TSH is able to induce cell cycle-related gene expression in rat thyroid cell.

Rat thyroid cells in culture (FRTL-5 strain) require thyrotropic hormone (TSH) for growth. TSH alone in serum free medium is able to induce DNA synthesis of FRTL-5 cells. DNA synthesis occurs 18-20 hours following TSH stimulation of quiescent cells. Here we demonstrate that two sets of genes, related to the entry of cells in the S phase, are induced by TSH: 1) immediate early genes, such as c-jun and a gene coding for a zinc-finger protein Xrox 20/Egr2, both having a pattern of expression similar to the c-fos oncogene; 2) early delayed genes such as ornithine decarboxylase (ODC), 2F-1, a gene that shows a strong similarity in aminoacid sequence to a mitochondrial ADP/ATP carrier, and the asparagine synthetase gene (TS11). Furthermore, an increased expression of the histone H3 gene, a typical marker of S phase, has been observed in TSH-treated FRTL-5 cells.     

Thyroid-stimulating hormone stimulates increases in inositol phosphates as well as cyclic AMP in the FRTL-5 rat thyroid cell line.

Studies were conducted to determine whether thyroid-stimulating hormone (TSH; thyrotropin), a hormone known to increase cytosol concentrations of cyclic AMP, also stimulates the formation of inositol phosphates in thyroid cells. TSH and noradrenaline both stimulated [3H]inositol phosphate formation in a concentration-dependent manner in the rat thyroid cell line, FRTL-5 cells, which had been prelabelled with [3H]inositol. The threshold concentration of TSH required to stimulate inositol phosphate formation was more than 20 munits/ml, which is approx. 10(3)-fold greater than that required for cyclic AMP accumulation and growth in these cells. We also demonstrate that membranes prepared from FRTL-5 cells possess a guanine nucleotide-activatable polyphosphoinositide phosphodiesterase, which suggests that activation of inositide metabolism in these cells may be coupled to receptors by the G-protein, Gp. Our findings suggest that two second-messenger systems exist to mediate the action of TSH in the thyroid.     

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.     

Astereological analysis of FRTL-5 cells as an effect of thyrotropin and irradiation

FRTL-5 cell line is a cloned epithelial follicular cell line from Fischer rat thyroids. This cell line expresses many of the thyroid differentiated markers in vitro. Their growth and function depend on thyrotropin (TSH) as the main regulatory hormone. In this astereological analysis, the authors concentrate on FRTL-5 nuclei as the most vulnerable part of the cell. Using morphometrical variables, they wished to discover the morphologically identifiable sign of transformation of FRTL-5 cells after irradiation and to study the effect of different TSH concentrations. FRTL-5 cells were grown in a medium of 4 different concentrations of TSH (0, 0.1, 1, 10 mU/ml) and irradiated with 0 Gy, 2 Gy, and 4 Gy. The results showed that the nuclear-cytoplasmic ratio decreases after irradiation with doses of 4 Gy or if TSH was included in the medium. The nuclear maximum diameter of FRTL-5 cells increased with higher concentrations of TSH more obviously after irradiation with 4 Gy than with 2 Gy. On the basis of astereological analysis, it was concluded that different concentrations of TSH and irradiation exert an effect especially upon FRTL-5 cell nuclei. The possible transformation of FRTL-5 cells after culturing in TSH medium and after irradiation could be confirmed by injection into an animal of the Fischer strain.     

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.     

Differential effects of growth factors on [3H]thymidine incorporation and [125I]iodine uptake in FRTL-5 rat thyroid cells

We studied the effect of several growth factors on DNA synthesis and function of FRTL-5 rat thyroid cells by simultaneous measurement of [3H]thymidine incorporation and [125I]iodide uptake. Endothelial cell growth factor, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor I stimulated thymidine incorporation in a dose-dependent manner without the parallel increase of [125I]iodide uptake. These growth factors had an additive effect with thyroid-stimulating hormone (TSH) on thymidine incorporation, but they inhibited TSH-stimulated iodide uptake. Bombesin stimulated thymidine incorporation and inhibited TSH-stimulated iodide uptake; epidermal growth factor and gastrin-releasing peptide 10 had neither effect. None of the growth factors studied affected iodide uptake in the absence of TSH. Of the growth factors tested, endothelial cell growth factor, fibroblast growth factor, insulin-like growth factor bombesin, and platelet-derived growth factor all share similar differential effects on FRTL-5 cells: stimulation of DNA synthesis, potentiation of the effects of TSH on DNA synthesis, and attenuation of the effects of TSH on cell function. The data suggest that these growth factors may play important roles in regulation of thyroid function.     

Thyrotropin modulates low density lipoprotein binding activity in FRTL-5 thyroid cells

FRTL-5 cells possess high affinity low density lipoprotein (LDL) receptors which bind, internalize, and degrade LDL. When FRTL-5 cells are deprived of thyrotropin (TSH) the binding of LDL increases more than 2-fold. Upon addition of TSH, at a concentration of 1 x 10(-10) M or greater, LDL binding decreases rapidly and within 24 h reaches the level which is typical of FRTL-5 cells chronically stimulated by TSH. The data available suggest that TSH-dependent down-regulation of LDL receptor activity is exerted through a reduction of the number of active LDL receptors, with no change in affinity. It is unlikely that the synthesis of LDL receptors is impaired, since LDL receptor messenger RNA is not decreased by TSH. The effect of the hormone on LDL receptor activity can be mimicked by 8-Br-cAMP and is completely abolished by the protein synthesis inhibitor cycloheximide but not by actinomycin D. TSH regulation of LDL receptor activity is lost in v-ras Ki-transformed FRTL-5 cells (Ki Mol) which also have lost TSH dependence for adenylate cyclase activation and growth. However, 8-Br-cAMP decreases LDL binding in Ki Mol FRTL-5 cells. The reduced availability of LDL receptor in TSH-stimulated FRTL-5 cells may be related to the increased membrane fluidity (Beguinot, F., Beguinot, L., Tramontano, D., Duilio, C., Formisano, S., Bifulco, M., Ambesi-Impiombato, F. S., and Aloj, S. M. (1987) J. Biol. Chem. 262, 1575-1582) or may reflect increased degradation of LDL receptors. We propose that a lower cholesterol uptake is needed in an actively proliferating cell population, to increase the production of isoprenoids whether it be for cholesterol biosynthesis or for the synthesis of other compounds requiring isoprenoid precursors.     

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.     

Thyroid peroxidase: rat cDNA sequence, chromosomal localization in mouse, and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells

A rat thyroid peroxidase cDNA has been isolated from a FRTL-5 thyroid cell library and sequenced. The cDNA is 2776 base pairs long with an open reading frame of 770 amino acids. By comparison to full-length human thyroid peroxidase cDNA and based on its identification of a 3.2 kilobase mRNA in rat thyroid FRTL-5 cell Northern blots, the rat peroxidase cDNA appears to lack 400-500 base pairs at the 5'-end of the mRNA. It exhibits only a 74% nucleotide and 77% amino acid sequence similarity to human thyroid peroxidase cDNA within the total aligned sequences, although the predicted active site regions are highly conserved (greater than 90-100%). The cDNA has been used to map the thyroid peroxidase gene in mice to chromosome 12 and to compare thyroid peroxidase and thyroglobulin gene expression in FRTL-5 rat thyroid cells. Despite the fact TSH action in both cases is duplicated, and presumably mediated, by cAMP, TSH-induced increases in thyroid peroxidase and thyroglobulin mRNA levels differ. Differences exist with respect to hormone concentration and time. The ability of TSH to increase thyroglobulin, but not thyroid peroxidase mRNA levels, requires insulin, 5% serum, or insulin-like growth factor-I. Insulin or insulin-like growth factor-I alone can increase thyroglobulin mRNA levels as well as or better than TSH but have only a small effect on thyroid peroxidase mRNA levels by comparison to TSH. The ability of TSH to increase thyroglobulin gene expression is readily detected in nuclear run-on assays but not the ability of TSH to increase thyroid peroxidase gene expression. Cycloheximide inhibits TSH-increased thyroglobulin but not peroxidase mRNA levels. Finally, methimazole and phorbol 12-myristate 13-acetate show different effects on TSH-induced increases in thyroglobulin and thyroid peroxidase mRNA levels.     

Modulation of the carbohydrate moiety of thyroglobulin by thyrotropin and calcium in Fisher rat thyroid line-5 cells.

Thyroglobulin secreted in the medium by Fisher rat thyroid line-5 (FRTL-5) cells cultured in the presence of thyroid stimulating hormone (TSH) shows a slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a higher density position in a CsCl gradient than thyroglobulin secreted by FRTL-5 cells cultured in the absence of TSH for 5-7 days. Such a TSH effect is much less or not evident when secreted thyroglobulin is digested with peptide N-glycohydrolase F or when intracellular thyroglobulin is compared. Intracellular thyroglobulin migrates faster than thyroglobulin secreted either in the presence or in the absence of TSH. Evaluation of the mannose and galactose content of thyroglobulin demonstrates that intracellular thyroglobulin has more mannose and less galactose than extracellular thyroglobulin; it also shows that TSH decreases the mannose content of thyroglobulin while increasing its galactose content. Bio-Gel P6 chromatography shows that TSH increases the complex type carbohydrate chains while decreasing the high mannose chains in the secreted thyroglobulin. High mannose type oligosaccharides were characterized by fast atom bombardment-mass spectrometry analysis. Treatment with the calcium ionophore A23187 (5 microM) of FRTL-5 cells cultured with or without TSH causes the appearance of a "fast" migrating form of thyroglobulinin in the culture medium. Bio-Gel P6 chromatography shows that A23187 causes a dramatic decrease of the complex carbohydrate chains of the secreted thyroglobulin.     

Peptide sequences from the hypervariable regions of two monoclonal anti-idiotypic antibodies against the thyrotropin (TSH) receptor are similar to TSH and inhibit TSH-increased cAMP production in FRTL-5 thyroid cells.

Monoclonal antibodies, D2 and 4G11, selected by the autoantiidiotypic approach following injection of thyrotropin (TSH) into mice, mimic TSH in binding to receptors on thyroid membranes. Based on TSH receptor transfection studies, D2 and 4G11 show unequivocal specificity for the TSH receptor. To see if the complementary determining regions (CDRs) of these antibodies share any primary sequence similarities to regions of TSH critical for receptor binding, we deduced the primary structure of the variable regions of D2 and 4G11 by sequencing the immunoglobulin mRNA. We found that CDR1 of 4G11K and CDR2 of D2 mu show sequence similarity to regions of TSH alpha and TSH beta that had been previously implicated in the interaction of the hormone with its receptor. We tested the inhibitory effects of synthetic peptides from D2 mu-CDR2 and 4G11K-CDR1 on the binding of the corresponding antibodies to rat thyroid FRTL-5 cells and found an EC50 of 0.1 and 1 microM, respectively. TSH-derived peptides with similarity to D2 mu-CDR2 and 4G11K-CDR1 showed a significant but lesser effect on the binding of 4G11 or D2 to thyroid cells. Additionally, we tested the effects of the CDR peptides and TSH-derived peptides on TSH-stimulated cAMP production in FRTL-5 cells and found that D2 mu-CDR2 and 4G11K-CDR1 inhibited this activity, D2 mu-CDR2 most strongly (EC50 10 microM). Thus, linear sequences from the CDRs of these autoantiidiotypic antibodies with similarity to sequences from both subunits of TSH appear to interact with the TSH receptor. These data support previous studies indicating the complexity of the interaction between TSH and its receptor and advance earlier findings that such immunologic approaches are useful in dissecting receptor-ligand interactions.     

Regulation of prostaglandin synthesis by thyrotropin, insulin or insulin-like growth factor-I, and serum in FRTL-5 rat thyroid cells.

The present report shows that thyrotropin (TSH) regulates all three steps involved in prostaglandin synthesis in FRTL-5 rat thyroid cells, i.e. arachidonic acid release from membrane phospholipids, cyclooxygenase (prostaglandin H synthase) action, and individual prostaglandin formation; however, its action at specific steps may require the presence of, or can be duplicated by, insulin, insulin-like growth factor-I (IGF-I), and/or a serum factor. Thus, TSH releases free arachidonic acid from rat FRTL-5 thyroid cells whose phospholipid fraction is radiolabeled with [3H]arachidonic acid; this action involves a pertussis toxin-sensitive G protein, is not cAMP mediated, and does not require insulin or 5% serum. To quantitate TSH effects on cyclooxygenase activity and on individual prostaglandin formation, a homogenate system and a rapid reversed-phase high pressure liquid chromatography procedure have been developed to measure cyclooxygenase metabolites. TSH increased cyclooxygenase activity in homogenates only if the cells were also exposed to insulin, IGF-I, and/or 5% calf serum; TSH alone had no apparent effect on the activity. Maximal activation, 4-fold over basal/micrograms of DNA, took 36 h to achieve and reflected, at least in part, an increase in cyclooxygenase gene expression. Like cyclooxygenase activity, induction of prostaglandin E2 production required 2 or more factors, i.e. TSH plus insulin/IGF-I or TSH plus insulin/IGF-I plus serum. Increased production of prostaglandin D2, could, however, be detected if cells were treated with TSH alone and the TSH activity could be duplicated by insulin, IGF-I, or calf serum alone.     

Somatostatin interferes with thyrotropin-induced G1-S transition mediated by cAMP-dependent protein kinase and phosphatidylinositol 3-kinase. Involvement of RhoA and cyclin E x cyclin-dependent kinase 2 complexes

cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.     

Ischemia-responsive protein (irp94) is up-regulated by endoplasmic reticulum stress

The expression of the ischemia-responsive protein (irp94) was enhanced by endoplasmic reticulum (ER) stress inducing drugs such as brefeldin A (BFA), calcium ionophor A23187, dithiothreitol (DTT) and tunicamycin in fisher rat thyroid epithelial cell line (FRTL-5 cells). In particular, irp94 mRNA expression was increased dose dependently by tunicamycin, and there was increased irp94 expression when the cells were incubated with the thyroid-stimulating hormone (TSH) together.     

Thyrotropin increases malic enzyme messenger ribonucleic acid levels in rat FRTL-5 thyroid cells

The addition of TSH to FRTL-5 thyroid cells induces a 7- to 8-fold increase in the steady state level of malic enzyme [L-malate-NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] mRNA, but does not alter beta-actin mRNA levels. Insulin alone or together with TSH has no effect on malic enzyme mRNA. The effect of TSH is not the result of thyroid hormone formation, since the addition of T3 in the presence or in the absence of TSH and the addition of 5% serum (which includes T3 and T4) have no effect. Forskolin (10(-6) M) reproduces the TSH effect, suggesting that cAMP is involved.     

Regulation of geranylgeranyl pyrophosphate synthase in the proliferation of rat FRTL-5 cells: involvement of both cAMP-PKA and PI3-AKT pathways

We have reported that geranylgeranyl pyrophosphate (GGPP), one of the isoprenoids in the mevalonate pathway, plays an essential role for cell growth through the geranylgeranylation of Rho small GTPases, which control the degradation of P27Kip1 at G1/S transition in rat thyroid FRTL-5 cells. Since GGPP is synthesized from isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP) by GGPP synthase, we analyzed the regulatory roles of GGPP synthase in the proliferation of FRTL-5 cells stimulated by thyrotropin and insulin in the presence of 5% calf serum (TSH+Ins). We found that: (1) GGPP synthase was activated at G1/S transition with increasing mRNA accumulation followed by protein expression, (2) pravastatin, an inhibitor of HMG-CoA reductase, did not suppress the increasing activity of GGPP synthase with its protein expression although it inhibits proliferation in growth-stimulated FRTL-5 cells, (3) forskolin stimulated proliferation with activation of GGPP synthase in FRTL-5 cells, and (4) LY294002, an inhibitor of phosphatidylinositol 3-kinase, inhibited proliferation with the decreasing activity of GGPP synthase in growth-stimulated FRTL-5 cells. These data indicated that growth stimulation by TSH+Ins increased the activity of GGPP synthase with its increasing protein expression from G1/S transition, in which both cAMP-PKA and PI3-kinase pathways are involved in the proliferation of FRTL-5 cells.