals stimulated for 20-21 days where major motor seizures were produced, the phosphorylation of a protein having a molecular weight of 45,000 ( 45K ) was markedly increased. The phosphorylation of this protein was increased in both the right (unstimulated) and left (stimulated) amygdala. Major motor seizures induced by electroconvulsive shocks, however, did not alter phosphorylation of this protein. Phosphorylation of the 45K protein was stimulated by calcium and calmodulin. The 45K protein is a major phosphoprotein of amygdala, representing 3.2% of the total particulate phosphoproteins in control animals and 7.4% in the kindled animals. In the presence of calcium-calmodulin, 16.2% of net protein phosphorylation was accounted for by the 45K protein.