TSH/cAMP up-regulate sarco/endoplasmic reticulum Ca2+-ATPases expression and activity in PC Cl3 thyroid cells

OBJECTIVE: We recently reported that the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2b is the SERCA form preferentially expressed in rat thyroid. Moreover, SERCA2b expression dramatically decreases in virally transformed, highly tumorigenic, PC Cl3 thyroid cells. These results suggest that, in the thyroid, SERCA2b, in addition to its housekeeping role, is linked to differentiation and is a regulated gene. We therefore sought to study the effect of TSH, the main regulator of thyroid function, on SERCA2b expression and activity. METHODS: PC Cl3 cells were hormone starved in low-serum medium and stimulated for long (48 h) or short (1, 2 and 4 h) times. SERCA2b expression and activity were evaluated by Northern and Western blots, Ca2+-ATPase activity and Ca2+ store content. RESULTS: In PC Cl3 cells, SERCA2b mRNA and protein were induced twofold by a 48-h long treatment with TSH. Long-term elevation (48 h) of intracellular cAMP levels, by forskolin or 8-Br-cAMP, had similar effects on SERCA2b mRNA and protein. We also measured Ca2+-ATPase activity and Ca2+ store content. Both long (48 h) and short (0.5-1 h) treatments with TSH, forskolin or 8-Br-cAMP induced a marked increase of SERCA2b activity. This effect was completely abolished by H89, a specific inhibitor of cAMP-dependent protein kinase A (PKA). TSH and 8-Br-cAMP increased Ca2+ store content after both long (48 h) and short (1-2 h) treatments. CONCLUSIONS: These data suggested that TSH/cAMP acts as an important regulator of both SERCA2b expression and activity in the thyroid system, through PKA activation.

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The expression of the sarco/endoplasmic reticulum Ca2+-ATPases in thyroid and its down-regulation following neoplastic transformation

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0300399 ◽

2003 ◽

Vol 30 (3) ◽

pp. 399-409 ◽

Cited By ~ 30

Author(s):

F Pacifico ◽

L Ulianich ◽

S De Micheli ◽

S Treglia ◽

A Leonardi ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Northern Blot ◽

Blot Analysis ◽

Thyroid Tissue ◽

Neoplastic Transformation ◽

Thyroid Cell ◽

Rt Pcr ◽

Down Regulation ◽

Thyroid Cells ◽

Rat Thyroid

Maintaining a high Ca(2+) concentration in the lumen of the endoplasmic reticulum (ER), by the action of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs), is important in many cellular processes, such as Ca(2+)-mediated cytosolic signaling in response to extracellular stimuli, cell growth and proliferation, and synthesis, processing and folding of ER-translated proteins. In the thyroid gland, SERCAs have not been studied yet, and there is little information available on general problems such as the expression of SERCAs following neoplastic transformation. In this study we investigated the expression of SERCA2b and SERCA3 in rat thyroid tIssue and, in addition, in normal and transformed rat thyroid cell lines. RT-PCR and Northern blot assays showed that SERCA2b is the SERCA form preferentially expressed in the thyroid. In rat thyroid, SERCA2b mRNA was expressed at a higher level than that of other non-muscle tIssues such as liver or spleen, but at much lower level than in brain. On the other hand, SERCA3 mRNA was not detected in thyroid by Northern blot analysis, or barely detected by RT-PCR assays. We also studied the SERCA2b expression pattern in PC Cl3 thyroid cells transformed by several oncogenes that induce different degrees of malignancy and dedifferentiation. RT-PCR and Northern blot assays showed that SERCA2b mRNA expression dramatically decreased in highly tumorigenic thyroid cells, while expression of glyceraldehyde-3-phosphate dehydrogenase mRNA, a housekeeping gene used as internal control, exhibited no variations. The dramatic down-regulation of SERCA2b expression in fully transformed thyroid cells was also evident by Western blot analysis. Also, following neoplastic transformation of thyroid cells, the enzymatic activity of SERCA2b was reduced in a measure which correlated with the mRNA and protein levels. Therefore, rat thyrocytes expressed intermediate levels of SERCAs, mostly the SERCA2b isoform. This pattern of expression was basically reproduced in fully differentiated thyroid cells in culture and was sensitive to neoplastic transformation.

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cAMP-Dependent Activation of Mammalian Target of Rapamycin (mTOR) in Thyroid Cells. Implication in Mitogenesis and Activation of CDK4

Molecular Endocrinology ◽

10.1210/me.2010-0087 ◽

2010 ◽

Vol 24 (7) ◽

pp. 1453-1468 ◽

Cited By ~ 43

Author(s):

Sara Blancquaert ◽

Lifu Wang ◽

Sabine Paternot ◽

Katia Coulonval ◽

Jacques E. Dumont ◽

...

Keyword(s):

Protein Kinase ◽

Glycogen Synthase ◽

Mammalian Target Of Rapamycin ◽

Cyclin D3 ◽

Target Of Rapamycin ◽

Thyroid Cells ◽

Dependent Protein ◽

Rat Thyroid ◽

Amp Activated Protein Kinase

Abstract How cAMP-dependent protein kinases [protein kinase A (PKA)] transduce the mitogenic stimulus elicited by TSH in thyroid cells to late activation of cyclin D3-cyclin-dependent kinase 4 (CDK4) remains enigmatic. Here we show in PC Cl3 rat thyroid cells that TSH/cAMP, like insulin, activates the mammalian target of rapamycin (mTOR)-raptor complex (mTORC1) leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMP-activated protein kinase and glycogen synthase kinase 3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate protein kinase B or induce tuberous sclerosis complex 2 phosphorylation at T1462 and Y1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity that was associated with augmented 4E-BP1 binding to raptor. This could be caused in part by T246 phosphorylation of PRAS40, which was found as an in vitro substrate of PKA. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and retinoblastoma protein phosphorylation induced by TSH and insulin. Although rapamycin reduced cyclin D3 accumulation, the abundance of cyclin D3-CDK4 complexes was not affected. However, rapamycin inhibited the activity of these complexes by decreasing the TSH and insulin-mediated stimulation of activating T172 phosphorylation of CDK4. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 T172-phosphorylation.

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Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1162 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1162-1168 ◽

Cited By ~ 35

Author(s):

N al-Alawi ◽

D W Rose ◽

C Buckmaster ◽

N Ahn ◽

U Rapp ◽

...

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Dna Synthesis ◽

Signaling Pathways ◽

Cellular Growth ◽

Intracellular Camp ◽

Thyroid Cells ◽

Kinase Cascade ◽

Rat Thyroid ◽

Stimulation Of

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.

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The sarcoplasmic–endoplasmic reticulum Ca2+ ATPase 2b regulates the Ca2+ transients elicited by P2Y2 activation in PC Cl3 thyroid cells

Journal of Endocrinology ◽

10.1677/joe.1.06455 ◽

2006 ◽

Vol 190 (3) ◽

pp. 641-649 ◽

Cited By ~ 4

Author(s):

Luca Ulianich ◽

Maria Giovanna Elia ◽

Antonella Sonia Treglia ◽

Antonella Muscella ◽

Bruno Di Jeso ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Thyroid Cell ◽

Myristate Acetate ◽

Thyroid Cells ◽

Pkc Inhibitor ◽

Kinase C ◽

Thyroid Cell Line ◽

Rat Thyroid ◽

Stimulation Of

In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y2 purinoceptor activation provoked a transient increase of [Ca2+]i, followed by a decreasing sustained phase. The α and β1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca2+]i level and increased the peak of Ca2+ entry of the P2Y2-provoked Ca2+transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn2+ and Ba2+ uptake were not changed by Gö 6976. Similarly, the Na+/Ca2+ exchanger was not implicated, since the rate of decrement to the basal [Ca2+]i level was equally decreased in physiological and Na+-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic–endoplasmic reticulum Ca2+ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca2+]i level after P2Y2 stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca2+ transients caused by P2Y2 stimulation.

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Anoctamin-1/TMEM16A is the major apical iodide channel of the thyrocyte

AJP Cell Physiology ◽

10.1152/ajpcell.00126.2014 ◽

2014 ◽

Vol 307 (12) ◽

pp. C1102-C1112 ◽

Cited By ~ 41

Author(s):

L. Twyffels ◽

A. Strickaert ◽

M. Virreira ◽

C. Massart ◽

J. Van Sande ◽

...

Keyword(s):

Cell Lines ◽

Apical Membrane ◽

Specific Inhibitor ◽

Anion Channel ◽

Thyroid Cell ◽

Anoctamin 1 ◽

Thyroid Cells ◽

Rat Thyroid

Iodide is captured by thyrocytes through the Na+/I− symporter (NIS) before being released into the follicular lumen, where it is oxidized and incorporated into thyroglobulin for the production of thyroid hormones. Several reports point to pendrin as a candidate protein for iodide export from thyroid cells into the follicular lumen. Here, we show that a recently discovered Ca2+-activated anion channel, TMEM16A or anoctamin-1 (ANO1), also exports iodide from rat thyroid cell lines and from HEK 293T cells expressing human NIS and ANO1. The Ano1 mRNA is expressed in PCCl3 and FRTL-5 rat thyroid cell lines, and this expression is stimulated by thyrotropin (TSH) in rat in vivo, leading to the accumulation of the ANO1 protein at the apical membrane of thyroid follicles. Moreover, ANO1 properties, i.e., activation by intracellular calcium (i.e., by ionomycin or by ATP), low but positive affinity for pertechnetate, and nonrequirement for chloride, better fit with the iodide release characteristics of PCCl3 and FRTL-5 rat thyroid cell lines than the dissimilar properties of pendrin. Most importantly, iodide release by PCCl3 and FRTL-5 cells is efficiently blocked by T16Ainh-A01, an ANO1-specific inhibitor, and upon ANO1 knockdown by RNA interference. Finally, we show that the T16Ainh-A01 inhibitor efficiently blocks ATP-induced iodide efflux from in vitro-cultured human thyrocytes. In conclusion, our data strongly suggest that ANO1 is responsible for most of the iodide efflux across the apical membrane of thyroid cells.

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Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4477-4484.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4477-4484

Author(s):

E Kupperman ◽

W Wen ◽

J L Meinkoth

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Dna Synthesis ◽

Kinase Inhibitor ◽

Dependent Protein Kinase ◽

Thyroid Cells ◽

Heat Stable ◽

Heat Stable Protein ◽

Dependent Protein ◽

Rat Thyroid

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

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Organization of the thyroglobulin polysome, studied in Endoplasmic Reticulum surface views of rough endoplasmic reticulum in cultured rat thyroid cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160285 ◽

1990 ◽

Vol 48 (3) ◽

pp. 546-547

Author(s):

A. Kent Christensen ◽

Hayden G. Coon

Keyword(s):

Endoplasmic Reticulum ◽

Thyroid Gland ◽

Thyroid Hormones ◽

Cross Section ◽

Electron Micrographs ◽

Rough Endoplasmic Reticulum ◽

Thyroid Cells ◽

En Face ◽

Face View ◽

Rat Thyroid

Thyroglobulin is synthesized in the thyroid gland and is subsequently degraded to provide thyroid hormones. Rat thyroglobulin is made up of two identical 330 kD subunits, and the mRNA for each subunit contains about 8,500 nucleotides. Since polysomes have approximately one ribosome for each 90-100 nucleotides of mRNA, a polysome of about 85-95 ribosomes would be expected for thyroglobulin. We have been interested in how this very large polysomes is organized on the membranes of the rough endoplasmic reticulum (RER).It is well known that bound polysomes assume characteristic shapes on the surface of the RER, resembling beads on a string arranged in circles, spirals, loops, hairpins or other forms. These polysomal shapes can be observed in conventional electron micrographs when the membranes of the RER are seen in surface or en face view, rather than in the usual cross section. Clearcut surface views are infrequent, but the likelihood of seeing them is greatly improved when flattened cells in culture are sectioned in the plane of the cell, since the RER in a flattened cell tends to be oriented in that plane.

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Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level

Acta Endocrinologica ◽

10.1530/eje.0.1450477 ◽

2001 ◽

pp. 477-483 ◽

Cited By ~ 4

Author(s):

M Marino ◽

L Chiovato ◽

S Lisi ◽

A Pinchera ◽

RT McCluskey

Keyword(s):

Low Density Lipoprotein ◽

Specific Inhibitor ◽

Density Lipoprotein ◽

Inhibitory Effect ◽

Thyroid Cells ◽

Microbial Product ◽

Phosphoinositide 3 Kinase ◽

Rat Thyroid ◽

Upper Chamber

BACKGROUND: Phosphoinositide 3-kinase (PI3-K) is implicated in various cellular processes involving signaling, including intracellular trafficking. PI3-K has been shown to play a part in both receptor- and non-receptor-mediated transcytosis across cultured kidney cells and undifferentiated thyroid cells. OBJECTIVE: To investigate the role of PI3-K in transcytosis of thyroglobulin (Tg) across differentiated cultured Fisher rat thyroid cells (FRTL-5 cells) - a process known to be mediated by megalin, a member of the low-density lipoprotein receptor family. DESIGN: We studied the effect of the microbial product wortmannin, a specific inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells. METHODS: Transcytosis experiments were performed using FRTL-5 cells cultured as tight layers on filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper cell surface. Tg was added to the upper chamber and cells were incubated at 37 degrees C. Transcytosed Tg was measured in fluids collected from the lower chamber. To study the role of PI3-K, cells were pre-incubated with wortmannin. RESULTS: Pre-incubation of FRTL-5 cells with wortmannin did not affect Tg binding and uptake, but resulted in a considerable increase in Tg transcytosis (by 40-75%, depending on the concentration of wortmannin), suggesting that PI3-K exerts an inhibitory effect on Tg transcytosis. In experiments in which a monoclonal antibody against megalin was used to reduce Tg transcytosis, pre-incubation with wortmannin did not increase Tg transcytosis from its reduced levels, indicating that PI3-K is involved in the megalin-mediated pathway. Wortmannin did not affect the extent of release of tri-iodothyronine from exogenously added Tg by FRTL-5 cells, which was used as a measure of Tg degradation in the lysosomal pathway, indicating that the effect of PI3-K on transcytosis occurs after diversion of Tg from the lysosomal pathway. CONCLUSIONS: PI3-K exerts an inhibitory role on megalin-mediated Tg transcytosis across cultured thyroid cells. PI3-K action takes place at a post-sorting level, after Tg bypassing of the lysosomal pathway.

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PHOSPHATASES OF RAT THYROID AND ANTERIOR PITUITARY GLANDS DURING VARIOUS PHASES OF SECRETORY ACTIVITY. A CYTOCHEMICAL STUDY

Journal of Endocrinology ◽

10.1677/joe.0.0300323 ◽

1964 ◽

Vol 30 (3) ◽

pp. 323-NP ◽

Cited By ~ 12

Author(s):

H. J. SOBEL

Keyword(s):

Endoplasmic Reticulum ◽

Anterior Pituitary ◽

Plasma Membranes ◽

Secretory Activity ◽

Thyroid Cells ◽

Cytoplasmic Staining ◽

Pituitary Glands ◽

Inosine Diphosphatase ◽

Guanosine Triphosphatase ◽

Rat Thyroid

SUMMARY 1. The cytochemical localization and changes of various phosphatase activities were studied in rat thyroid and anterior pituitary glands in various phases of secretory activity. The procedures used, made possible light microscopic study of the Golgi apparatus, plasma membranes and sinusoids, lysosomes, endoplasmic reticulum, nuclei and nucleoli; reactive sites many of which up to the present were in the realm of ultrastructure and could only be visualized with very specialized techniques or the electron microscope. The enzymatic make up and changes, and changes in size, shape and distribution of these structures were also studied in the conditions described. 2. Nucleoside phosphatase techniques visualized sinusoids, but this staining did not vary with secretory activity. The alkaline phosphatase procedure did not stain sinusoids of the anterior pituitary. However, the sinusoidal staining of the thyroid with this technique paralleled secretory activity. 3. Thiamine pyrophosphatase, inosine diphosphatase and guanosine triphosphatase staining visualized the Golgi lamellae in the two glands and this staining paralleled secretory activity. The adenosine monophosphatase technique stained occasional Golgi lamellae. 4. The adenosine monophosphatase procedure stained the plasma membranes of the less active cells of the anterior pituitary and granular cytoplasmic particles (lysosomes) of more active pituitary and thyroid cells. This granular cytoplasmic staining was abolished by sodium fluoride and d-tartaric acid as was acid phosphatase staining. 5. The inosine diphosphatase and guanosine triphosphatase techniques also revealed some diffuse cytoplasmic and cell membrane staining which was thought to represent staining of endoplasmic reticulum. 6. The adenosine diphosphatase procedure invariably stained nuclei and nucleoli.

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