Biological effect of anti-fucosyl GM1 ganglioside antibody on cyclic adenosine 3',5'-monophosphate production in FRTL5 rat thyroid cells

Addition of specific anti-fucosyl GM1 antibody raised in a rabbit caused dose-dependent inhibition of endogenous and thyrotropin (TSH)- or thyroid stimulating antibody-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in cultured FRTL5 rat thyroid cells. Further, the antibody inhibited the cAMP increase induced by prostaglandin E1 and forskolin. However, anti-fucosyl GM1 antibody did not affect the binding of [125I]bovine TSH to solubilized porcine thyroid TSH receptor or to FRTL5 cells. In conclusion, fucosyl GM1 is one of the specific membrane components of thyrocytes and appears to be involved in adenylate cyclase stimulation or cAMP generation. Further, the biological effects of the ganglioside do not seem to be mediated by the TSH receptor, suggesting a post receptor mechanism.     

Pseudopod membrane in TSH-stimulated thyroid cells: a specialized domain in the neighboring apical plasma membrane

Summary Pseudopod formation in response to thyrotropin can be obtained with porcine thyroid cell monolayers attached to floating collagen gels or collagen-coated Millipore filters, a model system that allows free access to ligands and antibodies to the apical plasma membrane. To obtain new insight concerning the molecular composition of the pseudopod membrane, (1) ligands were used allowing identification of anionic sites (ruthenium red, cationized ferritin) or carbohydrate units (wheat germ agglutinin, WGA) and (2) antibodies elicited against isolated porcine thyroid membranes or dog intestinal aminopeptidase were employed.Wheat germ agglutinin-binding sites, detected by fluorescence and electron microscopy, were heterogeneously dispersed on the apical membrane. In TSH-stimulated cells, the absence of WGA-binding sites was showed on the pseudopod membrane of thyroid cells, in addition to the previously reported absence of anionic sites. This absence of binding appeared to be independent of the conditions of incubation and/or times of stimulation. Aminopeptidase, which is an apical marker in thyroid cells, was redistributed and clustered on the pseudopod membrane in the cells exposed to TSH stimulation.These present findings support the view that the pseudopod surface constitutes a highly specialized microdomain within the thyroid apical plasma membrane during TSH acute stimulation.With the technical assistance of Brigitte Nguyen Than Dao, Laboratoire de Neuroendocrinologie A, U.S.T.L., Montpellier. Preliminary accounts of this study were presented at the XXI-Vème Colloque de la Société Française de Biologie Cellulaire, Montpellier, 1984     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions

Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.     

Similar triiodothyronine releasing activity of thyroid stimulating antibodies in human and porcine thyroid slices

In an approach to addressing species specificity of thyroid stimulating antibodies (TSAb) stimulation of T3 release by Graves' sera was comparatively studied in human and porcine thyroid slices. A high sensitivity and specificity was found for the T3 bioassay independently on the use of human or porcine thyroid. Moreover, activity indices of the individual sera in both tissues were significantly correlated to each other and to circulating hormone levels in untreated disease. In conclusion, we suppose a lack of functionally relevant differences between target antigens, brought about probably by the TSH receptor itself and other membrane components, in human and porcine thyroid. Thus, for clinically applicable T3 releasing bioassay porcine thyroid may be alternatively used. In addition, this bioassay renders the advantage of reflecting the activity of disease.     

Thyrotropin stimulation of cultured thyroid cells increases steady state levels of the messenger ribonucleic acid for thyroid peroxidase

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.     

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Lysosomal membrane recovery following labilization by thyroid stimulators

Lysosomal membrane permeability was assessed by measuring freely available naphthylamidase activity in intact preparations of guinea pig thyroid follicular cells following exposure of thyroid tissue to sequential stimulation by two thyroid stimulators, thyrotrophin (TSH) and thyroid stimulating immunoglobulins (TSI). These investigations showed that following labilization by TSH, the lysosomal membranes recovered and were capable of responding to a second thyroid stimulator (TSI). That such recovery represented restabilization of lysosomal membranes was confirmed by the finding that latent naphthylamidase activity was restored without a change in total activity of the enzyme.     

Functional roles of the bone morphogenetic protein system in thyrotropin signaling in porcine thyroid cells

We uncovered a new regulation of thyrocyte function by bone morphogenetic protein (BMP) under the influence of thyrotropin (TSH) using primary culture of porcine thyrocytes. The BMP type I receptors, ALK-2 (ActRIA), -3 (BMPRIA), and -6 (BMPRIB), were expressed in porcine thyrocytes, while ALK-6 was not detected in human thyroid. Treatment with BMP-2, -4, -6, -7, and TGF-beta1 exhibited a dose-dependent suppression of DNA synthesis by porcine thyrocytes. BMP-2, -4, -6, -7, and TGF-beta1 suppressed TSH receptor mRNA expression on thyrocytes, which was consistent with their suppressive effect on TSH-induced cAMP synthesis and TSH-induced insulin-like growth factor-1 expression. Activin exhibited minimal suppression of thyrocyte DNA synthesis and did not exhibit suppressive effects on TSH receptor mRNA expression. Phosphorylated Smad1/5/8 was detected in the lysates of porcine thyrocytes treated with BMP-2, -4, -6, and -7. However, in the presence of TSH, BMP-6 and -7 failed to activate Smad1/5/8 phosphorylation and 3TP-reporter activity, whereas BMP-2 and -4 maintained clear activation of the BMP signaling regardless of the presence of TSH. This diverged regulation of thyroid BMP system by TSH is most likely due to the reduction of ALK-6 expression caused by TSH. Thus, the thyroid BMP system is functionally linked to TSH actions through modulating TSH receptor expression and TSH, in turn, selectively inhibits BMP signaling. Given that BMP system is present in human thyroid and the expression pattern of ALK-2 and BMPRII is different between follicular adenomas and normal thyroid tissues, the endogenous BMP system may be involved in regulating thyrocyte growth and TSH sensitivity of human thyroid adenomas.     

Thyroid follicular morphogenesis mechanism: organ culture of the fetal gland as an experimental approach

The morphological and physiological changes induced by organ culture and thyroid-stimulating hormone (TSH) stimulation in the rat fetal thyroid gland were studied. Organ culture increased Golgi activity which was further enhanced by TSH, subsequently facilitating the formation of intracellular lumina. TSH also raised the intracellular cAMP level. The intracellular lumina observed during follicular morphogenesis are structurally comparable to typical intracellular cavities formed in adult thyroid cells, which are considered as being the result of increased Golgi activity. The intracellular lumen, therefore, is probably not a physiologically significant step in thyroid morphogenesis.     

Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: A quantitative confocal microscopic study

The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1–50 μM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface-located TPO was detected by fluorescein-labeled antibodies on saponin-treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2-fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 μM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation (1) increased 2-fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and (2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells. J. Cell. Physiol. 174:160–169, 1998. © 1998 Wiley-Liss, Inc.     

Differential regulation of thyroid cell-cell and cell-substrate adhesion by thyrotropin

Preservation of cell aggregation is necessary for thyroid follicular differentiation in vitro and requires stimulation by thyrotropin (TSH). We have tested the hypothesis that TSH preferentially increases thyroid cell-cell adhesion relative to cell-substrate adhesion. Cell-cell adhesion was measured in short-term suspension cultures by the decrease in the fraction of single cells remaining in culture (free cell ratio, FCR). When incubated in medium alone freshly isolated cells showed a progressive fall in FCR but this was accelerated by TSH and the cyclic AMP analog, 8-(4-chlorophenylthio)cyclic AMP. Aggregation was dependent upon extracellular Ca2+ and also promoted by a cell-free membrane extract. In contrast, attachment of cells to plastic dishes treated for tissue culture was not affected by TSH. We conclude that thyroid cells possess a TSH-sensitive cell adhesion system. The preferential increase in cell-cell adhesion may be one mechanism by which TSH stimulates the formation and preservation of follicles in vitro.     

Immunolocalization of a spectrin-like protein (fodrin) in pancreatic acinar cells

A spectrin-like protein (fodrin) was localized in porcine pancreas using an immunoperoxidase procedure with antibodies raised against erythrocyte spectrin. Fodrin was primarily associated with the cell plasma membrane although some was also detectable in the cytoplasm of the acinar cells. The membrane labelling of the acinar cells was uneven such that the lateral and basal membranes were strongly labelled by anti-spectrin antibodies whereas the apical membranes were poorly labelled. The implications of the results to secretion and to the occurrence of specific membrane domains are discussed.     

Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by epidermal growth factor

We studied the effects of epidermal growth factor (EGF), thyroid-stimulating hormone (TSH) and amiloride on cytoplasmic pH (pHi) in cultured porcine thyroid cells. We used 2',7'-bis(2-carboxyethyl)-5- (and 6-)carboxyfluorescein (BCECF), an internalized fluorescent pH indicator, to measure pHi. EGF stimulated thyroid cell alkalinization and proliferation, which were blocked by amiloride. EGF-stimulated thyroid cell alkalinization depended on extracellular Na+ concentrations. EGF stimulation resulted in an activation of Na+/H+ exchange, which alkalinized the cells. The results indicated that Na+/H+ exchange or cell alkalinization might function as a transmembrane signal transducer in the action of EGF. In the present system, TSH did not stimulate alkalinization or proliferation.     

The value of plasma TSH estimation and TRH stimulation test in the course of treatment of differentiated thyroid cancers.

Plasma TSH levels were measured on 114 occasions in 96 patients treated for differentiated thyroid cancer. Prior to thyroid surgery, plasma TSH levels were within the range of normal. Plasma TSH levels increased slightly following partial thyroid resection and definitely after total thyroid ablation. In patients where the removal of normal thyroid induced hormonogenesis in thyroid tumours, plasma TSH levels were dependent on the hormonal secretion of the tumour as shown by inverse relationship between TSH and both PBI and 131I uptake. The increase of radioiodide uptake following stimulation by exogenous bovine TSH was inversely related to the plasma thyrotropin levels. The suppressibility of enhanced thyrotropin levels was complete with individually adjusted doses of synthetic thyroid hormones. With the exception of patients on suppressive treatment, TRH administration induced increase in plasma TSH levels. The findings are discussed with regard to the role played by TSH in the induction of hormonogenesis in thyroid tumours. The practical values of TSH estimation and TRH stimulation seem to be low; the measurements of thyrotropin levels may be important for the estimation of the suppressive effect in the course of and following withdrawal of treatment with thyroid hormones.     

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

Summary 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 the 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.     

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.     

Impairment of the TSH signal transduction system in human thyroid carcinoma cells

In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.     

cAMP and Ca(2+)-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton

The potential involvement of actin and fodrin (brain spectrin) in secretory events has been assessed in primary cultured guinea pig parotid acinar cells, using as a tool affinity purified anti-alpha-fodrin antibody, phalloidin, and immunofluorescence techniques. In resting parotid acinar cells fodrin and actin appeared as a continuous ring under the plasma membrane of most of the cells. Upon stimulation with secretagogues fodrin and actin labeling at the level of the plasma membrane disappeared almost completely. To establish a correlation between secretion and cytoskeletal changes at the individual cell level, anti-alpha-amylase-antibodies were used to label secreted amylase exposed at the surface of secreting cells. The number of cells expressing alpha-amylase on their surface followed bulk secretion of alpha-amylase. A strict correlation between secretion and alteration of the actin-fodrin labeling was observed at the individual cell level. The cytoskeletal changes occurred in parallel with secretion independently of the secretagogue used (carbamoylcholine in the presence of Ca2+, isoproterenol in presence or absence of Ca2+, forskolin, or dibutyryl-cyclic-AMP). The changes were reversible upon removal of the secretagogue. Since Ca2+, as well as cAMP-mediated secretion, was associated with the same kind of cytoskeletal changes, a reorganization of the cytoskeleton may play an essential part in regulated secretion.