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.     

Thyrotropin stimulation of lysosomal tyrosine transport in rat FRTL-5 thyroid cells

Tyrosine countertransport was used to demonstrate the hormonal stimulation of neutral amino acid transport across the lysosomal membrane of FRTL-5 cells. Cells grown with thyrotropin (1 X 10(-10) M) had 7-fold (+/- S.E.) higher tyrosine countertransport activity in their lysosomes than cells grown without thyrotropin. Thyrotropin also stimulated the uptake into tyrosine-loaded lysosomes of other neutral amino acids recognized by the tyrosine carrier, namely, phenylalanine (3-fold) and leucine (6-fold). In contrast lysosomal cystine countertransport was not affected by thyrotropin. Addition of thyrotropin to cells grown without thyrotropin showed that the stimulation of tyrosine counter-transport (a) required at least 48 h to reach the level of the thyrotropin-supplemented cells, (b) depended upon protein synthesis, since cycloheximide (20 microM) was inhibitory, and (c) depended upon RNA synthesis, since actinomycin D (1 nM) was inhibitory. Cells grown without thyrotropin but with dibutyryl cyclic AMP (1 mM) or cholera toxin (1 nM) exhibited enhanced lysosomal countertransport of tyrosine, suggesting that cyclic AMP may act as a messenger. This represents the first demonstration of hormonal responsiveness in a lysosomal transport system and may reflect the importance of salvage and reutilization of lysosomal degradation products for the thyroid epithelial cell.     

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.     

Thyrotropin and norepinephrine stimulate the metabolism of phosphoinositides in FRTL-5 thyroid cells

Hormone-induced changes in phospholipid metabolism were examined in a functioning rat thyroid cell line (FRTL-5). Stimulation of FRTL-5 cells, prelabeled with 32P, with TSH or NE resulted in a rapid decrease in the radioactivity of both phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-monophosphate (PIP). The effects of TSH on phospholipid metabolism and calcium mobilization are independent of those on adenylate cyclase. This suggests that the TSH receptor may be unique in that it activates enzyme cascades involved in cAMP production and Ca2+ mobilization.     

Thyrotropin effect on the availability of Ni regulatory protein in FRTL-5 rat thyroid cells to ADP-ribosylation by pertussis toxin

Incubation of FRTL-5 rat thyroid cell membranes with [32P]NAD and pertussis toxin results in the specific ADP-ribosylation of a protein of about 40 kDa. This protein has the same molecular mass of the alpha i subunit of the adenylate cyclase regulatory protein Ni and is distinct from proteins ADP-ribosylated by cholera toxin in the same membranes. Prior treatment of FRTL-5 cells with pertussis toxin results in the ADP-ribosylation of Ni, as indicated by the loss of the toxin substrate in the ADP-ribosylation assay performed with membranes prepared from such cells. Preincubation of FRTL-5 cells with thyrotropin causes the same loss; cholera toxin has no such effect. Pertussis toxin, as do thyrotropin and cholera toxin, increases cAMP levels in FRTL-5 cells. Forskolin together with thyrotropin, cholera toxin or pertussis toxin causes a further increase in cAMP levels. Pertussis toxin and thyrotropin are not additive in their ability to increase adenylate cyclase activity, whereas both substances are additive with cholera toxin. A role of Ni in the thyrotropin regulation of the adenylate cyclase activity in thyroid cells is proposed.     

Secretion of immunoreactive ANF from FRTL-5 rat thyroid cells: regulation by calcium ionophore A23187.

Atrial natriuretic factor-like immunoreactivity (ir-ANF) was characterized in a continuous line of rat thyroid follicular cells (FRTL-5) and the influence of the calcium ionophore A23187 on ir-ANF secretion was examined. Ir-ANF was identified by immunohistochemical staining as primarily reticular and juxtanuclear in short-term cultures, and more peripheral and granular in longer-term cultures, suggesting a process of ir-ANF packaging into secretory granules. The accumulation of ir-ANF granules was dependent upon the presence of thyrotropin (TSH) in the medium. Secreted ir-ANF was characterized using reversed-phase, high-performance liquid chromatography (RP-HPLC) and radioimmunoassay as a single peak eluting one fraction earlier than 125I-labeled rat ANF (99-126) (i.e., circulating atrial ANF) included as an internal standard. A23187 treatment of cells exhibiting primarily reticular ir-ANF caused a change to a pattern of more distinct, peripherally localized granules. This change occurred within 1 h after A23187 treatment and was dependent on the presence of Ca2+ in the medium. In cultures containing primarily ir-ANF granules, A23187 (0.5 micrograms/ml) induced a peripheral translocation of the granules at 30 min and a complete degranulation by 7 h. Enzyme-linked immunoadsorbent assay (EIA) confirmed a dose-dependent effect of A23187 on ir-ANF release into the medium. These results suggest that some of the effects of Ca2+ in the thyroid could be ascribed to its mobilization and release of ir-ANF, which in turn may have autocrine effects on thyroid follicular cells.     

Purinergic agonists stimulate the secretion of endothelin-1 in rat thyroid FRTL-5 cells

The aim of the present study was to investigate the mechanisms regulating endothelin-1 (ET-1) secretion in rat thyroid FRTL-5 cells. ET-1 was found to be secreted after stimulation with adenosine and ATP. The release of ET-1 was sensitive to pertussis toxin, indicating a role of G-proteins in the stimulus-secretion coupling. The stimulation evoked by ATP or adenosine was inhibited by the P₁-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and in the presence of adenosine deaminase the adenosine- and ATP-mediated ET-1 secretion was abolished. These evidences suggest a role of a P₁-adenosine receptor in the secretion of ET-1. Increasing cyclic AMP with forskolin decreased the adenosine-mediated secretion. In addition, the intracellular calcium chelator BAPTA or inhibition of calcium entry with Ni²⁺ prevented the response. Protein kinase C (PKC) is also partly involved in ET-1 secretion in FRTL-5 cells. Activation of PKC with the phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated the secretion of ET-1 in a time- and dose-dependent manner. Furthermore, downregulation of PKC decreased the secretion of ET-1 stimulated by adenosine. In conclusion, ET-1 secretion in FRTL-5 cells is stimulated via a pertussis toxin-sensitive P₁-receptor pathway which is modulated by several signal transduction mechanisms including cAMP, Ca²⁺, and PKC. © 1996 Wiley-Liss, Inc.     

Thyrotropin regulation of membrane lipid fluidity in the FRTL-5 thyroid cell line. Its relationship to cell growth and functional activity

The mitogenic effect of thyrotropin on functional rat thyroid cells of the line FRTL-5 is correlated with membrane lipid fluidity as evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Continued exposure of FRTL-5 cells to a medium lacking thyrotropin causes cessation of cell proliferation and a decrease in membrane lipid fluidity which reaches its minimum in approximately 8 days. The change in lipid fluidity is due to an absolute increase (greater than 2-fold) of membrane cholesterol, with an increased cholesterol/phospholipid ratio and an increased ratio of saturated to unsaturated fatty acids of the membrane phospholipids, contributed primarily by a nearly 4-fold increase in the ratio of saturated to unsaturated C16 fatty acids. It is also associated with a variation of the relative proportions of the major membrane phospholipids; thus, phosphatidylinositol and phosphatidylethanolamine decrease while phosphatidylcholine increases. Both membrane fluidity and lipid composition can be restored by thyrotropin to their original levels, i.e. levels measured under continuous exposure to the hormone. Complete reversal requires at least 48 h, i.e. approximately the same time required for resumption of growth when FRTL-5 cells, starved in thyrotropin, are re-exposed to the hormone. Changes in lipid composition and fluidity can be prevented or can be reversed if FRTL-5 cells are exposed to dibutyryl cAMP while being deprived of thyrotropin. Dibutyryl cAMP has only a modest direct effect on growth; however, this pretreatment eliminates the 48-h lag phase with respect to thyrotropin stimulation. It is proposed that the effects of thyrotropin on growth of FRTL-5 cells requires a modification of the molecular structure and the physical state of cell membranes, which can be mediated by cAMP, although cAMP is not sufficient by itself to promote growth.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.     

Purinergic agonist ATP is a comitogen in thyroid FRTL-5 cells

Several growth factors may stimulate proliferation of thyroid cells. This effect has, in part, been dependent on calcium entry. In the present study using FRTL-5 cells, we show that in addition to its effect on calcium fluxes, ATP acts as a comitogen in these cells. In medium containing 5% serum, but no TSH, ATP stimulated the incorporation of ³H-thymidine in a dose- and time-dependent manner in the cells. At least a 24-h incubation with ATP was necessary to observe the enhanced (30–50%) incorporation of ³H-thymidine and an increased (30%) cell number. The effect of ATP was dependent on insulin in the incubation medium. Furthermore, ATP enhanced the TSH-mediated incorporation of ³H-thymidine. The effect of ATP was apparently mediated via a G-protein dependent mechanism, as no stimulation of thymidine incorporation was observed in cells treated with pertussis toxin. The effect of ATP was not dependent on the activation of protein kinase C (PKC), as ATP was effective in cells with downregulated PKC. ATP rapidly phosphorylated mitogen activated protein (MAP) kinase in FRTL-5 cells. In addition, ATP stimulated the expression of a 62 kDa c-fos dependent protein in a dose- and time-dependent manner. Our results thus suggest that extracellular ATP, in the presence of insulin, may be a cofactor in the regulation of thyroid cell proliferation, probably by phosphorylating MAP kinase and stimulating the expression of c-fos. © 1996 Wiley-Liss, Inc.     

High-efficient nonviral transfection of the rat thyroid cell line FRTL-5

FRTL-5 cells are used in many laboratories as an in vitro system of thyroid follicular cells since they share many properties of human thyrocytes. However, the use of FRTL-5 cells for experimental modifications is limited by low transfection efficiencies of lipid-based transfections and the need for cumbersome viral transduction protocols. A new technology - nucleofection - has become available for cell lines that are difficult to transfect. Here, we report the application and optimization of this method in FRTL-5 cells. Using the green fluorescent protein (GFP) as a reporter gene, FRTL-5 cells were easily transfectable with efficiencies over 60%. In addition, the simultaneous transfer of siRNA against GFP was feasible and allowed suppression of GFP over at least 4 days. Furthermore nucleofection was successful for establishing stable FRTL-5 cell clones. In conclusion, this optimized fast and efficient nucleofection protocol offers new properties for the experimental use of FRTL-5 cells.     

Protein tyrosine phosphorylation and calcium signaling in thyroid FRTL-5 cells

We examined the importance of tyrosine kinase(s) on the ATP-evoked Ca²⁺ entry and DNA synthesis of thyroid FRTL-5 cells. ATP rapidly and transiently tyrosine phosphorylated a 72-kDa protein(s). This phosphorylation was abolished by pertussis toxin and by the tyrosine kinase inhibitor genistein, and was dependent on Ca²⁺ entry. Pretreatment of the cells with genistein did not affect the release of sequestered Ca²⁺, but the capacitative Ca²⁺ or Ba²⁺ entry evoked by ATP or thapsigargin was attenuated. Pretreatment of the cells with orthovanadate enhanced the increase in intracellular free Ca²⁺ ([Ca²⁺]_i), whereas the Ba²⁺ entry was not increased. Phorbol 12-myristate 13-acetate (PMA) phosphorylated the same protein(s) as did ATP. Genistein inhibited the ATP-evoked phosphorylation of MAP kinase and attenuated both the ATP- and the PMA-evoked DNA synthesis. However, genistein did not inhibit the ATP-evoked expression of c-fos. Furthermore, genistein enhanced the ATP-evoked release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca²⁺-dependent mechanism. A genistein-sensitive mechanism participates, in part, in the ATP-evoked activation of DNA synthesis. Genistein inhibits only modestly capacitative Ca²⁺ entry in FRTL-5 cells. J. Cell. Physiol. 175:211–219, 1998. © 1998 Wiley-Liss, Inc.     

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

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

Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells

We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell.     

Nutritional and hormonal regulation of malic enzyme synthesis in rat mammary gland.

Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was constant during the first 6 h of starvation. When lactating rats (maintained with their pups) were starved for 24 h and then re-fed, the relative rate of enzyme synthesis increased 2.5-, 4-, and 4.5-fold at 3 h, 6 h and 18 h respectively after initiation of re-feeding. The relative rate of malic enzyme synthesis was about 50% of normal at 15 h after weaning, whereas the rate of synthesis of soluble proteins did not change. Administration of bromocriptine or adrenalectomy of lactating rats decreased the relative rate of synthesis of malic enzyme by 40% or 30% respectively; these effects were counteracted by hormone supplementation. Hormone therapy also caused an increase in the rate of incorporation of [3H]leucine into soluble proteins and in malic enzyme activity.