Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models

TSH via cAMP, and various growth factors, in cooperation with insulin or IGF-I stimulate cell cycle progression and proliferation in various thyrocyte culture systems, including rat thyroid cell lines (FRTL-5, WRT, PC Cl3) and primary cultures of rat, dog, sheep and human thyroid. The available data on cell signaling cascades, cell cycle kinetics, and cell cycle-regulatory proteins are thoroughly and critically reviewed in these experimental systems. In most FRTL-5 cells, TSH (cAMP) merely acts as a priming/competence factor amplifying PI3K and MAPK pathway activation and DNA synthesis elicited by insulin/IGF-I. In WRT cells, TSH and insulin/IGF-I can independently activate Ras and PI3K pathways and DNA synthesis. In dog thyroid primary cultures, TSH (cAMP) does not activate Ras and PI3K, and cAMP must be continuously elevated by TSH to directly control the progression through G(1) phase. This effect is exerted, at least in part, via the cAMP-dependent activation of the required cyclin D3, itself synthesized in response to insulin/IGF-I. This and other discrepancies show that the mechanistic logics of cell cycle stimulation by cAMP profoundly diverge in these different in vitro models of the same cell. Therefore, although these different thyrocyte systems constitute interesting models of the wide diversity of possible mechanisms of cAMP-dependent proliferation in various cell types, extrapolation of in vitro mechanistic data to TSH-dependent goitrogenesis in man can only be accepted in the cases where independent validation is provided.     

Thyrotropin (TSH)-induced production of vascular endothelial growth factor in thyroid cancer cells in vitro: evaluation of TSH signal transduction and of angiogenesis-stimulating growth factors

Thyroid tumor growth requires angiogenesis, and vascular endothelial growth factor (VEGF) has been shown to be the most important endothelial mitogen. TSH is the major thyrotropic hormone, but its impact to modulate VEGF production has not yet been studied. Several other growth factors have also been shown to affect thyroid cancer cell growth and function in vitro. Therefore, the aim of the current study was to 1) establish the effect of TSH on VEGF as well as 2) evaluate the TSH signal transduction of this effect, and 3) screen other growth factors for the ability to modulate VEGF in thyroid cancer cell lines. HTC, a follicular cancer cell line lacking endogenous TSH receptor (TSHr), its receptor positive variant (HTC TSHr), and a cell line of Huerthle cell origin (XTC) were used. After stimulation with growth factors in vitro [TSH; epidermal growth factor (EGF), IGF, placenta growth factor, TGF-alpha, TGF-beta1, fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor] cells were analyzed for VEGF gene expression by Northern blotting and for VEGF protein by enzyme immunoassay. TSHr signal transduction was evaluated by analyzing the effect of stimulators (cholera toxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate) and inhibitors (2',5'-dideoxyadenosine and staurosporine) on VEGF protein levels under basal and TSH-stimulated conditions. TSH increased VEGF mRNA and protein in a dose-dependent manner in HTC TSHr and XTC cells by up to 40%. The effects of TSH were mediated by protein kinase C (PKC), rather than protein kinase A (PKA), stimulation, because inhibition of PKC by staurosporine resulted in a decrease in VEGF production of up to 65%, whereas inhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine) resulted in only a minor decrease. TSH was not the most powerful stimulator of VEGF production. TGF-beta1 and EGF were 1.5- to 2-fold more potent. Placenta growth factor and TGF-alpha did not induce VEGF production in TSHr-positive HTC cells, whereas they did induce VEGF production in TSHr-negative HTC cells. In thyroid cancer cell lines, TSH induces VEGF production involving the PKC, rather than the PKA, pathway. However, EGF and TGF-beta increase the capacity of thyroid cancer cells to provide VEGF more effectively than TSH. In the absence of a functioning TSHr, additional growth factors, such as TGF-alpha, increase capacity for VEGF stimulation.     

Independent and interactive effects of tetradecanoyl phorbol acetate on growth and differentiated functions of FRTL5 cells

In studies of regulation of the growth and differentiated function of the thyroid follicular cell, we have employed the FRTL5 cell line to evaluate both the effects of agents that activate protein kinase-C (PKC) and their interaction with other agents that influence the growth and/or function of the FRTL5 cell. The PKC activator tetradecanoyl-phorbol acetate (TPA) alone induced a time- and concentration-dependent stimulation of the incorporation of [3H]thymidine into the DNA of quiescent FRTL5 cells, an effect anteceded by an increase in the levels of the mRNAs of the proto-oncogene c-myc and associated with a stimulation of cell replication. TPA also produced a dose-dependent inhibition of the low levels of radioiodine uptake in quiescent FRTL5 cells. These effects of TPA were unaccompanied by any change in the cellular cAMP concentration. TPA also modified a variety of responses to TSH, attenuating the TSH-induced stimulation of [3H]thymidine incorporation into DNA, cell replication, cAMP generation, and iodine uptake. Inhibition of TSH-stimulated growth and iodine uptake by TPA could not be ascribed solely to a decrease in cAMP generation, since TPA also inhibited the increase in [3H]thymidine incorporation and iodide uptake induced by the cAMP analog (Bu)2cAMP. In contrast, the independent stimulatory effects of TPA and insulin-like growth factor I (IGF-I) on [3H]thymidine incorporation and cell replication were at least additive when the two stimulators were added together. We have previously reported that both TSH and (Bu)2cAMP amplify the enhancement of DNA synthesis and cell replication in FRTL5 cells induced by IGF-I, and that the response of DNA synthesis to IGF-I is also enhanced if cells are preincubated with either TSH or (Bu)2cAMP. Both the former amplification of mitogenesis and the latter priming effect were decreased by exposing cells to TPA concomitant with their exposure to TSH or (Bu)2cAMP. The effects of TPA were mimicked by other activators of PKC, but not by a phorbol ester that fails to activate this enzyme. In general, we conclude that in the FRTL5 cell, regulation of cell growth is extremely complex; there are at least three mitogenic pathways that are separate from but interact with one another. The first is the well known cAMP-dependent pathway, which is activated by TSH. The second is activated by IGF-I and is cAMP independent. These two pathways interact to produce a marked amplification of their individual mitogenic effects. The third pathway is that stimulated by TPA and involves activation of PKC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells

In previous studies, we showed that pretreatment of rat FRTL-5 thyroid cells with TSH, or other agents that increased intracellular cAMP, markedly potentiated DNA synthesis in response to insulin-like growth factor-I (IGF-I). In addition, we found that TSH pretreatment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kDa protein that was well correlated with the TSH-potentiating effect on DNA synthesis induced by IGF-I. These results suggested that cAMP amplified IGF-I-dependent signals for cell growth through changes of cAMP-dependent tyrosine phosphorylation. The present studies were undertaken to determine how tyrosine kinase activation followed by an increase in tyrosine phosphorylation is required for cAMP-dependent potentiation of DNA synthesis induced by IGF-I in this cell line. First of all, we measured tyrosine kinase or protein-tyrosine phosphatase activities in the cell lysates by the in vitro assay. Chronic treatment with TSH or (Bu)2-cAMP stimulated tyrosine kinase activity in the particulate fraction and protein-tyrosine phosphatase activity in the soluble fraction, suggesting that tyrosine kinase plays more important roles for a cAMP-dependent increase in tyrosine phosphorylation of intracellular proteins. The increased tyrosine kinase activity was sensitive to genistein, a potent tyrosine kinase inhibitor. Genistein abolished both the cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa protein and the enhanced DNA synthesis induced by IGF-I in a similar concentration-dependent manner. The only tyrosine-phosphorylated protein associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in response to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity bound to p85 subunit significantly increased after (Bu)2cAMP treatment. These results suggested that cAMP stimulates PI 3-kinase through tyrosine phosphorylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)2cAMP in the absence or presence of LY294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h. Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abolished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyrosine kinases that in turn activate PI 3-kinase activity. These mechanisms appear to be necessary for cAMP-dependent potentiation of the DNA synthesis induced by IGF-I.     

Iodine inhibits the proliferation of rat thyroid cells in culture

We have explored the mechanisms whereby iodine inhibits thyroid growth using as models both the FRTL5 line of rat thyroid follicular cells that require TSH for growth and the M12 line of mutant cells that grow in the absence of TSH. Between 0.01-1.0 mM, NaI produced a dose-dependent inhibition of TSH stimulation of [3H]thymidine incorporation and replication in FRTL5 cells as well as spontaneous growth in M12 cells. Iodide also inhibited the cAMP-dependent growth of FRTL5 cells induced by forskolin and (Bu)2cAMP, as well as the cAMP-independent mitogenesis induced by insulin-like growth factor-I. The effect of iodide to inhibit both TSH- and insulin-like growth factor-I-stimulated growth in FRTL5 cells was abolished by concomitant culture with methimazole, and no iodide inhibition of growth was observed in L6 myoblasts and BRL 30E hepatocytes. Exposure of cells to iodide under conditions that resulted in inhibition of TSH-stimulated growth did not significantly alter the ability of TSH to increase the intracellular cAMP concentration, nor did iodide alter two responses to TSH in FRTL5 cells that depend upon an increase in cAMP concentration: down-regulation of TSH receptor and cytoskeletal reorganization. We conclude that iodide exerts its inhibitory action on the growth of thyroid cells at multiple loci related to both the cAMP-dependent and cAMP-independent pathways of mitogenic regulation.     

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin.

We investigated the glucose transporter mRNAs expressed in FRTL5, a rat thyroid cell line, and their regulation by TSH by means of the polymerase chain reaction. FRTL5 cells as well as rat thyroid tissue expressed three types of glucose transporter mRNAs: GLUT1 or erythrocyte/HepG2/brain isoform, GLUT2 or pancreatic beta-cell/liver isoform, and GLUT4 or muscle/fat isoform. GLUT1 mRNA predominated, GLUT4 mRNA was minor, and GLUT2 mRNA expression was faint. Incubation of FRTL5 cells with TSH induced a time- and concentration-dependent increase in GLUT1 mRNA levels, while GLUT4 mRNA levels were decreased. The response of GLUT1 mRNA to TSH was evident at 3 h, and the maximal response was achieved at 12 h. TSH at a dose of 1 mU/ml elicited an approximately 3-fold increase in GLUT1 mRNA levels. (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and forskolin (50 microM) mimicked the effect of TSH on GLUT1 and GLUT4 mRNA levels. The increase in GLUT1 mRNA by TSH was correlated with the increase in GLUT1 protein and the increase in 2-deoxyglucose transport activity. These observations suggest that in thyroid cells, TSH stimulates glucose transport at least in part by enhancing GLUT1 gene expression, and that the effect of TSH on GLUT1 and GLUT4 mRNA levels is mediated by a cAMP-dependent pathway.     

Direct effect of methimazole on rat thyroidal cell growth induced by thyrotropin and insulin-like growth factor I

The direct effect of methimazole (MMI) on FRTL-5 cell growth was examined. TSH, (Bu)2cAMP, calf serum, insulin-like growth factor I, and Graves' immunoglobulin (IgG) increased [3H]thymidine incorporation into DNA during 72-h incubation. MMI (10(-3) M), which does not damage cell viability, significantly enhanced the increase in [3H]thymidine incorporation induced by TSH and (Bu)2cAMP. In contrast, MMI suppressed the increase in [3H]thymidine incorporation induced by calf serum, insulin-like growth factor I, and Graves' IgG. MMI had no effect on the production of cAMP by TSH. Accordingly, we concluded that MMI has opposite effects on cAMP- and non-cAMP-dependent cell growth pathways. Moreover, Graves' IgG, which has a modest effect on cAMP production, is believed to induce cell growth via the non-cAMP dependent cell growth pathway.     

Differential involvement of the actin cytoskeleton in differentiation and mitogenesis of thyroid cells: inactivation of Rho proteins contributes to cyclic adenosine monophosphate-dependent gene expression but prevents mitogenesis

In thyroid epithelial cells, TSH via cAMP induces a rounding up of the cells associated with actin stress fiber disruption, expression of differentiation genes and cell cycle progression. Here we have evaluated the role of small G proteins of the Rho family and their impact on the actin cytoskeleton in these different processes in primary cultures of canine thyrocytes. TSH and forskolin, but not growth factors, rapidly inactivated RhoA, Rac1, and Cdc42, as assayed by detection of GTP-bound forms. Using toxins that inactivate Rho proteins (toxin B, C3 exoenzyme) or activate them [cytotoxic necrotizing factor 1 (CNF1)], in comparison with disruption of the actin cytoskeleton by dihydrocytochalasin B (DCB) or latrunculin, two unexpected conclusions were reached: 1) inactivation of Rho proteins by cAMP, by disorganizing actin microfilaments and inducing cell retraction, could be necessary and sufficient to mediate at least part of the cAMP-dependent induction of thyroglobulin and thyroid oxidases, but only partly necessary for the induction of Na(+)/I(-) symporter and thyroperoxidase; 2) as indicated by the effect of their inhibition by toxin B and C3, some residual activity of Rho proteins could be required for the induction by cAMP-dependent or -independent mitogenic cascades of DNA synthesis and retinoblastoma protein (pRb) phosphorylation, through mechanisms targeting the activity, but not the stimulated assembly, of cyclin D3-cyclin-dependent kinase 4 complexes. However, at variance with current concepts mostly derived from fibroblast models, DNA synthesis induction and cyclin D3-cyclin-dependent kinase 4 activation were resistant to actin depolymerization by dihydrocytochalasin B in canine thyrocytes, which provides a first such example in a normal adherent cell.     

Adenosine 3',5'-monophosphate and phorbol ester induce transforming growth factor-beta 1 messenger ribonucleic acid levels in choriocarcinoma cells.

Transforming growth factor-beta 1 (TGF-beta 1), a regulator of growth and differentiation of many cell types, has previously been purified from the human placenta, and the messenger (m) RNA is abundantly expressed there. We found that the approximate 2.5-kilobase TGF-beta 1 mRNA is expressed in JEG-3 human choriocarcinoma cells, which have been widely used as a model system for studying the regulation of trophoblast hormone secretion. Cholera toxin (CT) elevates the cellular levels of the second messenger cAMP and increases the secretion of CG and steroids in these cells, thus being a potent inducer of trophoblast-differentiated functions. We show that CT also stimulates TGF-beta 1 mRNA levels in JEG-3 cells in a concentration- and time-dependent manner as studied by Northern and dot blotting. The maximal effect (about 5-fold increase above basal levels) occurs within 12-48 h of induction with a CT concentration of 1.0 ng/ml. The cell-permeable cAMP-analog 8-bromo-cAMP stimulates the accumulation of TGF-beta 1 mRNA in JEG-3 cells as well. Furthermore, this cAMP analog also induces TGF-beta 1 mRNA levels in normal cultured term placental cytotrophoblasts. 12-O-Tetradecanoyl phorbol-13-acetate, an active phorbol ester protein kinase C regulator and inducer of TGF-beta 1 mRNA in many cells, increases TGF-beta 1 mRNA accumulation in JEG-3 cells with a similar time course as cAMP analogs but to a lesser extent. Human HT-1080 fibrosarcoma and A-549 lung carcinoma cells exhibit up-regulation of TGF-beta 1 mRNA in response to TGF-beta 1 itself, but we show that activation of the cAMP-dependent pathway does not affect TGF-beta 1 mRNA levels in these cells. Cycloheximide, an inhibitor of protein synthesis, prevents the effect of CT and 12-O-tetradecanoyl phorbol-13-acetate on TGF-beta 1 mRNA expression in JEG-3 cells, suggesting that a protein mediator may be involved in the transduction of their effects. Our finding of a cAMP-dependent induction pathway for TGF-beta 1 mRNA expression in JEG-3 cells provides a new mechanism for the regulation of the synthesis of this ubiquitous growth and differentiation factor and suggests that TGF-beta 1 may have a role in trophoblast differentiation.     

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.     

Transferrin in FRTL5 cells: regulation of its receptor by mitogenic agents and its role in growth

Transferrin, a serum iron-binding protein, delivers iron to the cell after binding to specific receptors on the cell surface and is an important component of culture medium for virtually all cell lines, including the FRTL5 line of rat thyroid follicular cells. Therefore, we undertook studies in FRTL5 cells to examine the regulation of the transferrin receptor, the effects of transferrin on growth and differentiated functions, and the interactions of transferrin with several mitogenic pathways. FRTL5 cells possess one class of saturable transferrin receptors (Ka, 0.7 x 10(9) M-1). Binding of 125I-labeled transferrin was highest in actively growing cells and declined progressively, reaching minimal values when confluence was achieved. Removal of transferrin from culture medium caused a rapid increase in transferrin binding. TSH, acting within 5 min, induced a modest increase in transferrin binding, due to a cycloheximide-resistant increase in binding sites. Binding of transferrin after a 24-h incubation was also increased by other mitogenic agents, (Bu)2cAMP, forskolin (FK), insulin, insulin-like growth factor-I (IGF-I), and the phorbol ester TPA. Transferrin alone stimulated growth only minimally, but enhanced the mitogenic effect of TSH, (Bu)2cAMP, and FK, all of which act through the cAMP pathway. In contrast, transferrin did not alter the cAMP-independent mitogenic effects of insulin and IGF-I. Transferrin did not affect TSH-induced cAMP generation. Desferoxamine, an iron chelator, inhibited the mitogenic effects of all of the agents tested. Desferoxamine had no significant effect on TSH-induced cAMP accumulation. We conclude that FRTL5 cells contain saturable receptors for transferrin whose abundance varies with the rate of cell replication. Transferrin down-regulates its own receptors, while stimulation of growth by various mitogens is accompanied by increased binding of transferrin. Transferrin enhances the mitogenic effect of the cAMP-dependent mitogens, TSH, (Bu)2cAMP, and FK, without modifying basal or stimulated cAMP generation. In contrast, transferrin fails to affect the mitogenic responses to IGF-I and insulin, which are cAMP independent. Iron is required for the mitogenic response to various mitogens, especially those that are cAMP dependent.     

Effects of rat gamma- and non-gamma-interferons on the expression of Ia antigen, growth, and differentiated functions of FRTL5 cells

We undertook the present studies with several objectives in mind: 1) to determine whether recombinant rat gamma-interferon (r gamma IFN) would induce expression of the class II major histocompatibility antigen (Ia) in rat thyroid follicular cells (FRTL5) in culture as human gamma IFN does in cultured human thyrocytes; 2) to characterize the properties of this response, if it does indeed occur; 3) to ascertain whether r gamma IFN has any effect on the growth or differentiated function of FRTL5 cells; and 4) to determine how, if at all, effects of r gamma IFN on the growth and function of FRTL5 cells might be related to expression of the Ia antigen. At concentrations between 1 and 30 U/ml, r gamma IFN induced expression of Ia antigen in a concentration-dependent manner. With a supramaximal concentration of r gamma IFN, Ia antigen first appeared between 4 and 16 h and reached a maximum concentration at about 36 h. After removal of r gamma IFN, the Ia antigen concentration remained constant for about 24 h and then declined, becoming undetectable by 72 h. Induction could not be detected in FRTL5 cells cultured with human gamma IFN, rat non-gamma IFN, Concanavalin-A, phytohemagglutinin, or bovine TSH (bTSH). Over the same range of concentrations that induced the Ia antigen, r gamma IFN proved to be a potent inhibitor of the growth of FRTL5 cells induced by a variety of agents. It produced a concentration-dependent inhibition of the stimulation of [3H] thymidine incorporation and cell replication in FRTL5 cells induced by bTSH. This effect was unaccompanied by any inhibition of either the binding of bTSH to FRTL5 cells or the bTSH-induced increase in cellular cAMP concentration induced therein. However, r gamma IFN did inhibit the stimulation of [3H] thymidine incorporation into DNA induced by (Bu)2cAMP. r gamma IFN also inhibited the stimulation of DNA synthesis and cell replication induced by insulin-like growth factor I (IGF-I) without affecting the specific binding of IGF-I, and decreased the extent of stimulation of [3H]thymidine incorporation induced by the phorbol ester tetradecanoyl phorbol acetate (TPA). Thus, r gamma IFN inhibited both the cAMP-dependent pathway of growth activated by TSH, doing so at some post-cAMP locus, and the cAMP-independent pathways of growth regulation that are activated by IGF-I and TPA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mitogenic effects of thyrotropin and adenosine 3',5'-monophosphate in differentiated normal human thyroid cells in vitro

Previous studies of human thyroid cells in culture (mostly from pathological tissues) failed to demonstrate a mitogenic effect of TSH, leading to the proposal that the growth effect of TSH in vivo might be indirect. To reexamine the influence of TSH on DNA synthesis and cell proliferation, we established primary cultures of normal thyroid tissue from nine subjects. When seeded in a 1% serum-supplemented medium, thyroid follicles released by collagenase/dispase digestion developed as a cell monolayer that responded to TSH by rounding up and by cytoplasmic retraction. When seeded in serum-free medium, the cells remained associated in dense aggregates surrounded by few slowly spreading cells. In the latter condition, the cells responded to TSH and other stimulators of cAMP production, such as cholera toxin and forskolin, by displaying very high iodide-trapping levels. Exposure to serum irreversibly abolished this differentiated function. TSH stimulated the proliferation (as shown by DNA content per culture dish) of 1% serum cultured cells (doubling times were reduced from 106 to 76 h) and increased by 100% the [3H]thymidine labeling indices. In serum-free cultured cells (dense aggregates or cell monolayers after initial seeding with serum), control levels of DNA synthesis were lower, and up to 8-fold stimulation of DNA synthesis occurred in response to 100 mU/L TSH (stimulation was consistently detected with 20 mU/L), based on measurements of [3H]thymidine incorporation into acid-precipitable material and counts of labeled nuclei on autoradiographs (up to 40% labeled nuclei within 24 h). The mitogenic effect of TSH required a high insulin concentration (8.3 X 10(-7) mol/L) or a low insulin-like growth factor I concentration. The mitogenic effects of TSH were mimicked in part by cholera toxin, forskolin, and dibutyryl cAMP. Epidermal growth factor and phorbol myristate ester also stimulated thyroid cell proliferation and DNA synthesis, but they potently inhibited TSH-stimulated iodide transport. We conclude that TSH, acting at least in part through cAMP, is a potent growth factor for human thyroid cells and thus provide an experimental basis in vitro for the well established in vivo goitrogenic action of TSH.     

Effect of thyrotropin and cAMP on FRTL5 cell growth in a serum free medium

TSH-induced proliferation of FRTL5 cells was studied in a chemically serum-free, defined medium. FRTL5 cells incubated for several days in a medium lacking of serum and hormones were fully able to respond to TSH in terms of cAMP production and iodine uptake. In the same medium, TSH stimulated FRTL5 cell growth as assessed by thymidine incorporation, DNA content and cell count. In these experimental conditions the cellular doubling time was of 7.5 days as compared to 40 h when calf serum was present together with TSH. Cholera toxin, forskolin and (bu)2cAMP, substances able to selectively increase intracellular cAMP levels, were not as efficient as TSH in inducing FRTL5 cell growth. However, both TSH and (bu)2cAMP, in a similar way, induced c-myc gene expression and cellular progression through the prereplicative phase of cell division. These data demonstrate that cAMP-dependent mechanisms are only partially responsible for TSH-induced FRTL5 cell growth, thus suggesting a role of cAMP-independent mechanisms.     

hCG-induced TSH receptor activation and growth acceleration in FRTL-5 thyroid cells

Our previous studies have indicated specificity cross-over between LH/hCG and the TSH receptor. We have now analyzed the ability of the TSH-dependent Fisher Rat thyroid cell line (FRTL-5) to proliferate in a differentiated state under the influence of highly purified hCG (hCG-CR121). TSH receptor activation and growth induction were observed after 7 days suspension from a TSH-induced growth phase. The effect of 10 ug hCG-CR121 was equivalent to 500 +/- 43 (mean +/- SEM) uIU of human TSH (2nd IRP, 80/558) with reference to growth stimulation, as judged by 72-h [3H]-thymidine uptake and to approximately 15 +/- 35 uIU human TSH with reference to receptor activation as judged by cyclic AMP accumulation. These data demonstrate specificity cross-over by hCG for the TSH-dependent FRTL-5 cell line and suggest that hCG has greater growth-stimulating than thyroid-stimulating potential when compared with human TSH.     

Thyrotropin stimulates the generation of inositol 1,4,5-trisphosphate in human thyroid cells

CONTEXT: Dual activation by TSH of the phospholipase C and cAMP cascades has been reported in human thyroid cells. In contrast, Singh et al. reported convincing data in FRTL-5 thyrocytes arguing against such an effect in this model. Their data in FRTL-5 cells indicated no increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to TSH. Therefore, the authors questioned results previously obtained on human cells by cruder methodology. OBJECTIVE: We investigated the formation of inositol phosphates by HPLC techniques in human thyroid slices to separate the inositol phosphate isomers. RESULTS: Ins(1,4,5)P3, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate were increased after TSH stimulation. The effect of TSH in human thyroid cells was reproduced by recombinant TSH and prevented by antibodies blocking the TSH receptor. Thyroid-stimulating antibodies at concentrations eliciting a cAMP response equivalent to TSH failed to stimulate inositol phosphate generation. CONCLUSIONS: TSH, but not thyroid-stimulating antibodies, activates both cAMP and the phospholipase C cascade in human thyroid as now demonstrated by an increase in Ins(1,4,5)P3 and its inositol phosphate metabolites. Therefore, this effect cannot be extrapolated to the FRTL-5 cell line. The apparent discrepancy may be due to a difference between species (human vs. rat) or to the loss of the fresh tissue properties in a cell line. The dual effect of TSH in human cells, through cAMP on secretion of thyroid hormones and through the diacylglycerol, Ins(1,4,5)P3 Ca2+ pathway on thyroid hormone synthesis, implies the possible separation of these effects in thyroid disease.     

Decrease in cAMP-dependent protein kinase activity in suspension cultures of porcine thyroid cells exposed to TSH or forskolin

Suspension cultures of porcine thyroid cells were used to study the action of TSH and forskolin (Fk) on cAMP-dependent (PKa) and Ca2+-phospholipid-dependent (PKc) protein kinase--enzymes which represent the key step in the transduction of extracellular signals. The PKa activity in cells cultured for 2 days in the presence of TSH was decreased to about 50% of control level with a TSH dose of 0.1 mU/ml. This decrease is dose dependent; only traces of PKa activity remained at very high doses of TSH (50 mU/ml). Similar results were obtained with Fk (10(-5) M), the adenylate cyclase activator. It decreased the PKa activity to the level obtained with 0.1-1.0 mU/ml TSH. The loss of the PKa activity was parallel in cytosol and particulate fractions, suggesting that there is no translocation of enzymes under the action of either TSH or Fk. Neither TSH nor Fk had any effect on PKc, which became the predominant activity in cells exposed to either of the regulators. The cAMP-dependent phosphorylation of endogenous proteins was lower in TSH- or Fk-treated cells than in controls, and was dependent, like the PKa activity, on the dose of TSH. Polyacrylamide gel electrophoresis (PAGE) revealed the specific substrates of PKa in cultured thyroid cells. Proteins of 28, 30 and 33 kDa were regularly found, while 58 kDa protein was not present in all experiments. PAGE patterns showed that the decrease in endogenous phosphorylation in TSH- and Fk-treated cells was due to decreased labelling of PKa-specific substrates. The observed down-regulation of PKa activity could have an influence on the expression of thyroid cell differentiation.     

Control of thyroid secretion: effects of stimulators of protein kinase C, thyrotropin, and calcium mobilization on secretion of iodinated compounds from sheep thyroid cells.

We have compared and contrasted the abilities of TSH and agents capable of discretely activating the cAMP-dependent protein kinase, protein kinase C, or calcium mobilization to influence the secretion of iodinated compounds from cells prelabeled with iodide and blocked from further organification with methimazole. We found that calcium mobilization induced by A23187, protein kinase C activation induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) and TSH all stimulated the secretion of iodinated compounds. The effects of TSH were mimicked by forskolin and those of TPA by a synthetic diacylglycerol, sn-1,2-dioctanoylglycerol. The effects of TPA were partially inhibited by staurosporine whereas those of TSH were not. Epidermal growth factor and norepinephrine were without effect on thyroid secretion. The effects of A23187 and TPA were synergistic. The effects of TSH and TPA were not and the increased secretion induced by either agent was partially prevented by the combination. Preincubation of cells with TSH desensitized the cells to further stimulation by TSH but the stimulatory effects of TPA were unaffected. Exposure of cells to medium without calcium also induced loss of iodinated compounds which was partially prevented by TSH or forskolin but not TPA. TSH did not stimulate the rapid production of inositol trisphosphate production. We conclude that the mechanisms by which TSH (through stimulation of cAMP) and stimulators of other intracellular pathways exert their effects on secretion of iodocompounds, differ. Activation of protein kinase C and acute production of inositol trisphosphate do not appear to be involved in the mechanism of action of TSH in stimulating thyroid secretion but calcium mobilization is implicated.