FRTL-5 Thyroid Cells Research Articles

Hydrogen peroxide attenuates store-operated calcium entry and enhances calcium extrusion in thyroid FRTL-5 cells.

Redox modulation participates in the regulation of intracellular free calcium concentration ([Ca(2+)](i)) in several cell types. In thyroid cells, including FRTL-5 cells, changes in [Ca(2+)](i) regulate several important functions, including the production of H(2)O(2) (hydrogen peroxide). As H(2)O(2) is of crucial importance for the production of thyroid hormones, we investigated the effects of H(2)O(2) on [Ca(2+)](i) in thyroid FRTL-5 cells. H(2)O(2) itself did not modulate basal [Ca(2+)](i). However, H(2)O(2) attenuated store-operated calcium entry evoked by thapsigargin, both in a sodium-containing buffer and in a sodium-free buffer. The effect of H(2)O(2) was abrogated by the reducing agent beta-mercaptoethanol. H(2)O(2) also attenuated the thapsigargin-evoked entry of barium and manganese. The effect of H(2)O(2) was, at least in part, mediated by activation of protein kinase C (PKC), as H(2)O(2) enhanced the binding of [(3)H]phorbol 12,13-dibutyrate. H(2)O(2) also stimulated the translocation of the isoenzyme PKCepsilon from the cytosolic fraction to the particulate fraction. Furthermore, H(2)O(2) did not attenuate store-operated calcium entry in cells treated with staurosporine or calphostin C, or in cells with down-regulated PKC. H(2)O(2) depolarized the membrane potential in bisoxonol-loaded cells and when patch-clamp in the whole-cell mode was used. The depolarization was attenuated in cells with down-regulated PKC. This depolarization, at least in part, explained the H(2)O(2)-evoked inhibition of calcium entry. In addition, H(2)O(2) enhanced the extrusion of calcium from cells stimulated with thapsigargin and this effect was abolished in cells with down-regulated PKC and after treatment of the cells with the reducing agent beta-mercaptoethanol. In conclusion H(2)O(2) attenuates an increase in [Ca(2+)](i). As H(2)O(2) is produced in thyroid cells in a calcium-dependent manner, our results suggest that H(2)O(2) may participate in the regulation of [Ca(2+)](i) in these cells via a negative-feedback mechanism involving activation of PKC.

The characterization of phospholipase D in FRTL-5 thyroid cells

We previously demonstrated that TSH activates phospholipase D (PLD) in Fischer rat thyroid line (FRTL)-5 cells. To date, two types of mammalian phosphatidylcholine-specific PLD cDNAs, designated as PLD-1 and PLD-2, have been cloned. The present study determined the PLD isoform composition in FRTL-5 thyroid cells and which isoform is regulated by TSH. PLD-1 is activated by small molecular weight G-proteins, such as ADP-ribosylation factor (ARF) and RhoA family members, while PLD-2 is relatively independent of such stimuli. We established the presence of PLD-1 and PLD-2 by Western blot analysis and compared PLD activity in cytosol, membranes and combined fractions in the presence and absence of GTPγS. The membrane fraction showed very little activity in the absence of GTPγS, but this activity increased ≈5-fold ( P<0.05, ANOVA) in the presence of GTPγS. Maximal PLD activity was seen with the combination of membrane plus cytosolic fractions (which contained ARF and RhoA) where the addition of GTPγS increased PLD activity ≈8-fold ( P<0.05, ANOVA). To determine the relative activities of PLD-1 and PLD-2 in FRTL-5 thyroid cells, cell-free PLD assays were performed in the presence of GTPγS or GDPβS with varying concentrations of phosphatidylinositol 4,5-bisphosphate (PIP 2). PLD-2 contributed only ≈19% of the total amount of PLD activity in the membranes and PLD-1 was the predominant PLD isoform. TSH stimulated PLD-1 activity by up to 2.3-fold over control values ( P<0.01, ANOVA). To establish the dependence of PLD-1 on small molecular weight G-proteins, the translocations of ARF and RhoA to the membrane fractions was determined after stimulation by TSH. Both ARF and RhoA were maximally translocated to the membrane fraction after 10 min incubation with 100 μU/ml TSH by ≈1.7- and 2.3-fold over control values, respectively ( P<0.02 and P<0.03, ANOVA). It is concluded that TSH stimulates PLD-1 activity in FRTL-5 thyroid cells and this is accompanied by the translocation of ARF and RhoA to the membrane fraction.

The thyrotropin receptor is not involved in the activation of p42/p44 mitogen-activated protein kinases by thyrotropin preparations in Chinese hamster ovary cells expressing the human thyrotropin receptor.

We studied whether bovine pituitary thyrotropin (bTSH) or human recombinant thyrotropin (rhTSH) stimulated p42/p44 mitogen-activated protein kinases (MAPKs) in Chinese hamster ovary cells expressing human thyrotropin receptor (CHO-hTSHR cells). We show that p42/p44 MAPK phosphorylation was induced by both TSH preparations at similar levels in CHO-hTSHR cells and in wild-type CHO cells. In contrast, cyclic adenosine monophosphate (cAMP) production was stimulated by TSH only in CHO-hTSHR cells, demonstrating that p42/p44 MAPK stimulation was independent of the TSH receptor. Moreover, similar results were obtained with two other cell lines: the FRTL-5 thyroid cell line and the CCL39 fibroblast cell line. Maximal stimulation of p42/p44 MAPK phosphorylation was observed after a 5- to 10-minute incubation with bTSH and rhTSH preparations. At this time, the phosphorylation of GST-Elk1 was also increased in a time- and concentration-dependent manner by bTSH preparations. The phosphorylation of p42/p44 MAPKs was abolished by PD 98059 and GF 109203X, indicating the involvement of MAPK kinases (MEK 1/2) and protein kinase C. In contrast, the activation of p42/p44 MAPKs was insensitive to H89, to cholera toxin and to pertussis toxin. These data suggest that the protein kinase A pathway was not implicated in p42/p44 MAPK activation by TSH preparations. Moreover, Gs or Gi/Go proteins do not appear to participate in p42/p44 MAPK activation. We also showed that these TSH preparations failed to induce activation of c-Jun NH2 terminal kinase. We therefore conclude that the commercial TSH preparations used in this study contained factor(s) responsible for the specific activation of p42/p44 MAPKs by a TSH receptor-independent mechanism.

Retinoic acid induces sodium/iodide symporter gene expression and radioiodide uptake in the MCF-7 breast cancer cell line

The sodium/iodide symporter (NIS) stimulates iodide uptake in normal lactating breast, but is not known to be active in nonlactating breast or breast cancer. We studied NIS gene regulation and iodide uptake in MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line. All-trans retinoic acid (tRA) treatment stimulated iodide uptake in a time- and dose-dependent fashion up to approximately 9.4-fold above baseline. Stimulation with selective retinoid compounds indicated that the induction of iodide uptake was mediated by retinoic acid receptor. Treatment with tRA markedly stimulated NIS mRNA and immunoreactive protein ( approximately 68 kDa). tRA stimulated NIS gene transcription approximately 4-fold, as shown by nuclear run-on assay. No induction of iodide uptake was observed with RA treatment of an ER-negative human breast cancer cell line, MDA-MB 231, or a normal human breast cell line, MCF-12A. The iodide efflux rate of tRA-treated MCF-7 cells was slow (t(1/2) = 24 min), compared with that in FRTL-5 thyroid cells (t(1/2) = 3.9 min), favoring iodide retention in MCF-7 cells. An in vitro clonogenic assay demonstrated selective cytotoxicity with (131)I after tRA stimulation of MCF-7 cells. tRA up-regulates NIS gene expression and iodide uptake in an ER-positive breast cancer cell line. Stimulation of radioiodide uptake after systemic retinoid treatment may be useful for diagnosis and treatment of some differentiated breast cancers.

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.

Role of Peroxiredoxins in Regulating Intracellular Hydrogen Peroxide and Hydrogen Peroxide-induced Apoptosis in Thyroid Cells

Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. One mechanism for this action involves modulation of hydrogen peroxide (H(2)O(2))-mediated cellular responses. This report examines the expression of Prx I and Prx II in thyroid cells and their roles in eliminating H(2)O(2) produced in response to thyrotropin (TSH). Prx I and Prx II are constitutively expressed in FRTL-5 thyroid cells. Prx I expression, but not Prx II expression, is stimulated by exposure to TSH and H(2)O(2). In addition, methimazole induces a high level of Prx I mRNA and protein in these cells. Overexpression of Prx I and Prx II enhances the elimination of H(2)O(2) produced by TSH in FRTL-5 cells. Treatment with 500 micrometer H(2)O(2) causes apoptosis in FRTL-5 cells as evidenced by standard assays of apoptosis (i.e. terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling, BAX expression, and poly(ADP-ribose) polymerase cleavage. Overexpression of Prx I and Prx II reduces the amount of H(2)O(2)-induced apoptosis measured by these assays. These results suggest that Prx I and Prx II are involved in the removal of H(2)O(2) in thyroid cells and can protect these cells from undergoing apoptosis. These proteins are likely to be involved in the normal physiological response to TSH-induced production of H(2)O(2) in thyroid cells.

Thyrotropin modulates interferon-gamma-mediated intercellular adhesion molecule-1 gene expression by inhibiting Janus kinase-1 and signal transducer and activator of transcription-1 activation in thyroid cells.

TSH is known as an important hormone that plays the major role not only in the maintenance of normal physiology but also in the regulation of immunomodulatory gene expression in thyrocytes. The adhesion molecule intercellular adhesion molecule-1 (ICAM-1) was identified as one of the proteins that are abnormally expressed in the thyroid gland during autoimmune thyroid diseases. In this study we found that TSH inhibits interferon-gamma (IFNgamma)-mediated expression of the ICAM-1 gene, and we investigated the involved mechanisms in rat FRTL-5 thyroid cells. After exposure to IFNgamma, ICAM-1 expression is positively regulated at the level of transcription. This effect occurs via the IFNgamma-activated site (GAS) element in the ICAM-1 promoter as a consequence of the activation of STAT1 (signal transducer and activator of transcription-1), but not of STAT3. On the other hand, after exposure to TSH plus IFNgamma, ICAM-1 transcription is negatively modulated. We found that this inhibitory effect of TSH also occurs via the GAS element. Electrophoretic mobility shift assays confirmed that the IFNgamma-induced DNA-binding activities of STAT1 were reduced by TSH. Furthermore, our results showed that the inhibitory effect of TSH on IFNgamma signaling is caused by inhibition of tyrosine phosphorylation on STAT1, Janus kinase-1 (Jak1), and IFNgamma receptor a, but not Jak2. In conclusion, we have identified a novel mechanism in which TSH modulates the IFNgamma-mediated Jak/STAT signaling pathway through the inhibition of Jak1 and STAT1.

Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

Signalling pathways of insulin-like growth factor-I that are augmented by cAMP in FRTL-5 cells

We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotropin (TSH) markedly potentiates the mitogenic response to insulin-like growth factor-I (IGF-I). The present study was undertaken to determine whether the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of known IGF-I receptor substrates plays an important role in the cAMP-dependent potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or dibutyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). In contrast, cAMP pretreatment potentiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not affect the amount of IRS-2. We found that the IGF-I-dependent tyrosine phosphorylation of 66 kDa Shc (Src homology collagen) was markedly increased by cAMP pretreatment, and that this change was mainly due to an increase in the levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment significantly increased binding of Grb2 (growth-factor-receptor-bound protein 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-activated protein kinase) induced by IGF-I was also enhanced by cAMP. The presence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treatment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA synthesis in response to IGF-I. On the other hand, cAMP pretreatment increased binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to IRS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatment with and without TSH or dibutyryl cAMP. Our results suggest that the interaction between cAMP-dependent and IGF-I-dependent pathways leads to an augmentation of cell proliferation, which is mediated, at least in part, through the MAP kinase and PI 3-kinase signalling pathways. These effects are mediated by changes in tyrosine phosphorylation of IGF-I receptor substrates, including IRS-2 and Shc.

Thyrotropin regulates adenosine A(1) receptor expression in rat thyroid FRTL-5 cells.

The effect of thyrotropin (TSH), on adenosine A(1) receptor expression in thyroid FRTL-5 cells was examined by [(3)H]-1, 3-dipropyl-,8-cyclopentyl xanthine ([(3)H]-DPCPX) binding on cells in suspension and on membrane preparation, and by in situ mRNA labelling. The estimated K(D) for intact cells was 0.19 nM and about 47,000 binding sites per cell were found in cells constantly grown in the presence of TSH. Three days deprivation of TSH decreased the number of [(3)H]-DPCPX binding sites without any significant effect of K(D). Reintroduction of TSH to the cells returned the higher level of A(1) receptors both in suspension binding studies on whole cells and on membrane preparations. In situ hybridization revealed that TSH evoked an increase in the number of cells densely labelled with a probe against A(1) receptor mRNA. The potency of the A(1) receptor agonist N(6)-cyclohexyladenosine (CHA) as an inhibitor of cyclic AMP formation induced by forskolin was increased in TSH-treated cells, with a shift in the IC(50) from 2.05 nM in TSH-deprived cells to 0.14 nM in TSH-treated cells. Since the activation of A(1) receptors inhibits TSH-mediated cyclic AMP signalling, our results suggest a regulatory feedback mechanism between signalling via adenosine A(1) receptors and TSH receptors.

Somatostatin Interferes with Thyrotropin-induced G1-S Transition Mediated by cAMP-dependent Protein Kinase and Phosphatidylinositol 3-Kinase: INVOLVEMENT OF RhoA AND CYCLIN E·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.

Extracellular ATP-mediated phospholipase a2 activation in rat thyroid FRTL-5 cells: Regulation by a Gi/Go protein, Ca2+, and mitogen-activated protein kinase

We investigated the mechanism of phospholipase A2 (PLA2) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA2 activity was determined by measuring the release of [3H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a Gi/Go protein. The AA release was also diminished by chelating extracellular Ca2+ with EGTA or by inhibiting influx of Ca2+ using Ni2+. Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA2 activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA2 by a Gi/Go protein-dependent mechanism. Moreover, Ca2+, PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process. J. Cell. Physiol. 183:155–162, 2000. © 2000 Wiley-Liss, Inc.

Extracellular ATP-mediated phospholipase A(2) activation in rat thyroid FRTL-5 cells: regulation by a G(i)/G(o) protein, Ca(2+), and mitogen-activated protein kinase.

We investigated the mechanism of phospholipase A(2) (PLA(2)) activation in response to the P2 receptor agonist ATP in rat thyroid FRTL-5 cells. The PLA(2) activity was determined by measuring the release of [(3)H]-arachidonic acid (AA) from prelabeled cells. ATP evoked a dose- and time-dependent AA release. This release was totally inhibited by pertussis toxin (PTX) treatment, indicating the involvement of a G(i)/G(o) protein. The AA release was also diminished by chelating extracellular Ca(2+) with EGTA or by inhibiting influx of Ca(2+) using Ni(2+). Although the activation of protein kinase C (PKC) by 12-phorbol 13-myristate acetate (PMA) alone did not induce any AA release, the ATP-evoked AA release was significantly reduced when PKC was inhibited by GF109203X or by a long incubation with PMA to downregulate PKC. Both the ATP-evoked AA release and the mitogen-activated protein kinase (MAP kinase) phosphorylation were decreased by the MAP kinase kinase (MEK) inhibitor PD98059. Furthermore, the ATP-evoked MAP kinase phosphorylation was also inhibited by GF109203X and by downregulation of PKC, suggesting a PKC-mediated activation of MAP kinase. Inhibiting Src-like kinases by PP1 attenuated both the MAP kinase phosphorylation and the AA release. These results suggest that these kinases are involved in the regulation of MAP kinase and PLA(2) activation. Elevation of intracellular cAMP by TSH or by dBucAMP did not induce a phosphorylation of MAP kinase. Furthermore, neither the ATP-evoked AA release nor the MAP kinase phosphorylation were attenuated by TSH or dBucAMP. Taken together, our results suggest that ATP regulates the activation of PLA(2) by a G(i)/G(o) protein-dependent mechanism. Moreover, Ca(2+), PKC, MAP kinase, and Src-like kinases are also involved in this regulatory process.

Role of Insulin and Serum on Thyrotropin Regulation of Thyroid Transcription Factor-1 and Pax-8 Genes Expression in FRTL-5 Thyroid Cells

Thyrotropin (TSH), via its cyclic adenosine monophosphate (cAMP) signal, decreases thyrotropin receptor (TSHR) gene expression in FRTL-5 thyroid cells, whereas it increases expression of the thyroglobulin (Tg) gene. Despite the opposite effects of TSH on TSHR and Tg expression, both genes are positively controlled by thyroid transcription factor-1 (TTF-1) and evidence has accumulated that TSH can decrease TTF-1 mRNA levels. In this report, we further characterize the action of TSH on TTF-1 in order to understand its different activities on the TSHR and Tg genes better. The effect of TSH on the TSHR requires the presence of insulin and serum and we show here that also both factors are necessary for the TSH effect to decrease TTF-1 mRNA levels. The decrease is paralleled by a downregulation of TTF-1 protein levels as well as by a decrease in TTF-1/DNA complex when the TTF-1 site of the TSHR promoter was used as probe. Again, the decrease requires insulin and serum. The TSH downregulation of TTF-1 mRNA levels is due to a decrease in its transcription rate. Using a luciferase-linked chimera construct spanning 5.18 kb of the TTF-1 5'-flanking region, we show that TSH decreases TTF-1 promoter activity and that this effect depends on insulin and serum. These data contrast with the action of TSH on Tg and Pax-8 gene expression. TSH increases Pax-8 mRNA levels and the increase is evident whether insulin and serum are present or not. Moreover, this increase is paralleled by an increase in Pax-8 protein binding to an oligonucleotide derived from the C site of the Tg promoter, which can bind both TTF-1 and Pax-8. The present data thus show that TTF-1 gene expression is interdependently regulated by TSH and serum growth factors including insulin. They also show this interdependent-regulation is not duplicated in the case of Pax-8. We suggest that these differences may contribute to the distinct ability of TSH to regulate TSHR versus Tg gene expression in FRLT-5 thyroid cells.

Short Term Feedback Regulation of cAMP in FRTL-5 Thyroid Cells: ROLE OF PDE4D3 PHOSPHODIESTERASE ACTIVATION

Together with a transient accumulation of intracellular cAMP, thyrotropin (TSH) stimulation of the FRTL-5 thyroid cell induces phosphorylation and activation of a cAMP-specific phosphodiesterase (PDE4D3). Here we have investigated the impact of PDE4D3 activation on hormone responsiveness. Stimulation of FRTL-5 cells with TSH caused an increase in PDE activity within 3 min, with a maximal stimulation reached after 5 min. Preincubation with the protein kinase A (PKA) inhibitor H89 or (R(p))-cAMPS, but not with the inactive isomer H85, blocked this activation. Preincubation with PKA inhibitors also blocked the shift in mobility of the PDE4D3 protein. Under these conditions, H89, but not H85, potentiated the cAMP accumulation induced by TSH. Incubation of FRTL-5 cells with the PKA activator 8-(4-chlorophenylthio)adenosine-cAMP caused an increase in PDE activity and a decrease in the endogenous cAMP, confirming the presence of a PKA-PDE feedback loop. MA-10 Leydig tumor cells stably transfected with either a wild type PDE4D3 or a PDE4D3 with mutations in the PKA phosphorylation sites showed an increase in PDE activity when compared with control cells. Human choriogonadotropin or Bt(2)cAMP treatment induced a stimulation of PDE activity in cells transfected with wild type PDE4D3, whereas the activation was absent in mutant- and control-transfected cells. The increase in cAMP accumulation elicited by human choriogonadotropin was reduced in cells transfected with the wild type PDE4D3, but not in cells transfected with the mutant PDE. Rolipram, a specific inhibitor of PDE4, restored the cAMP accumulation in the PDE4D3-transfected cells. These data provide evidence that a rapid activation of PDE4D3 is one of the mechanisms determining the intensity of the cAMP signal.

Thyrotropin induces SOCS-1 (suppressor of cytokine signaling-1) and SOCS-3 in FRTL-5 thyroid cells.

TSH has multiple physiological roles: it is required for growth, differentiation, and function of the thyroid gland, and it regulates transcription of interferon-gamma (IFN-gamma)-responsive genes in thyrocytes, including genes for the major histocompatibility complex and intercellular adhesion molecule-1. This report demonstrates that TSH induces the expression of suppressor of cytokine signaling (SOCS)-1 and -3 proteins and alters the phosphorylation state of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3. The expression of SOCS-1 and SOCS-3 and the phosphorylation state of STAT1 and STAT3 were examined after treatment with TSH or IFN-gamma in either TSH-sensitive FRTL-5 thyroid cells or TSH-insensitive FRT and buffalo rat liver (BRL) cells, which lack functional TSH receptors. SOCS-1 and SOCS-3 are constitutively expressed in FRTL-5 cells, but not in FRT and BRL cells. IFN-gamma up-regulated SOCS-1 and SOCS-3 RNA and protein in FRTL-5 cells, as reported previously for nonthyroid cells. Interestingly, TSH also significantly induced SOCS-1 and SOCS-3 in FRTL-5 cells, but not in FRT and BRL cells. When SOCS-1 or SOCS-3 was overexpressed in FRTL-5 cells, STAT1 phosphorylation at Y701 and STAT1/DNA complex formation in response to IFN-gamma were reduced. Furthermore, overexpression of either SOCS-1 or SOCS-3 significantly inhibited the IFN-gamma-mediated transactivation of the rat ICAM-1 (intercellular adhesion molecule-1) promoter. TSH and IFN-gamma had different effects on STAT1 and STAT3 phosphorylation. The phosphorylation of Y701 in STAT1, which is responsible for homodimer formation, nuclear translocation, and DNA binding, was specifically stimulated by IFN-gamma, but not by TSH or forskolin. However, the phosphorylation of S727 in STAT1 was induced by IFN-gamma, TSH, and forskolin. TSH induced phosphorylation of both Y705 and S727 in STAT3, while IFN-gamma phosphorylated only the Y705. In addition, we found that SOCS-3 was associated with JAK1 and JAK2 and that these associations were stimulated by TSH. These findings demonstrate that TSH induces SOCS in thyroid cells and provides the evidence of signal cross-talk between TSH and cytokines in thyroid cells.

Thyrotropin Induces SOCS-1 (Suppressor of Cytokine Signaling-1) and SOCS-3 in FRTL-5 Thyroid Cells

Thyrotropin Induces SOCS-1 (Suppressor of Cytokine Signaling-1) and SOCS-3 in FRTL-5 Thyroid Cells

The role of adenosine A 1 receptors in the ATP-evoked Ca 2+ response in rat thyroid FRTL-5 cells

The effect of adenosine A 1 receptor activation on the ATP-induced increase in intracellular free Ca 2+ was studied in control and protein kinase C down-regulated Fisher rat thyroid (FRTL-5) cells. Long-term phorbol ester treatment, which leads to protein kinase C down-regulation, enhanced the ATP-evoked extracellular Ca 2+ influx. The increased Ca 2+ influx was antagonized by the adenosine A 1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). [ 3H]DPCPX binding studies revealed that phorbol ester-treatment increased the number of adenosine A 1 receptors. The adenosine A 1 receptor-mediated inhibition of the cyclic AMP formation was not affected by the increased receptor number. We conclude that the enhanced ATP-evoked Ca 2+ influx in protein kinase C down-regulated cells is mediated by adenosine formed by hydrolysis of ATP, and that this adenosine interacts with the increased number of A 1 receptors. The mechanism by which adenosine enhances Ca 2+ entry is not known. Thus, the larger number of adenosine A 1 receptors broadens the spectrum of adenosine A 1 receptor affected signaling systems in FRTL-5 cells.

Increased major histocompatibility complex (MHC) expression in nontoxic goiters is associated with iodide depletion, enhanced ability of the follicular thyroglobulin to increase MHC gene expression, and thyroid autoantibodies.

Recent studies suggest that thyroglobulin (TG) accumulated in the follicular lumen of colloid nodular goiters can increase major histocompatibility complex (MHC) class I gene expression in FRTL-5 thyrocytes. Iodide deficiency, also present in these patients, was separately suggested to enhance thyroidal MHC class I and class II gene expression in vivo and in vitro. To test the clinical relevance of these observations, we examined 41 nontoxic goiters surgically removed from patients who had compression problems. Northern analysis revealed that there was a mean 3.9-fold increase in MHC class I expression and a 8.3-fold increase in class II expression by comparison to 9 normal glands. In situ hybridization showed that thyrocytes were the main source of class I and class II transcripts; histological examination revealed that lymphocytic infiltration was minimal to non-existent. The iodine content of the 41 nontoxic goiters was significantly lower than in normal glands, consistent with increased MHC class I and class II. There is also a profound accumulation of TG in the follicles of the nontoxic goiters, and TG purified from the follicles of these glands increased MHC class I gene expression in FRTL-5 thyroid cells significantly more than TG from normal glands per mg protein. Nearly all patients with nontoxic goiter had low, but significantly elevated, levels of antibodies against thyroid peroxidase and/or against TG in their sera compared with those in normal individuals. Moreover, there was a positive correlation between the titer of the serum antibodies against thyroid peroxidase and against TG and MHC class I and class II expression in the thyroid. The data support the possibility that the TG accumulated in the follicular lumen of nontoxic goiters together with relative iodine deficiency contributes to increased MHC expression in thyroid cells in vivo and that increased MHC gene expression contributes to the ability of thyroid antigens to trigger an autoimmune reaction.

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 GTPγS, 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.