Thyroid Follicle Cells Research Articles

Thyroglobulin, the major and obligatory exportable protein of thyroid follicle cells, carries the lysosomal recognition marker mannose-6-phosphate.

Thyroglobulin (TG), the major exportable protein of thyroid follicle cells, is conveyed to lysosomes on a complex secretion, storage and recapture pathway by as yet unknown transport mechanisms. This report establishes that the dimeric porcine TG-molecule carries an average of six phosphate residues. Endoglycosidase digestion showed that two phosphate residues are bound to the high-mannose carbohydrate side chains (CHO), while two others are linked to the complex CHO. These four residues are also sensitive to alkaline phosphatase treatment, indicating their terminal linkage. Immunoprecipitation analyses showed that TG obtained from microsomal fractions is already phosphorylated. Most important, an enzymatic assay applied to hydrolysates of TG established that the two phosphate residues at the high mannose CHO are present as mannose-6-phosphate (M-6-P). Alkaline phosphatase treatment of biosynthetically radiophosphorylated CHO followed by hydrolysis and t.l.c. indicated that M-6-P is present at least in part in phosphomonoester linkage. Furthermore, porcine TG binds specifically to the M-6-P receptor of Chinese hamster ovary cells. It is concluded that the M-6-P residues of TG are exposed and able to operate as a ligand for the M-6-P receptor. It is unknown why the lysosomal recognition-marker M-6-P does not convey TG directly on an intracellular route to lysosomes. We propose that for the secretion of newly synthesized TG into the follicle lumen an additional export signal dominating over the M-6-P recognition-marker is required.

Location of epidermal growth factor receptors on porcine thyroid follicle cells and receptor regulation by thyrotropin.

By using a direct electron microscopic autoradiographical technique, we were able to demonstrate that the epidermal growth factor (EGF) receptor is asymmetrically located on polarized porcine thyroid follicle cells cultured on a collagen gel matrix. More than 80% of autoradiographical grains from [125I]EGF were associated with the basolateral cell surface compared to only about 2.5% at the apical cell margin. EGF stimulated growth only when the collagen gels were floating, indicating a basolateral location of functional EGF receptors. Preincubation with TSH or another stimulator of cAMP synthesis, forskolin, increased binding of [125I]EGF to the cells. Moreover, the subsequent mitogenic response to EGF was potentiated by such pretreatment. The results are in accordance with the hypothesis that TSH potentiates the response to EGF in the stimulation of thyroid growth.

Graded response in the individual thyroid follicle cell to increasing doses of TSH

The in vivo activation of rat thyroid follicle cells by increasing doses of TSH (1–100 mU) was studied by quantitative light and electron microscopy. The number of pseudopods induced after 20 min by 5, 20, 50 and 100 mU was the same but a dose-dependent increment of the individual pseudopod size (measured as profile area or membrane surface area) was observed, suggesting a graded response in the individual follicle cell. The number of pseudopods induced by 1 mU was only 10% of that in the other group. Another early effect of TSH, besides the induction of pseudopods, is stimulation of exocytosis. The number of exocytotic vesicles in pseudopod-bearing follicle cell profiles (the presence of a pseudopod taken as a sign of activation) decreased in a dose-dependent manner. This indicates that the follicle cell does not respond in an all-or-none mode (in which case a similar decrease in vesicle number in activated cells should be expected irrespective of the level of TSH) and that the response in the individual cell is graded depending on the level of TSH stimulation.

Ethylenethiourea and nabam induced alterations of function and morphology of thyroid gland in rats.

Male Wistar rats were given 0-200 mg/l of nabam and 0-300 mg/l of ethylenethiourea (ETU) in drinking water ad libitum for 28 d to study their effect on thyroid gland function and morphology. Blood samples for RIA hormone assays (T3, T4, TSH) were taken and thyroids of each rat were dissected out for routine light and electron microscopic analysis after sacrifice performed under CO2 anesthesia. Nabam (8.4-30.5 mg/kg bw/d) inhibited dose-dependently T4-secretion and increased T3-secretion, but had no effect on basal TSH-secretion. ETU (10.6-23.4 mg/kg bw/d) inhibited dose-dependently T4- and T3-secretion. ETU also increased basal TSH-secretion tenfold, obviously due to the lack of negative feedback by T4 and T3. Light microscopic analysis revealed neither nabam nor ETU induced morphological alterations in thyroids. Both but especially ETU induced, ultrastructural changes, namely increased number of myelin bodies, dilatation of the rough endoplasmic reticulum, and increased vacuolization in the epithelial cells of thyroid follicles. The functional alterations are obviously connected with, and preceed slight ultrastructural degenerative changes in thyroid follicle epithelial cells even at low doses of nabam, and ETU, after a short-term oral administration.

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, PGE 1, 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, PGE 1, 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 PGE, 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.

Thyrotropin modulates EGF receptor function in porcine thyroid follicle cells

Porcine thyroid follicle cells in monolayer cultures were shown to contain one single class of high-affinity EGF receptors with Kd = 4.5 × 10−10 M and approximately 20 000–25 000 receptors per cell. Suspension cultures of aggregated follicle cells, exposed to TSH for 3 days, showed a 3-fold increase in [125I]EGF binding. Scatchard analysis demonstrated that this was due to an increase in receptor number. Other cAMP-elevating agents (choleratoxin, dibutyryl cAMP, forskolin) induced a similar effect. In suspension cultures, preincubation with TSH or choleratoxin for 2 days reduced the subsequent [3H]thymidine incorporation. This inhibition was overcome by a low concentration of EGF (0.1 ng/ml). At higher concentrations of EGF (1–10 ng/ml) the incorporation of [3H]thymidine was potentiated 2–3 fold in cultures preexposed to TSH or choleratoxin. The results demonstrate the presence of a high-affinity EGF receptor in porcine thyroid follicle cells. Receptor expression, as well as responsiveness to the mitogenic action of EGF, is modulated in vitro by TSH, through a cAMP-dependent process.

Morphological and biochemical responses of cultured thyroid cells to thyrotropin.

In the thyroid gland, TSH stimulates cAMP formation, exocytosis of precursor (noniodinated) thyroglobulin, endocytosis of thyroglobulin, and proteolytic processing of the thyroglobulin to form thyroid hormones. In this report we describe TSH effects on cAMP levels, microtubules, microfilaments, myosin fibrils, and the morphology of cultured thyroid follicle cells. The cells were normally cultured in the presence of 10 mU/ml TSH, and fresh TSH produced no stimulation when assayed for cAMP production in a 15-min assay. When such cells were cultured for up to 72 h in the absence of TSH and then assayed for cAMP production, the basal levels were much reduced, but fresh TSH stimulated cAMP levels half-maximally at 1 mU/ml and up to 50-fold at 20 mU/ml. Microtubules, myosin fibers, and microfilaments were demonstrated by indirect immunofluorescent staining. Fluorescent staining of fibers was observed in cells fixed before lysis and in cells lysed before fixation. In control cells grown without hormone, microtubules originated near the nucleus and extended to the cell periphery. Myosin-containing fibrils traversed the cell or radiated from foci. Microfilaments spanned the cell in a stress fiber pattern. After incubation with 20 mU/ml TSH and 4 mM isobutylmethylxanthine (IBMX) for 10-20 min, the microtubules in up to half of the cells appeared altered and more granular, and the cell periphery was scalloped. After 15-30 min with TSH and IBMX, normal myosin fibers were replaced with a fine lattice-work, peripheral staining disappeared, and the proportion of nonfibrous myosin increased. Stress fibers demonstrated with antibody to actin also disappeared, and the peripheral structures observed in normal cells became fragmented. Incubation with forskolin or TSH and IBMX for 2-3 h resulted in arborization of 30-60% of the cells that contained bundles of microtubules, myosin fibers, or microfilaments into dendrite-like processes and increased staining near the nucleus. At 5 h, more than 80% of the cells were arborized. These morphological changes were less pronounced with IBMX alone and minimal with TSH alone. The time course of cAMP levels observed basally or after TSH, forskolin, or TSH and IBMX was consistent with the relative effects of these agents on arborization. These studies are consistent with effects of cAMP on microtubules, myosin-containing fibrils, and microfilaments and may provide a basis for the morphological response to TSH.

Effect of a second dose of thyrotropin on exocytosis and endocytosis in the rat thyroid gland.

The effect of TSH on exocytosis and endocytosis in thyroid follicle cells was studied with electron microscopic stereology. Groups of rats, pretreated with T4 for 2 d, were injected iv with TSH (500 mU) 20 min, 2, 4, 6, and 8 h before perfusion fixation. Parallel groups were given a second dose of TSH 2, 4, 6 and 8 h after the first one and the thyroids were fixed after 20 min. In controls, injected with saline, the membrane surface area (msa) of exocytotic vesicles was about the same as that of the apical plasma membrane. Pseudopods and colloid droplets were not present. TSH induced exocytosis. The msa of exocytotic vesicles was reduced by about 85% already after 20 min and was even more reduced after 2 h. At later times after injection of TSH the msa of exocytotic vesicles increased gradually to reach control levels after 8 h. TSH also induced endocytosis. After 20 min the increase in msa of endocytotic structures (pseudopods, colloid droplets and micropinocytotic vesicles) corresponded to the decrease in msa of exocytotic vesicles during the same time interval. The msa of endocytotic structures remained high at 2 h but decreased gradually at later time intervals and after 8 h no pseudopods and colloid droplets remained in the apical cell region. A second injection of TSH given 2 h after the first one when few exocytotic vesicles were present did not influence the msa of endocytotic structures. At later time intervals a second dose of TSH stimulated exocytosis as well as endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural studies of protein transport, exocytosis and iodination in thyroid follicle cells

Ultrastructural studies of protein transport, exocytosis and iodination in thyroid follicle cells

In vivo shedding of apical plasma membrane in the thyroid follicle cells of the mouse.

Clusters of luminal dense bodies, limited by a triple-layered membrane, were found in all follicle lumina in thyroid glands of mice. After thyroxine treatment the number of luminal dense bodies increased, especially in the periphery of the lumen, where the intraluminal bodies often displayed a striking resemblance to microvilli. In hyperplastic goiters, obtained by feeding mice with propylthiouracil, luminal dense bodies were replaced by intraluminal vesicles. During goiter involution the vesicles were gradually replaced by luminal dense bodies; the presence of intermediate forms suggests that vesicles and dense bodies are basically the same formations. Luminal dense bodies were observed in colloid droplets indicating their removal by endocytosis. As demonstrated by electron-microscopic cytochemistry, luminal dense bodies contain a membrane-bound peroxidase, and electron-microscopic autoradiography after administration of 125I indicate that they possess an iodinating capacity. Our observations on mouse thyroid glands suggest that the luminal dense bodies, which appear as vesicles in hyperplastic glands, are formed by shedding of the apical plasma membrane of the follicle cell. The shedding process might be of importance for the turnover of plasma-membrane material.

Influence of thyroid autoantibodies on thyroid cellular growth in vitro

Porcine thyroid follicle cells, cultured in suspension, were employed to investigate the effects of immunoglobulin preparations from patients with colloid goitre, Graves' disease or Hashimoto's thyroiditis on thyroid growth in vitro. Epidermal growth factor (EGF, 19 ng/ml) was used as a reference for maximum growth stimulation and produced a 9-fold increase in [ 3H]thymidine incorporation. Immunoglobulins (1000 μg/ml) were found to increase [ 3H]thymidine incorporation compared to control: from 10 normal individuals 32 ± 4% (mean ± SEM, % of EGF response), from 10 patients with colloid goitre 26 ± 4% (not significantly different from normal), from 10 patients with Graves' disease 19 ± 3% ( P < 0.05) and from 15 patients with Hashimoto's thyroiditis 11 ± 2% ( P < 0.001). No patient immunoglobulin preparation showed activity greater than that of normal individuals. The lower growth stimulatory activity in Graves' disease and Hashimoto's thyroiditis remained after heat inactivation of serum and is thought to reflect surface binding of thyroid autoantibodies.

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland.

A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37 degrees C in reaction medium containing 1.4 mM DAB (in 0.1 M Tris-HCl) and 0.15 mM hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10 mM. Stimulation of peroxidase in the thyroid was observed in vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.

甲状腺小胞上皮細胞表面におけるレクチン結合部位の変化 : フェリチン標識法による研究 : 主論文の要旨

甲状腺小胞上皮細胞表面におけるレクチン結合部位の変化 : フェリチン標識法による研究 : 主論文の要旨

Transcytosis in thyroid follicle cells.

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.

Functional properties of porcine-thyroid follicles in suspension

Porcine thyroid follicle cells were isolated (about 10 7 cells per gram of tissue) and cultured in small aggregates in agarose-coated culture dishes. The aggregates became arranged into follicle-like structures capable of iodide uptake and organification. In the presence of TSH (0.2 mU/ml), the aggregation of follicles was enhanced, and iodide uptake as well as TSH-stimulated organification of iodide was increased compared with that in the control. In culture, the active iodide metabolism was gradually lost over a 7-day period. This was not due to a disappearance of the TSH-adenylate cyclase system, since cAMP production was retained and stimulated by TSH (half-maximal effect at about 1 mU/ml). Acutely, TSH stimulated iodide efflux and iodide organification (half-maximal effect at about 20 μU/ml). The stimulatory effect on organification was transient: within an hour further organification proceeded as in the absence of hormone. The effects on efflux and organification were already maximal at low TSH concentrations, whereas cAMP production was stimulated with up to 50-fold higher TSH levels i.e. the findings were typical of spare receptors. In the continued presence of epidermal growth factor, a potent mitogen for thyroid cells, the follicles increased in size and contained one single large lumen. Their capability to take up and organify iodide was reduced.

Regional differences in surface anionic sites in the rat thyroid follicle cells shown by cationized ferritin labeling.

Regional differences in surface anionic sites in the rat thyroid follicle cells shown by cationized ferritin labeling.

Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells.

Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing rhe intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.

Morphology of the second kind of follicle in the guinea pig thyroid gland.

The second type of follicle (S follicle) was studied in the thyroid gland of young and mature guinea pigs. A marked activity to non-specific esterase was used as a marker system for the S follicle. Usually a single S follicle was situated in the upper central two-thirds of each lobe occupying 0.5--2.7 mm of the length of the lobe. The S follicle could in both animal groups be divided into three parts, an upper, a middle, and a lower part. The upper tubular part consisted of cuboidal and cylindrical, strong-reacting cells completely separated from the thyroid follicles by connective tissue. The middle part was further subdivided into an irregular follicle without contact to the thyroid follicles, and a distended follicle with several contact areas to the thyroid follicles. An increasing amount of flattened, weak-reacting cells interpreted as thyroid follicle cells were encountered in the S follicle wall adjacent to the contact areas. In young animals the lower part had a tubular appearance like that seen in the upper part, while the lower part of the S follicle in mature animals were more follicle like.

Fine structure of the second kind of thyroid follicles in mature guinea pigs.

The second kind of follicle (S-follicle) is in mature guinea pigs a morphologically heterogeneous structure located in the central part of the upper two-thirds of the thyroid lobe. An S-follicle may appropriately be divided in an upper, a middle, and a lower part. This paper deals with the ultrastructure of the upper tubular part and the middle part. The latter is further subdivided in an irregular follicle and a distended follicle. In the tubular part a single cell type, termed the S-follicle cell, is dominating. This cell is most often arranged in a single layer and is characterized by an indented nucleus and by presence of pinocytose vesicles and hemi-desmosomes along the basal cytoplasm membrane. In addition, C cells are frequently found. In the irregular follicle five different cell types are noticed: S-follicle cells, dark fibrillar cells, granulated cells, ciliated cells, and C cells. The S-follicle cells display an ultrastructure similar to those in the tubular part but are frequently arranged in two or more layers. Both the tubular part and the irregular follicle are surrounded by connective tissue. In the distended follicle, however, contact areas to thyroid follicles are found. Adjacent to these contact areas, cells with an ultrastructure similar to that of thyroid follicle cells proper are encountered in the wall of the S-follicle. This observation suggests that the distended part of an S-follicle in mature guinea pigs might be regarded as a kind of mixed follicle. The origin of thyroid follicle cells proper in the wall of the S-follicle is briefly discussed.

Membrane labeling with cationized ferritin in isolated thyroid follicles

Follicles, closed and open, isolated from rat, pig, and human thyroids, were incubated with polycationized ferritin (CF) in order to label the plasma membrane and follow the fate of internalized membrane. All parts of the plasma membrane, except the pseudopod membrane, were labeled. CF was rapidly (within 5 min) internalized from the apical surface by micropinocytosis. Macropinocytotic vacuoles, formed from pseudopods, were not primarily labeled but received CF by fusion with micropinocytotic vesicles. CF was early present in phagolysosomes and most of the CF was eventually accumulated in these structures. After 30 min CF was found in minute amounts in some Golgi saccules. In all labeled intracellular structures only some of the CF was clearly membrane associated. The labeling of Golgi saccules, although minimal, might indicate a recycling of membrane. However, since CF was not a pure marker of intracellular membranes in thyroid follicle cells, it could not be used to demonstrate the extent or route(s) of membrane recycling.