Regulation of thyroperoxidase, thyroglobulin and iodide levels in sheep thyroid cells by TSH, tumor promoters and epidermal growth factor

Using sheep thyroid cells in culture, we have studied the effects of thyroid stimulating hormone (TSH), epidermal growth factor (EGF) and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the activity and expression of both thyroglobulin (Tg) and thyroid peroxidase (TPO) and on the ability of cells to trap and organify iodide. Using Western blotting techniques, we found that TSH increased the absolute cellular levels of Tg. The optimum TSH concentration for Tg mRNA production was between 0.1-1.0 mU/ml. Thyroglobulin mRNA levels were stimulated by TSH but detectable levels were also present in cultures grown in its absence containing cortisol, insulin, transferrin, somatostatin and glycyl-lysyl-histidyl acetate. Unlike Tg, TPO protein levels were found to be completely dependent upon TSH. A time course of TSH stimulation of TPO mRNA showed increases after 8 h of TSH stimulation, whereas induction of Tg mRNA by TSH was seen at 24 h. Iodide trapping and organification were also TSH-dependent processes, showing maximum activities at 300-500 muU/ml of TSH. The addition of 10 nM TPA caused a biphasic decrease in radiolabeled pertechnetate uptake, with complete inhibition being seen at 14 h. Inhibition of iodide organification occurred more rapidly. TPA and EGF (1 nM) reduced the amount of newly synthesized Tg in TSH-stimulated cells by 50% but the absolute amount of Tg within the cells was not markedly inhibited at these early times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological and functional differentiation of human thyroid cells in collagen gel culture

In order to study the expression of the morphological and functional characteristics of human thyroid cells, 3-dimensional cultures were carried out in collagen gel. This substrate allows the cells to retain their organization in follicles with a normal polarity. Cellular polarities appeared normal at the time of collagen embedding, but there was a delay of 4-5 days in culture before the maximal TSH stimulation of 125I- uptake and of cAMP accumulation occurred. In normal and adenoma-derived cells, 125I- uptake, which could be increased by TSH, was demonstrated. cAMP accumulated in the culture medium and thyroglobulin was secreted into the follicle lumen. Of the 4 differentiated carcinomas for which the 72-hr uptake of 125I- was measured, only 2 displayed slight 125I- uptake and response to TSH. Thus, human thyroid cells exhibit better morphological and functional differentiation in collagen gel culture than in monolayer culture. Furthermore, in a variety of pathological cases studied, the expression of specific characteristics in culture varied in a fashion similar to differences observed in vivo.     

Variations in the response of enzyme-dissociated rat pituitary cells to thyrotropin releasing hormone.

While exploring the interaction between thyrotropin releasing hormone (TRH) and normal rat anterior pituitary cells in monolayer culture we observed that cells dissociated with the use of trypsin did not respond to TRH with an increase in either TSH or prolactin (PRL) release. The dissociated cells were cultured for 3 days, then washed to remove serum proteins and exposed to 10^?6M TRH for 3 hours. TSH and PRL secretion from stimulated and unstimulated cultures was determined by radio-immunoassay and normalized using cell protein. When such trypsin-dissociated cells were exposed to 0.5 mM dibutyryl cyclic AMP the release of both TSH and PRL doubled indicating that the intracellular secretory machinery was functional and that the block to TRH was proximal to the formation of cyclic AMP and presumably at the level of a TRH surface receptor. Previous studies have shown that such trypsin-dissociated cells respond to LHRH and a crude hypothalamic extract with a dose dependent increase in LH, FSH and ACTH release. This rules out a non-specific effect of trypsin. When pituitary cells were dissociated with a non-trypsin technique, the unstimulated release of both TSH and PRL was comparable to that found with the trypsin-dissociated cultures. However, these cultures did respond to TRH with an increase in TSH release although again no effect was seen with PRL. The susceptibility of the cells to trypsin suggests the possibility that a protein moiety may be closely associated with the function of the receptor.     

The growth and morphology of FRTL-5 thyroid epithelial cells grown as multicellular spheroids in vitro

Summary FRTL-5 cells, a diploid line of differentiated rat thyroid epithelial cells, have been grown as multicellular spheroids in spinner culture. Spheroids were initiated by seeding FRTL-5 cells either into Lab-Tek dishes or culture flasks with a 0.5% agar base. Thyroid stimulating hormone (TSH, >1.0 mU/ml) was required for initial cell aggregation and spheroid growth. After 1 wk cellular aggregates were transferred to suspension culture in spinner flasks. As with FRTL-5 monolayer cultures, continued spheroid growth required the addition of TSH to the culture medium. The most unique characteristic of the FRTL-5 spheroids was the development of central lumina similar to thyroid follicles in vivo. Follicular structures were absent from spheroids not stimulated with TSH. In the presence of TSH epithelial cells seem metabolically active with morphological evidence of biosynthesis of thyroglobulin-like material and basal laminar-like components. In contrast, all evidence of cellular metabolic activity is absent from cells in spheroids maintained in the absence of TSH. Thus, nontransformed FRTL-5 cells grown as three-dimensional multicellular spheroids responded to hormonal manipulation in a manner comparable to follicular epithelial cells in vivo. This spheroid model might therefore prove to be a very effective tool for investigating aspects of thyroid physiology and pathology in vitro. This work was supported by Grant CA-11198 and CA-20329 awarded by the National Institutes of Health, and a Biomedical Research Support Grant awarded to R. T. Mulcahy.     

Thyrotropin regulation of plasminogen activator activity in primary cultures of ovine thyroid cells

In primary cultures of ovine thyroid cells, a high level of plasminogen activator (PA) activity was detected in the culture media. This level is much higher than in primary cultures of Sertoli cells, granulosa cells, and pituitary cells. PA activity increased with time in culture and was regulated by TSH and insulin. Activity gel analysis of the culture media revealed a major band of 43,000 daltons and a minor one of 70,000 daltons, suggesting the presence of both of the urokinase-type and the tissue-type PA in the media.     

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.     

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.     

Specificity of the cell-to-cell transmission of hormonal imprinting in cell cultures

Chang liver cells and Chinese hamster ovary (CHO) cells were imprinted either with insulin or with thyrotropin (TSH). Chang liver cells responded to insulin but not to TSH. As an effect of imprinting evoked by insulin administration the binding of insulin administered for the second time was enhanced. In the mixed culture of imprinted and intact cells the extent of the binding was similar to that seen in the cultures of the cells having received imprintatory treatment alone. CHO cells also responded to TSH, imprinting developed and was transmitted to the cells which were not in interaction with the hormone (intact cells). In CHO cells also insulin gave rise to imprinting for insulin, whereas TSH gave rise to moderate binding imprinting for insulin. On the other hand, insulin imprinting did not enhance the binding of TSH. The obtained results indicate that both the imprinting itself and the specificity of the transmission of imprinting depend on the characteristics of the cell-type in question. The extent of the transmission, however, is always proportional to the extent of imprinting.     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calpain inhibitors regulate the conversion of thyroid gland follicles in rats and humans

Effects of calpain inhibitors on thyroid follicle conversion into monolayer was investigated. Human and rat primary thyroid cultures require the follicular structure after enzyme disaggregation of native tissue fragments. Removal of thyroid-stimulating hormone (TSH) from the culture medium causes migration of thyrocytes from follicles into monolayer, some differences were noted in conversion of rat and human thyroid follicles. The locomotion of rat thyrocytes is observed after preliminary incubation with TSH, but migration of thyrocytes from human thyroid follicles does not require such a preincubation. Phorbol esters induce migration of rat and human thyroid cells. Calpain inhibitors exert a significant influence on locomotion of the thyroid gland cells induced by the removal of TSH from the culture medium. Human thyrocyte migration is markedly inhibited by calpain inhibitor I or II. Likewise, addition of calpain inhibitor I into primary culture of rat thyrocytes decreased the number of migrating cells by 52%, and shortened average migration distance by 34%. Also, calpain inhibitors reduced the speed of phorbol ether-induced conversion of rat and human thyroid follicles into monolayer.     

促甲状腺激素对甲状腺细胞胞内游离钙和钙调蛋白的影响

本文研究TSH和forskolin对原代培养的猪甲状腺细胞[Ca~(2+)]_i和钙调蛋白的影响。结果表明,TSH可引起甲状腺细胞[Ca~(2+)]_1急性升高。此反应是剂量依赖关系,而与细胞外钙的存在与否无关。其反应性在细胞单层高于细胞是液,近汇合细胞单层高于汇合细胞单层。TSH作用3天,可使甲状腺细胞的钙调蛋白含量增高,此作用与TSH对甲状腺细胞数的影响无关。Forskolin对甲状腺细胞的[Ca~(2+)]_i和钙调蛋白均无明显的影响。     

Studies of the in vitro sensitivity of iodothyronine synthesis to methimazole in normal human thyroid cells

The comparative effects of methimazole (MMI) on resting and thyrotropin (TSH) — stimulated human thyroid cell cultures were investigated in terms of the release of iodoprotein and newly — synthesised iodothyronine hormones into the culture medium during a 48h period of incubation.Iodoprotein recovery was increased after TSH, but both basal and TSH — enhanced iodoprotein release were depressed by MMI. TSH increased the release of tri-iodothyronine (T3) and thyroxine (T4), and although the TSH — enhanced T3 and T4 levels were depressed after MMI, (i) the basal levels found in control cultures were not attained, and (ii) T3 was more susceptible than T4 to MMI suppression, at high TSH levels.These findings indicate a retention of the $\text{in vivo}$ thyroidal sensitivity to MMI, under basal conditions and moderate TSH stimulation $\text{in vitro}$. The system may therefore facilitate further investigation into the mode of MMI suppression of peroxidase systems involved in iodothyronine hormone synthesis within the intact human thyroid cell.     

Thyrotropin-releasing hormone stimulates the activity of the rat thyrotropin beta-subunit gene promoter transfected into pituitary cells

In order to investigate the molecular mechanism(s) by which TRH regulates the biosynthesis of TSH, we are studying the effects of TRH on the expression of the TSH subunit genes (alpha and TSH beta). To study the structure-function relation of TRH stimulation of the activity of the single rat TSH beta gene, chimaeric plasmids were constructed. The 5'-flanking region of the rat TSH beta gene including exon 1 (5'-untranslated region) was inserted into a promoterless, modified pBR, chloramphenicol acetyltransferase (CAT) expression vector. After transfection, specific TSH beta promoter activity was evident in both TRH-responsive pituitary-derived GH3 and primary pituitary cell cultures. To determine potential regulation of TSH beta promoter-directed activity in these cells by TRH, cells were incubated with media containing TRH (10(-7) to 10(-11) M) for 1 to 48 h. TRH stimulated a 1.5- to 3-fold increase in TSH beta promoter activity. Concomitant with an increase in CAT activity was an anticipated increase in PRL synthesis in the GH3 cells in response to TRH. The TRH effect on the TSH beta gene was specific; no increase in CAT activity was detected for TKCAT (thymidine kinase of herpes simplex virus promoter), pBRCAT (no promoter), or TSH beta CAT (3'-5'-orientation). Similar results were obtained using primary pituitary cell cultures. Deletion mutation analysis indicated that TRH sensitivity was detected in a 1.1 kilobase, but not in a 0.38 kilobase TSH beta gene fragment suggesting that the TRH responsive element(s) resides at least in part within the 700 base pairs of the 5'-flanking sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structural aspects of phagocytotic activity in inverted follicle cells of cultured porcine thyroids

Inversion of thyroid follicles took place when they were isolated by collagenase and trypsin and cultured in suspension in Eagle's medium supplemented with 10% fetal calf serum without TSH. The apical surface facing the culture medium contained numerous microvilli and a central cilium, while the luminal surface became flattened. Phagocytotic activity by pseudopods was promoted after addition of TSH to the culture medium. When the inverted follicles were incubated in culture medium containing TSH (50 mU/ml) and human red blood cells, or TSH and polystyrene latex beads (2.02 micron in diameter) for 1-3 h, numerous red blood cells or latex beads respectively were observed to be taken up by the epithelial follicle cells by scanning electron microscopy, as well as conventional thin-section electron microscopy. These results show that the apical surface (culture medium side) of the epithelial cell of the cultured thyroid follicle whose polarity is reversed phagocytoses red blood cells and latex beads.     

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways.     

Effects of thyroid-stimulating hormone and phorbol ester on glycosaminoglycan synthesis in porcine thyroid epithelial cells in primary culture

The effects of thyroid-stimulating hormone (TSH) and a tumor promoter: 12-0-tetradecanoyl-phorbol-13-acetate on glycosaminoglycan (GAG) synthesis were studied in porcine thyroid epithelial cells in primary culture. TSH is known to involve cyclic AMP mechanism and phorbol ester to act by protein kinase C pathway. Chronic treatment of cells with TSH increased the synthesis of heparan sulphate associated with the cell layer and hyaluronic acid in the culture medium. Phorbol ester increased the radioactivity of total GAGs in the culture medium but had no effect on GAGs associated with the cell layer. It inhibited the positive effect of TSH on heparan sulphate synthesis. These results suggest that in thyroid epithelial cells the synthesis of the GAGs associated with the cell layer and those secreted into the culture medium are regulated by different intracellular mechanisms.     

Polarization of thyroid cells in culture: evidence for the basolateral localization of the iodide "pump" and of the thyroid-stimulating hormone receptor-adenyl cyclase complex

When cultured in collagen gel-coated dishes, thyroid cells organized into polarized monolayers. The basal poles of the cells were in contact with the collagen gel, whereas the apical surfaces were facing the culture medium. Under these culture conditions, thyroid cells do not concentrate iodide nor respond to acute stimulation by thyroid-stimulating hormone (TSH). To allow the free access of medium components to the basal poles, the gel was detached from the plastic dish and allowed to float in the culture medium. After release of the gel, the iodide concentration and acute response to TSH stimulation were restored. Increased cAMP levels, iodide efflux, and formation of apical pseudopods were observed. When the thyroid cells are cultured on collagen-coated Millipore filters glued to glass rings, the cell layer separates the medium in contact with the apical domain of the plasma membrane (inside the ring) from that bathing the basolateral domain (outside the ring). Iodide present in the basal medium was concentrated in the cells, whereas no transport was observed when iodide was added to the luminal side. Similarly, an acute effect of TSH was observed only when the hormone was added to the basal medium. These results show that the iodide concentration mechanism and the TSH receptor-adenylate cyclase complex are present only on the basolateral domain of thyroid cell plasma membranes.     

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.