TSH stimulates the activity of the c-fos promoter in FRTL5 rat thyroid cells

C-fos promoter activity was examined in FRTL5 rat thyroid cells transiently transfected with a chimeric gene containing the c-fos promoter region linked upstream of the receptor gene chloroamphenicol acetyl transferase (CAT). Thyroid-stimulating hormone (TSH) stimulation of transfected cells increased CAT activity 2- to 3-fold. TSH did not stimulate CAT activity driven by the Rous sarcoma virus (RSV) promoter. Both forskolin and 8-Br-cyclic AMP also stimulated CAT activity, and were not additive with TSH stimulation. The kinetics of c-fos-driven CAT activity in response to TSH and cyclic AMP inducers were similar, and stimulation was reversible. These data therefore provide the first evidence that TSH and cyclic AMP stimulate the activity of the c-fos promoter in FRTL5 cells.     

Immunoglobulins from Graves' patients stimulate phospholipase-A2 in FRTL5 thyroid cells.

The well documented ability of immunoglobulins G (IgGs) from Graves' patients to stimulate cAMP production is believed to be involved in the pathophysiology of this disease. It is still under discussion whether other intracellular messengers known to regulate thyroid function might play a similar role. This study shows that phospholipase-A2, a signal pathway unrelated to cAMP, is activated by Graves' IgGs. The IgGs from 67 patients with active Graves' disease, 8 patients with Graves' disease in remission, 5 patients with idiopathic myxedema, 2 patients with Hashimoto's thyroiditis, 57 patients with nonautoimmune thyroid disease, and 65 normal subjects were tested for their ability to stimulate phospholipase-A2 activity, as measured by arachidonic acid release from FRTL5 thyroid cells. The IgGs from patients with active Graves' disease caused a significant increase in arachidonic acid release compared to those from normal subjects, patients with nonautoimmune thyroid diseases, and patients with Graves' disease in remission (P less than 0.0001). The IgGs from active Graves' patients were also able to increase cAMP accumulation in FRTL5 cells. This effect did not correlate with the ability of the same IgGs to induce arachidonic acid release, suggesting that Graves' IgGs stimulate these two pathways by separate mechanisms. Moreover, a subgroup of IgGs that stimulated phospholipase-A2 did not increase the cAMP levels in FRTL5 cells. Our data suggest a novel mechanism of action of Graves' IgGs, the activation of phospholipase-A2, well distinguishable from the known effect on cAMP accumulation. The assay we describe could be helpful in improving the diagnosis and therapy of Graves' disease and in distinguishing it from nonautoimmune thyroid diseases. It also supplies the basis for a prospective subclassification of the Graves' patients, which might become useful to clarify the pathophysiology of this disease.     

3-Iodothyronamine metabolism and functional effects in FRTL5 thyroid cells

3-Iodothyronamine (T(1)AM), produced from thyroid hormones (TH) through decarboxylation and deiodination, is a potent agonist of trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor belonging to the family of TAARs. In vivo T(1)AM induces functional effects opposite to those produced on a longer time scale by TH and might represent a novel branch of TH signaling. In this study, we investigated the action of T(1)AM on thyroid and determined its uptake and catabolism using FRTL5 cells. The expression of TAAR1 was determined by PCR and western blot in FRTL5 cells, and cAMP, iodide uptake, and glucose uptake were measured after incubation with increasing concentrations of T(1)AM for different times. T(1)AM and its catabolites thyronamine (T(0)AM), 3-iodothyroacetic acid (TA(1)), and thyroacetic acid (TA(0)) were analyzed in FRTL5 cells by HPLC coupled to tandem mass spectrometry. The product of amplification of TAAR1 gene and TAAR1 protein was demonstrated in FRTL5 cells. No persistent and dose-dependent response to T(1)AM was observed after treatment with increasing doses of this substance for different times in terms of cAMP production and iodide uptake. A slight inhibition of glucose uptake was observed in the presence of 100 μM T(1)AM after 60 and 120 min (28 and 32% respectively), but the effect disappeared after 18 h. T(1)AM was taken up by FRTL5 cells and catabolized to T(0)AM, TA(1), and TA(0) confirming the presence of deiodinase and amine oxidase activity in thyroid. In conclusion, T(1)AM determined a slight inhibition of glucose uptake in FRTL5 cells, but it was taken up and catabolized by these cells.     

Influence of cytokines on growth and differentiated function of FRTL5 cells

A functioning rat thyroid cell line (FRTL5) was used to study interactions of TSH with interferon-gamma (IFN gamma) and tumor necrosis factor-alpha (TNF alpha). We examined effects on growth and differentiated function. Growth was assessed by DNA, incorporation of [3H]thymidine ([3H]Tdr) into DNA, and cell number; uptake of 125I (I- uptake) and the concentration of cAMP were measured simultaneously with growth assessment. IFN gamma stimulated the 30-min I- uptake and enhanced the effect of TSH. TNF alpha had minimal effects on growth indices (slight increase in [3H]Tdr incorporation) and had no influence on I- uptake; it inhibited TSH stimulation of both growth and I- uptake. When combined, IFN gamma and TNF alpha synergized in inhibiting TSH-stimulated growth. By itself TNF alpha inhibited stimulation of I- uptake by TSH, but augmented the enhancement seen with IFN gamma. The influence of calf serum (CS) was to increase the rate of incorporation of [3H]Tdr, but a similar qualitative pattern for the actions of the cytokines remained. A reverse profile (stimulation by IFN gamma, inhibition by TNF alpha, and stimulation by the combination) was seen for I- uptake, with CS increasingly diminishing all values. TSH stimulation of growth was progressively effective with increments of CS in the medium, but consistently there was inhibition that was greater with IFN gamma than with TNF alpha and was almost total with the combined cytokines. Stimulation of I- uptake by TSH was enhanced by IFN gamma, reduced by TNF alpha, and, when serum was present, increased to a degree that was greater than additive by the combined cytokines. Growth stimulation by insulin or insulin-like growth factor-I was inhibited partially by the individual cytokines and completely by the combination. Both insulin and insulin-like growth factor-I inhibited TSH stimulation of I- uptake, but similar stimulation by the cytokines was not affected. Simultaneous with inhibition of TSH-stimulated growth, both IFN gamma and TNF alpha enhanced cAMP accumulation. The mechanism of these multiple effects of IFN gamma and TNF alpha, especially on the actions of TSH, may not currently be fully explained, but they almost certainly reflect differing modes of action. The relevance to thyroid function in man is conjectural. Especially in Graves' disease, where thyroid infiltration with cells that secrete these cytokines is common, it seems probable that both IFN gamma and TNF alpha will have significant influences on both growth and differentiated cell function.     

肿瘤坏死因子对鼠甲状腺细胞钙离子的作用

目的　观察 Ｔ Ｎ Ｆα对甲状腺细胞肌醇脂质／ 钙离子（ Ｉｎｓ／ Ｃａ２ ＋ ） 系统的作用。方法　利用激光扫描共聚焦显微镜（ Ｌ Ｓ Ｃ Ｍ） 观察 Ｔ Ｎ Ｆα对鼠甲状腺 Ｆ Ｒ Ｔ Ｌ５ 细胞内 Ｃａ２ ＋ 的影响。实验前细胞以不含 Ｔ Ｓ Ｈ 的５ Ｈ（５ 种激素） 培养液培养３ 天，将待测细胞以 Ｆｌｕｏ３ 负载，分别加入不同浓度的 Ｔ Ｎ Ｆα，以 Ｌ Ｓ Ｃ Ｍ 扫描（４８８ｎｍ ） 测定细胞内 Ｃａ２ ＋ ，观察 Ｔ Ｎ Ｆα的作用。结果　１ ． Ｔ Ｎ Ｆα可降低 Ｆ Ｒ Ｔ Ｌ５ 细胞内基础的 Ｃａ２ ＋ 水平，其效应随 Ｔ Ｎ Ｆα浓度的增加而增强。２ ． Ｔ Ｎ Ｆα可抑制 Ｔ Ｓ Ｈ 刺激甲状腺 Ｆ Ｒ Ｔ Ｌ５ 细胞 Ｃａ２ ＋ 升高的效应。结论　 Ｔ Ｎ Ｆα可以通过抑制甲状腺细胞 Ｉｎｓ／ Ｃａ２ ＋ 系统发挥其抑制效应。     

Properties and regulation of the thyrotropin receptor in the FRTL5 rat thyroid cell line

Despite extensive use of FRTL5 cells in studies of responses to TSH and anti-TSH receptor antibodies, almost nothing is known of the properties of their TSH receptors, possibly because binding of TSH by these cells is negligible when studied in their usual culture medium. In the present studies, we have demonstrated that specific binding of TSH can readily be demonstrated in confluent monolayers of FRTL5 cells if their culture medium is replaced by Krebs-Ringer bicarbonate (KRB) buffer. In keeping with previous observations concerning the effects of cations on the binding of TSH in other thyroid systems, binding of TSH to FRTL5 was far greater when the medium used was a modified KRB in which an isosmotic substitution of sucrose for NaCl had been made. Kinetic studies of TSH binding in both types of medium suggested the presence of two binding sites, one with a higher affinity and lower maximum binding capacity than the other. The influence of NaCl was to decrease the capacity of both sites, that of the low affinity site to a greater extent than that of the high affinity site, whereas the affinities of the two sites remained unchanged. Correlative studies indicated that physiological responses to TSH were associated mainly with occupancy of the higher affinity sites. Experiments in which TSH binding was studied in cells grown to confluence in the presence of TSH from which TSH was then withdrawn and in cells maintained in the absence of TSH to which TSH was then added demonstrated the occurrence of up-and down-regulation, respectively, of receptor concentrations without a change in their affinities. The reduction in maximum binding capacity induced by TSH was proportionately greater in the case of the high affinity than the low affinity receptor. Down-regulation by TSH was concentration dependent and was demonstrable at a TSH concentration of 10(-11) M, considered to be physiological. Further, maximum down-regulation was induced by 10(-9) M TSH, the approximate concentration at which other responses to TSH in these cells reach their peak. Therefore, down-regulation of TSH receptors can be considered to be one of the physiological responses that TSH elicits.     

Modulation of differentiated function in cultured thyroid cells: thyrotropin control of thyroid peroxidase activity

The activity of thyroid peroxidase (TPO) in primary dog thyroid cell cultures was measured by both guaiacol oxidation and iodide oxidation assays. Whether cultures were initiated in the absence or presence of 50 mU/ml TSH, TPO activity fell in the first 24 h of culture to approximately 10% of the activity in freshly isolated follicles. After 5 days in culture, TPO activity almost completely disappeared in the absence of TSH, whereas in the presence of TSH, TPO activity rebounded to approximately 30% of that in freshly isolated follicles. TSH similarly induced TPO activity in cells that had lost this activity during a 1- to 6-day preincubation period in the absence of hormone. The half-time for the induction of TPO activity was approximately 3 days. Whether TSH was present from the start of culture or added after 5 days of culture without TSH, the half-maximal dose for reinduction of TPO activity was 0.3-0.4 mU TSH/ml. (Bu)2cAMP, 8-bromo-cAMP, forskolin, and cholera toxin all mimicked, either completely or in part, the ability of TSH to induce TPO activity in cells preincubated without hormone. We conclude that, in cultured dog thyroid cells, TPO activity is modulated by chronic TSH stimulation, and that this effect is mediated by cAMP. However, even though TSH stimulates TPO activity in cultured thyroid cells, the fact that there is no comparable restoration of organic iodine formation (as found in previous studies) makes it likely that other aspects of the iodide organification mechanism are altered.     

Evidence that adenosine 3',5'-monophosphate mediates stimulation of thyroid growth in FRTL5 cells

Thyroid function, including growth, is TSH dependent, and most metabolic functions of TSH are thought to be mediated by cAMP. Recently, it has been suggested by several groups that growth may be an exception and that it may not be related to cAMP action. In addition, evidence has accrued indicating that the thyroid-stimulating antibody (TSAb) of Graves' disease, the metabolic actions of which are also cAMP mediated, may not be the goitrogenic agent in that syndrome. To evaluate these concepts, we used functioning rat thyroid cells (FRTL5) in monolayer culture and, as indices of growth, the incorporation of [3H]thymidine ([3H]Tdr) into DNA, the concentration of DNA measured directly, and the percentage of cells in S phase, as assessed by flow cytometry, all studied over 72 h of incubation. TSH, forskolin, and cholera toxin enhanced growth by each criterion and increased the concentration of cAMP in parallel; the effect on cAMP occurred rapidly and was maximal well in advance of influences on growth. In all instances, measures of growth promotion were minimal at 24 h and maximal at 48 h, except for [3H]Tdr incorporation, which was greater at 72 h than at 48 h. 3-Isobutyl-1-methylxanthine (IBMX) and (Bu)2 cAMP were also tested. Both enhanced all indices of growth and were as effective as TSH. Maximal responses to TSH were obtained at 100-200 microU/ml, maximal responses to both IBMX and (Bu)2cAMP occurred at 5 X 10(-4) M, and all three stimulators increased the DNA concentration and [3H]Tdr uptake and induced S phase in at least 20% of all cells in culture. The peak effect on DNA and S phase was consistently at 48 h. Epidermal growth factor (EGF) was shown to increase [3H]Tdr incorporation in a nondose-dependent fashion (10(-10) to 5 X 10(-9) M gave approximately 250% of control) over 1, 2, 3, 5, and 7 days, with no increase in DNA and a slight decrement in the concentration of cAMP. A laboratory standard TSAb-immunoglobulin G was shown to parallel TSH in both increasing cAMP (over 2 h of incubation) and growth stimulation (over 72 h). The data are entirely consistent with the view that TSH-stimulated thyroid growth is mediated by cAMP and that the established action of TSAb on adenylate cyclase is sufficient to explain goiter as well as hyperthyroidism in Graves' disease.     

Thyroid hormone receptors and 3,5,3'-triiodothyronine biological effects in FRTL5 thyroid follicular cells.

Specific thyroid hormone (T3) receptors are present in thyroid follicular cells, including the rat FRTL5 clonal line, but little is known about the effects of T3 on the growth and differentiated function of the thyroid. Unlike primary cultures of animal or human thyroid cells, FRTL5 do not secrete appreciable amounts of thyroid hormones. We now have studied the effects of T3 by itself and in combination with TSH and insulin-like growth factor-I (IGF-I) on [3H]thymidine incorporation into DNA, iodide uptake, and cAMP production in FRTL5. We also have investigated the expression of different c-erbA mRNAs in these cells. Specific binding of T3 to FRTL5 cell nuclei in intact cells occurred with a binding capacity of 0.1-0.15 ng T3/mg DNA and an apparent Kd of 0.4 nM. Using an RNase protection assay on total cellular FRTL5 RNA and specific cRNA probes, we demonstrated the presence of c-erbA alpha and -beta mRNAs, both encoding T3 receptors. Biological effects were assessed in serum-free medium or buffer containing 0.1% BSA after maintaining quiescent culture of cells for at least 5 days in hormone-free medium containing 5% calf serum. T3 alone stimulated a dose-dependent increase in [3H]thymidine incorporation that reached a plateau at 188% of the control value at 10 nM T3. At 10(-11) M TSH, T3 potentiated TSH-stimulated [3H]thymidine incorporation (2.2-fold), but at TSH concentrations greater than 5 x 10(-11) M, T3 had no effect or reduced the response to TSH. T3 potentiated the [3H]thymidine response to 2 and 10 ng/ml IGF-I by 1.5- to 1.7-fold. T3 alone had no effect on iodide uptake, but attenuated iodide uptake stimulated by TSH. T3 was more potent in inhibiting TSH-stimulated iodide uptake than in enhancing TSH-stimulated DNA synthesis. T3 did not affect either basal or TSH-stimulated cAMP accumulation. Thus, in FRTL5 thyroid follicular cells 1) T3 receptors are expressed, as measured by direct binding assays and by the expression of c-erbA mRNAs; and 2) T3 acts as a growth factor and weak antidifferentiation factor. We suggest that T3 may modulate the actions of TSH and growth factors in thyroid epithelium.     

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)     

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)     

Iodothyronine deiodinase activities in FRTL5 cells: predominance of type I 5'-deiodinase

FRTL5 cells, a thyroid follicular cell line derived from normal rat thyroid, has been extensively used as a model system to study various aspects of the physiology of the thyroid epithelium. The capacity of these cells to metabolize iodothyronines and to generate T3 from T4 has not been previously examined. Here we studied the deiodination of T4, T3, and rT3 in homogenates of FRTL5 cells. By far, these homogenates were more potent catalyzing the 5'-deiodination (outer ring) of T4 and rT3 than the inner ring deiodination of T4 or T3. Both the production of rT3 and the degradation of newly formed T3 from T4 were very limited. Thus, when T4 was used as a substrate, T3 and iodide accumulated in a linear fashion with time, and initially the amounts of iodide and T3 were approximately equal. rT3 and 3,3'-diiodothyronine were rapidly deiodinated by these homogenates, with the 3'-deiodination of 3,3'-diiodothyronine occurring at a slower rate than the 5'-deiodination of rT3. The iodothyronine 5'-deiodinase activity corresponded to type I, as indicated by the following: the Km for T4 and T3 was in the micromolar range; rT3 was a better substrate than T4 (maximum velocity = 101 vs. 19 pmol/min.mg protein; Km = 0.83 vs. 3.1 microM, respectively); and the kinetics of inhibition by 6n-propyl-2-thiouracil were uncompetitive and substrate dependent, suggesting ping-pong kinetics. The type I 5'-deiodinase activity of FRTL5 cells was distinctly stimulated by TSH. This stimulation seems to be mediated by cAMP and requires serum as a permissive factor. In conclusion, 1) FRTL5 cells exhibit both inner and outer ring iodothyronine-deiodinating activities; 2) iodothyronine 5'-deiodination is by far more active; 3) the 5'-deiodination has been characterized as type I deiodinase based on substrate preference, enzyme kinetics, and inhibitors; 4) in all respect iodothyronine deiodination by FRTL5 cell homogenates proceeded with marked similarity to that in homogenates or microsomes of thyroid glands from several species; and 5) the FRTL5 type I deiodinase is more active than that reported in thyroid tissue and as active as that reported in liver and kidney, the prototype of type I deiodinase-containing tissues. The present studies indicate that FRTL5 cells are an excellent model system to study cellular and biochemical aspects of the regulation of this enzyme as well as its regulation by TSH and putative serum factors.     

The effects of lysophosphatidate on thyrotropin-mediated differentiated thyroid function in FRTL-5 thyroid cells.

Lysophosphatidate (LPA; 1-acyl-sn-glycero-3-phosphate) is a novel lipid mediator with diverse biological activity. The intracellular mechanisms that mediate the actions of LPA include activation of phospholipase C and protein kinase C (PKC), increases in intracellular Ca2+, inhibition of adenylyl cyclase, and activation of phospholipase D (PLD). We have shown that thyrotropin (TSH) mediated PLD activation involves both the cyclic adenosine monophosphate (cAMP) and PKC pathways. We determined the effects of LPA (10 or 50 microM; 30 minutes) on TSH- and forskolin-mediated cAMP production in FRTL-5 thyroid cells. Basal cAMP was unaffected by LPA. However, both 10 microM and 50 microM LPA inhibited TSH-mediated cAMP production by 66% and 64%, respectively (p < 0.01, ANOVA). A similar inhibition of forskolin-mediated cAMP production was observed following LPA (p < 0.01, ANOVA). After 30-minutes exposure to 50 microM LPA, TSH-mediated iodide uptake (IU) was unaffected. However, 50 microM LPA enhanced TSH-IU after 24-hour exposure by 23%+/-8% (p < 0.03, ANOVA) and inhibited TSH-IU following 72-hour exposure by 43%+/-10% (p < 0.02, ANOVA). There was no effect of LPA on basal IU. To determine whether PLD activation mediated the effects of LPA, PLD activity was examined in FRTL-5 thyroid cells 30 minutes after LPA exposure. While PLD was increased 3.5-fold compared to control values following 50 microM LPA (p < 0.05, ANOVA), no increase in PLD activation was seen following treatment with 10 microM LPA. Preliminary evidence revealed no effect of a protein kinase C inhibitor on LPA inhibition of cAMP generation. To examine the products of PLD activation, we measured the production of phosphatidate (PA) and diacylglycerol (DAG) in FRTL-5 thyroid cells following treatment with 50 microM LPA or 100 microU/mL TSH. Within 1 minute following LPA, a rapid spike of DAG production was observed (1.5- +/- 0.2-fold above basal, p < 0.05, ANOVA). No similar increases in PA or bisPA were demonstrated. However, TSH caused a steady increase in PA and DAG that reached a maximum after 30 minutes. In summary, the effects of LPA on differentiated thyroid function in FRTL-5 thyroid cells are complex. LPA inhibits TSH- and forskolin-mediated cAMP generation most likely via a direct inhibition of adenylyl cyclase, whereas its effects on TSH-IU involve other mechanisms, possibly including PLD activation.     

Antiphosphotyrosine immunoprecipitation of an insulin-stimulated receptor phosphatase activity from FRTL5 cells.

Phosphotyrosine-containing proteins (PYPs) from FRTL5 epithelial cells were immunoprecipitated with Sepharose-linked phosphotyrosine antibody and phosphorylated in vitro with insulin and insulin-like growth factor-I (IGF-I) receptor kinases. Insulin preactivation of the kinases resulted in increased phosphorylation of a single 175,000 mol wt PYP (p175) beside insulin and IGF-I receptors. Phosphorylation of both p175 and receptors exhibited almost identical time courses and insulin dose responses, suggesting that p175 may serve as a substrate for the insulin and IGF-I receptor kinases. Preincubation of autophosphorylated receptors with PYPs derived from insulin-stimulated cells decreased 32P-labeled receptor precipitation by antiphosphotyrosine-Sepharose by 50-70% compared to that obtained upon preincubation with PYPs from unstimulated cells. This effect was not the result of PYP-receptor competition, was observed on both in vivo and in vitro phosphorylated receptors, and was almost completely blocked by 100 microM sodium vanadate, suggesting PYP copurification of a phosphotyrosine phosphatase. No phosphatase was recovered in lysates from insulin-unstimulated cells. Maximum activity was detected after 2 min of insulin stimulation, decreasing by more than 50% after 30 min. Antiphosphotyrosine recovery of p175 from cell lysates exhibited almost identical insulin dependency. Complete recovery of both p175 and phosphatase activity was obtained after receptor immunodepletion of the extracts. Thus, a receptor phosphatase activity from insulin-stimulated FRTL5 cells is retained on an antiphosphotyrosine affinity matrix and correlates with phosphorylation of the p175 substrate for the insulin and IGF-I receptor kinases.     

Identification of exchange mechanisms for the regulation of intracellular pH in rat thyroid FRTL-5 cells.

pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO3- ions, FRTL-5 cells regulate intracellular pH (pHi) by an Na+/H+ antiport but HCO3(-)-dependent exchangers cannot operate. We have investigated pHi regulation (by microfluorimetry and the pH sensitive dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in Krebs-Ringer buffer (pH 7.4) with or without HCO3- ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na+/H+ antiporter and the stilbene derivative disodium 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) for HCO3(-)-dependent mechanisms), ionic substitution and by NH4+/NH3 (10 mM) acid loading. Basal pHi was lower in the presence (7.3 +/- 0.058, mean +/- S.D., n = 14) than in the absence (7.59 +/- 0.078, n = 10) of HCO3- ions. In HCO3(-)-free media, cells recovered from acid load by 0.34 +/- 0.04 pH units in the first 2 min and finally reached a pHi of 7.35 +/- 0.06. This recovery was Na(+)-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO3(-)-containing media, cells recovered from an acid load at a similar rate, but reached 99 +/- 10% (n = 9) of the baseline pH; this recovery was also dependent on Na+ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0.06 +/- 0.02 and 0.18 +/- 0.04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of malignantly transformed rat thyroid (FRTL) cells with thyrotropin receptors and their growth inhibition by 3',5'-cyclic adenosine monophosphate

Differentiated rat thyroid cells, designated FRTL, are totally dependent on TSH for their growth. We continuously cultured FRTL cells in the absence of TSH and found that another type of cell appeared in the culture. The new cells were large, flattened and epithelial-like, and none of them exhibited thyroglobulin immunoreactivity. Since they grew independently of TSH, we further cloned these mutated cells by the limited dilution method in the absence of TSH. cAMP production in the cloned cells (FRTL-Tc) was stimulated dose-dependently by TSH. The TSH concentration that produced a maximal level of cAMP in FRTL-Tc cells was 1 order of magnitude higher than in FRTL cells. A [125I]TSH binding study confirmed that the FRTL-Tc cells had TSH receptors with the same binding capacity but a higher Kd than those of FRTL cells. A [125I]cyanopindolol binding study revealed that the FRTL-Tc cells had acquired beta 2-adrenergic receptors and that isoproterenol or epinephrine could stimulate cAMP production in the cells. TSH or beta-adrenergic agonists inhibited the growth of these cells, as did (Bu)2cAMP. When FRTL-Tc cells were transplanted into the sc tissue in Fisher rats, they grew as a tumor in all of the animals (n = 10). Metastasis of the tumors to the lung and liver occurred. These results indicate that FRTL-Tc cells are malignantly transformed cells with TSH receptors derived from thyroid epithelial cells and also suggest that the role of cAMP in the proliferation of the transformed cells might be different from that in normal cells.     

Insulin stimulates cell growth of a new strain of differentiated rat thyroid cells

A new strain, named WRT cells, has been generated from primary cultures of rat thyroids. The primary culture was grown in Coon's modified Ham's F12 medium with 5% calf serum, insulin, hydrocortisone, transferrin, somatostatin, glycyl-L-histidyl-L-lysine and thyrotropin (TSH). On the basis of the following facts, the WRT cell strain, cloned from the primary culture, was considered 'normal': the cells are euploid, not carcinogenic, not able to grow in soft agar, and show contact inhibition. Their differentiated functions consist of the ability to synthesize thyroglobulin and to take up iodide, and they have a TSH-dependent adenylate cyclase system. TSH increases cellular adenosine 3',5'-cyclic monophosphate (cAMP) levels and [3H]thymidine incorporation in WRT cells from a concentration similar to that active on another clonal rat cell line (FRTL-5), even though the cell replication appears to be differently regulated in the two cell strains. In fact, the WRT cell doubling time is 42 h and they are also able to grow in the absence of TSH, though more slowly. In the same conditions, FRTL-5 cells have a population doubling time of 38 h, but they are not able to grow in the absence of TSH. When the effect of the other growth factors of the medium was studied, insulin appears to be a growth stimulus by itself, while it is only a facilitative step for TSH action in FRTL-5 cells. WRT cells, unlike FRTL-5 cells, can grow with a population doubling time of 80 h, when cultured for prolonged periods in a medium with a low serum concentration (0.5%), but containing insulin plus TSH. In conclusion, the WRT cell strain is a new and interesting experimental model for studying growth factors at the level of the thyroid, especially for their mechanism of action on the TSH receptor.