Margaret C. Eggo

The effects of growth factors and serum on DNA synthesis and differentiation in thyroid cells in culture.

The effects of three putative growth factors and serum on [Me-3H]thymidine and Na125I incorporation into thyroid cell cultures have been examined. We found that serum and EGF could stimulate radioactively labelled thymidine incorporation into confluent cultures. However, both factors completely inhibited iodine uptake and organification at low concentrations. Insulin also stimulated [Me-3H]thymidine incorporation but had no adverse effect on thyroid differentiated function. TSH examined under the same conditions was not a growth factor but was essential to maintain differentiated functions. We conclude that thyroid growth and differentiation are not mutually exclusive processes. However, EGF and serum inhibit thyroid differentiated function at very low concentrations. Elucidation of the physiological role of these factors and their mechanism of action may lead to a greater understanding of thyroid hormone biosynthesis.

Interaction of TSH, insulin and insulin-like growth factors in regulating thyroid growth and function.

Primary cultures of sheep thyroid cells have been used to study regulation of thyroid growth and function by growth factors and TSH. Cells were plated at low density to minimize contributions from the endogenously produced insulin-like growth factors and their binding proteins and other proteins or hormones secreted by thyroid cells in culture. Growth of the cells was followed for 7-11 days in medium without serum. We found that TSH by itself was unable to stimulate thyroid growth. However, the ability of insulin and IGF-I to stimulate thyroid cell growth was markedly potentiated by TSH. Thyroid function was assayed by measurement of uptake of pertechnetate and organification of iodide and also by synthesis of thyroglobulin mRNA. TSH alone was unable to stimulate thyroid function appreciably. Insulin and IGF-I were ineffective by themselves at stimulating thyroid differentiated function, but in the presence of TSH, all indices were stimulated markedly. We conclude that TSH by itself is not a growth factor for thyroid cells. However, in the presence of insulin or IGF-I, TSH potentiates the growth-stimulating properties of this hormone. Similarly, TSH by itself does not stimulate thyroid function but requires the presence of insulin or IGF-I. These data show the cooperative interactions between growth factors and TSH in regulating both thyroid growth and function.

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

SummaryHuman thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10−9M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells.

Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.

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)

Epidermal growth factor inhibits thyrotropin-mediated synthesis of tissue-specific proteins in cultured ovine thyroid cells

Primary cultures of ovine thyroid cells were induced to differentiate by addition of thyrotropin (TSH). This was demonstrated as an accumulation of 2 thyroid-specific proteins, thyroglobulin and thyroid peroxidase, using immunofluorescent staining methods and immunoprecipitation of biosynthetically labeled cultures. As an additional measure of differentiation, cells exhibited a morphological response to TSH and regained the ability to incorporate radioactive iodide. Epidermal growth factor (EGF) markedly inhibited differentiation when added together with TSH. Thyroglobulin synthesis was reduced to low levels and peroxidase synthesis was reduced to levels that were undetectable by the methods used. Morphological changes in response to TSH were also diminished by EGF. The antagonistic interaction between TSH and EGF in regulating differentiation in cultured thyroid cells may reflect the type of control that exists in vivo.

Thyrotropin inhibits while insulin, epidermal growth factor and tetradecanoyl phorbol acetate stimulate insulin-like growth factor binding protein secretion from sheep thyroid cells.

Six insulin-like growth factor binding proteins (IGFBP) have been identified in the conditioned medium from sheep thyroid cells cultured under serum-free conditions. IGFBPs of 32, 28, 23 and 19 kDa were secreted by cells cultured for 14 days in serum-free and hormone-free medium. The constitutive secretion of IGFBP was inhibited by thyrotropin (TSH, 0.3 mU per mL). The effect was most marked on the secretion of the 28 kDa BP. High insulin concentrations stimulated the secretion of this IGFBP. The stimulatory effects of insulin were inhibited by TSH. Growth hormone treatment decreased the secretion of the 28 kDa protein. Tetradecanoylphorbol-13 acetate (TPA) and epidermal growth factor (EGF) both of which stimulate thyroid cell growth but inhibit differentiated function, markedly stimulated IGFBP secretion and induced the appearance of a 46 and a 150 kDa IGFBP. The effects of EGF and TPA were not identical. A rat IGFBP-2 cDNA reacted with sheep thyroid RNA of approximate size 1.6 kb. TPA treatment increased IGFBP-2 mRNA. Other hormones used to enhance differentiation and growth in thyroid cells in culture i.e. transferrin, somatostatin, cortisol and glycyl-histidyl-lysine acetate had no marked effects on IGFBP secretion nor on TSH-dependent, insulin-mediated iodide uptake and organification and cell growth. We show a correlation between secretion of high molecular weight IGFBP with enhanced growth but decreased function. Conversely, we find a correlation between decreased secretion of the 28 kDa BP and increased growth and function.

Thyrotropin-induced formation of functional follicles in primary cultures of ovine thyroid cells.

In primary cultures of ovine thyroid cells, TSH induced the expression of several differentiated functions including the formation of follicles, and synthesis and storage of iodinated thyroglobulin in the follicular lumen. In the present report, these follicles were shown by transmission (TEM) and scanning electron microscopy (SEM) to be intact, comprised of two or more cells and to possess numerous microvilli on the inner cell membranes facing the follicular lumen. The TSH-induced formation of follicles was reversible and dynamic, with the kinetics of formation preceding that of iodination. The follicles were further demonstrated to be functional in terms of thyroglobulin storage and iodination.

Synthesis, membrane insertion and glycosylation of thyroglobulin in a completely heterologous system

Abstract Thyroglobulin has been synthesized, inserted into membranes and glycosylated in a completely heterologous, reconstituted system consisting of a protein synthesizing extract, stripped dog pancreas microsomal membranes and calf thyroid RNA.

REGULATION OF GROWTH AND DIFFERENTIATION IN FOLLICLE CELLS

Hypertrophy and hyperplasia follow chronic TSH stimulation in vivo. An early study by Matovinovic and Vickery1 showed that the total number of cells in guinea pig thyroid glands was increased 2.5 times following 14 daily injections of TSH. Another study showed that the volume of the nucleus was increased threefold in guinea pigs within 2h of an injection of TSH although mitotic activity was not increased until the third day.2,3 Data from rats suggest that in adults there is a very slow turnover of the thyroid cell population. Growth of endothelial and mesenchymal cells was observed to accompany that of thyroid follicular cells when rats were given goitrogens.4 In man there are few in vivo studies examining direct effects of TSH on follicular cell growth. In patients expressing autoantibodies capable of stimulating the TSH receptor, hyperplasia rather than growth may account for the modest increase in size of the gland observed in some patients. The ability to culture cells in a hormone-free and serum-free environment has allowed a more minute examination of the growth process. In FRTL5 cells, a rat thyroid cell line maintaining some aspects of thyroid differentiated function, TSH was found to stimulate growth only when insulin or IGF-I was included in the incubation medium.5 In dog and human thyroid cells insulin is required with TSH in order for optimum expression of thyroid growth.6,7 In this paper we shall examine the synergism between insulin and IGF’s in stimulating sheep thyroid cell growth and function. The sheep thyroid cell culture system differs significantly from FRTL5, dog and human thyroid cell culture systems in that cells synthesize and secrete physiologic quantities of thyroid hormones de novo.8 In addition we have shown that these cultures condition the medium with relevant concentrations of both types of insulin-like growth factors,9,10,11, as well as the insulin-like growth factor binding proteins. The production of these proteins is regulated by hormones and in particular growth factors.10,11,12