FRTL-5 Cells Research Articles

The Presence of Adenosine A2a Receptor in Thyrocytes and Its Involvement in Graves' IgG-Induced VEGF Expression

Goitrogenesis in Graves' disease (GD) has been attributed to anti-TSH receptor antibody stimulation. Recently, a role for adenosine A2a receptor (A2aR) in goiter formation was reported in the thyroglobulin-A2aR transgenic mice. However, it is unclear whether A2aR is expressed in the thyroid and whether it is associated with the pathogenesis of goiter in GD. Here, we confirmed the expression of A2aR in FRTL-5 cells, primary normal human thyrocytes (both sexes were used without regard to sex), and thyroid tissue (both sexes were used without regard to sex) by PCR, Western blotting, immunohistochemistry, and immunofluorescence. After treatments with A2aR-specific agonist 2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine or GD IgG, the mRNA and protein levels of vascular endothelial growth factor (VEGF), a growth factor related to goitrogenesis, were evaluated along with upstream signaling pathways. A2aR activation and GD IgG promoted the expression of VEGF in thyrocytes, which was accompanied by the activation of cAMP/protein kinase A/phosphorylated-cAMP-response element-binding protein, peroxisome proliferator-activated receptor γ coactivator-1α, and hypoxia-inducible factor-1α. The changes induced by GD IgG were partially abrogated by A2aR small interfering RNA and an A2aR antagonist. These results were supported by data on the goiter samples from the thyrotropin receptor adenovirus-induced GD mouse model (female). These data demonstrate that GD IgG could up-regulate the VEGF expression through A2aR, indicating a potential mechanism for goitrogenesis in GD.

Concurrent overexpression of RET/PTC1 and TTF1 confers tumorigenicity to thyrocytes

A variant located on 14q13.3 nearest to thyroid transcription factor-1 (TTF1) predisposes individuals to thyroid cancer, but whether this variant is related to the RET/PTC rearrangement associated with human papillary thyroid carcinomas (PTCs) is unknown. The aims of this study were to investigate the effects of RET/PTC1 on the expression of thyroid-specific genes in thyrocytes and their relationship with malignant transformation of the thyrocytes. In the absence or presence of TSH, an extracellular signal-regulated kinase was phosphorylated in FRTL5 cells that stably expressed RET/PTC1, and these cells grew independently of TSH. FRTL (RET/PTC1) cells produced 566% more thyroglobulin mRNA and 474% more Na+/I− symporter mRNA than did the control FRTL (pcDNA) cells. FRTL (RET/PTC1) cells expressed 468% more Ttf1 mRNA than did FRTL (pcDNA) cells, but these two cell types did not differ significantly with respect to Pax8 or Ttf2 mRNA levels. When FRTL (RET/PTC1) cells and FRTL (pcDNA), cells were injected into each of nine nude mice, each mouse developed a single tumor at the site of FRTL (RET/PTC1) cell injection; in contrast, tumor formation never occurred at sites of FRTL (cDNA) cells injection. Tumors resulting from FRTL (RET/PTC1) cells retained 125I-uptake activity; moreover, the cells invaded into surrounding skeletal muscle. When overexpression of Ttf1 in FRTL (RET/PTC1) cells was silenced, the cells completely lost their tumorigenic potential. Exogenous TTF1 cDNA enhanced the tumorigenicity of BHP18-21v cells, human PTC cells that express RET/PTC1, in nude mice. These results indicated that concurrent overexpression of RET/PTC1 and TTF1 confers tumorigenicity to FRTL5 and BHP18-21v cells in nude mice.

Ca2 +-binding protein expression in primary human thyrocytes

We recently identified several Ca2+-binding proteins (CaBP) from the S100 and annexin family to be regulated by TSH in FRTL-5 cells. Here, we study the regulation of S100A4, S100A6 and ANXA2 in primary human thyrocytes (PHT) derived from surrounding tissues (ST), cold benign thyroid nodules (CTN) and autonomously functioning thyroid nodules (AFTN). We investigated the expression and regulation of CaBP and the effect of their expression on Ca2+ and TSHR signaling. We used an approach that accounts for the potential of an individual PHT culture to proliferate or to express thyroid differentiation features by assessing the expression of FOS and TPO. We found a strong correlation between the regulation of CaBP and the proliferation-associated transcription factor gene FOS. PKA and MEK1/2 were regulators of ANXA2 expression, while PI3-K and triiodothyronine were additionally involved in S100 regulation. The modulated expression of CaBP was reflected by changes in ATP-elicited Ca2+ signaling in PHT. S100A4 increased the ratio of subsequent Ca2+ responses and showed a Ca2+ buffering effect, while ANXA2 affected the first Ca2+ response to ATP. Overexpression of S100A4 led to a reduced activation of NFAT by TSH. Using S100A4 E33Q, D63N, F72Q and Y75K mutants we found that the effects of S100A4 expression on Ca2+ signaling are mediated by protein interaction. We present evidence that TSH has the ability to fine-tune Ca2+ signals through the regulation of CaBP expression. This represents a novel putative cross-regulating mechanism in thyrocytes that could affect thyrocyte signaling and physiology.

Reactive Oxygen Species and Extracellular Signal-Regulated Kinase 1/2 Mediate Hexachlorobenzene-Induced Cell Death in FRTL-5 Rat Thyroid Cells

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the environment. We have previously shown that chronic HCB exposure triggers apoptosis in rat thyroid follicular cells. This study was carried out to investigate the molecular mechanism by which the pesticide causes apoptosis in FRTL-5 rat thyroid cells exposed to HCB (0.005, 0.05, 0.5, and 5µM) for 2, 6, 8, 24, and 48h. HCB treatment lowered cell viability and induced apoptotic cell death in a dose- and time-dependent manner, as demonstrated by morphological nuclear changes and the increase of DNA fragmentation. The pesticide increased activation of caspases-3, -8, and full-length caspase-10 processing. HCB induced mitochondrial membrane depolarization, release of cytochrome c and apoptosis-inducing factor (AIF), from the mitochondria to the cytosol, and AIF nuclear translocation. Cell death was accompanied by an increase in reactive oxygen species (ROS) generation. Blocking of ROS production, with a radical scavenger (Trolox), resulted in inhibition of AIF nuclear translocation and returned cells survival to control levels, demonstrating that ROS are critical mediators of HCB-induced apoptosis. The pesticide increased ERK1/2, JNK, and p38 phosphorylation in a time- and dose-dependent manner. However, when FRTL-5 cells were treated with specific MAPK inhibitors, only blockade of MEK1/2 with PD98059 prevented cell loss of viability, as well as caspase-3 activation. In addition, we demonstrated that HCB-induced production of ROS has a critical role in ERK1/2 activation. These results demonstrate for the first time that HCB induces apoptosis in FRTL-5 cells, by ROS-mediated ERK1/2 activation, through caspase-dependent and -independent pathways.

Effects of Ultraviolet Radiation on FRTL-5 Cell Growth and Thyroid-Specific Gene Expression

During space missions, radiation represents a major hazard for human health and involves all body organs and tissues. Regarding thyroid function, it has been shown that ultraviolet radiation (UVC) has dose-dependent apoptotic effects on FRTL-5 cells, a normal strain of rat thyrocytes. We examined the effects of a sublethal dose of UVC on FRTL-5 cell growth and gene expression. Cells exposed to 10 J/m(2) UVC showed no differences in viability compared to control cells after 24 h, but the BrdU incorporation was reduced, indicating a cytostatic effect. Quantitative RT-PCR carried out at 24 and 48 h after irradiation demonstrated that the mRNA levels of thyroglobulin (Tg), thyroperoxidase (Tpo), and sodium/iodide symporter (Nis) were transiently decreased at 24 h in treated cells, while the mRNAs of the thyroid transcription factors TTF1, Foxe1, and Pax8 were not affected. In cells cultured with TSH-free medium, the basal transcription of Tg, Tpo, and Nis genes was equally impaired by radiation and no longer stimulated by TSH. Overall, the results demonstrate that a sub-apoptotic dose of UVC compromises not only thyrocyte proliferation but also the expression of genes involved in thyroid hormone production. These findings might contribute to explaining the histological, biochemical, and clinical features of hypothyroidism observed in both animals and humans during spaceflight, and suggest that free thyroxine levels of astronauts during prolonged space missions should be monitored.

Complexation of C6-Ceramide with Cholesteryl Phosphocholine – A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca2+ influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca2+ homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides.

Effects of the Smad4 C324Y mutation on thyroid cell proliferation

Smad4 is a key mediator of the transforming growth factor-β (TGF-β) superfamily that is involved in the control of cell proliferation and differentiation. We recently demonstrated that a Smad4 mutation, Smad4 C324Y, isolated from nodal metastases of papillary thyroid carcinoma, causes an increase of TGF-β signaling, responsible for the acquisition of transformed phenotype and invasive behaviour in thyroid cells stably expressing this mutation. In this paper, we demonstrate that the stable expression of Smad4 C324Y mutation in FRTL-5 cells is responsible for TSH-independent growth ability. Our data show that the Smad4 C324Y mutation interacts with P-Smad3 more strongly than Smad4 wt, already in basal condition; this interaction is responsible for TGF-β signaling and PKA activation that, in turn, determines an increased phosphorylation of CREB, necessary for the mitogenic actions of TSH. The expression of cyclin D1 also increases in all cells overexpressing the Smad4 C324Y mutation. All together, these data demonstrate that Smad4 C324Y mutation, interacting with the PKA pathway, gives cells the ability to proliferate independently from TSH.

Significance of the transient receptor potential canonical 2 (TRPC2) channel in the regulation of rat thyroid FRTL-5 cell proliferation, migration, adhesion and invasion

Mammalian transient receptor potential (TRP) channels are involved in many physiologically important processes. Here, we have studied the significance of the TRPC2 channel in the regulation of rat thyroid FRTL-5 cell proliferation, migration, adhesion and invasion, using stable TRPC2 (shTRPC2) knock-down cells. In the shTRPC2 cells, proliferation was decreased due to a prolonged G1/S cell cycle phase. The tumor suppressor p53 and the cyclin-dependant kinase inhibitors p27 and p21 were upregulated. Cell invasion, adhesion and migration were also attenuated in shTRPC2 cells, probably due to decreased activity of both Rac and calpain, and a decreased secretion and activity of matrix metalloproteinase 2. The attenuated proliferation, migration, invasion and ATP-evoked calcium entry was mimicked by overexpressing a non-conducting, truncated TRPC2 (TRPC2-DN) in wild type cells, and was reversed by overexpression of TRPC2-GFP in shTRPC2 cells. In conclusion, TRPC2 is an important regulator of rat thyroid cell function.

Sterol Regulatory Element-Binding Proteins Are Regulators of the NIS Gene in Thyroid Cells

The uptake of iodide into the thyroid, an essential step in thyroid hormone synthesis, is an active process mediated by the sodium-iodide symporter (NIS). Despite its strong dependence on TSH, the master regulator of the thyroid, the NIS gene was also reported to be regulated by non-TSH signaling pathways. In the present study we provide evidence that the rat NIS gene is subject to regulation by sterol regulatory element-binding proteins (SREBPs), which were initially identified as master transcriptional regulators of lipid biosynthesis and uptake. Studies in FRTL-5 thyrocytes revealed that TSH stimulates expression and maturation of SREBPs and expression of classical SREBP target genes involved in lipid biosynthesis and uptake. Almost identical effects were observed when the cAMP agonist forskolin was used instead of TSH. In TSH receptor-deficient mice, in which TSH/cAMP-dependent gene regulation is blocked, the expression of SREBP isoforms in the thyroid was markedly reduced when compared with wild-type mice. Sterol-mediated inhibition of SREBP maturation and/or RNA interference-mediated knockdown of SREBPs reduced expression of NIS and NIS-specific iodide uptake in FRTL-5 cells. Conversely, overexpression of active SREBPs caused a strong activation of the 5'-flanking region of the rat NIS gene mediated by binding to a functional SREBP binding site located in the 5'-untranslated region of the rat NIS gene. These findings show that TSH acts as a regulator of SREBP expression and maturation in thyroid epithelial cells and that SREBPs are novel transcriptional regulators of NIS.

Demonstration of Innate Immune Responses in the Thyroid Gland: Potential to Sense Danger and a Possible Trigger for Autoimmune Reactions

Autoimmune thyroid disease is an archetypal organ-specific autoimmune disorder that is characterized by the production of thyroid autoantibodies and lymphocytic infiltration into the thyroid. However, the underlying mechanisms by which specific thyroid antibodies are produced are largely unknown. Recent studies have shown that innate immune responses affect both the phenotype and the severity of autoimmune reactions. Moreover, it appears that even non-immune cells, including thyroid cells, have an ability to launch such responses. The aim of this study was to conduct a more detailed analysis of innate immune responses of the thyroid upon stimulation with various "non-self" and "self" factors that might contribute to the initiation of autoimmune reactions. We used rat thyroid FRTL-5 cells, human thyroid cells, and mice to investigate the effects of various pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and iodide on gene expression and function that were related to innate immune responses. RT-PCR analysis showed that both rat and human thyroid cells expressed mRNAs for Toll-like receptors (TLRs) that sensed PAMPs. Stimulation of thyrocytes with TLR ligands resulted in activation of the interferon-beta (IFN-β) promoter and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-dependent promoter. As a result, pro-inflammatory cytokines, chemokines, and type I interferons were produced. Similar activation was observed when thyroid cells were stimulated with double-stranded DNA, one of the typical DAMPs. In addition to these PAMPs and DAMPs, treatment of thyroid cells with high concentrations of iodide increased mRNA expression of various cytokines. We show that thyroid cells express functional sensors for exogenous and endogenous dangers, and that they are capable of launching innate immune responses without the assistance of immune cells. Such responses may relate to the development of thyroiditis, which in turn may trigger autoimmune reactions.

Rab1b overexpression modifies Golgi size and gene expression in HeLa cells and modulates the thyrotrophin response in thyroid cells in culture

Rab1b belongs to the Rab-GTPase family that regulates membrane trafficking and signal transduction systems able to control diverse cellular activities, including gene expression. Rab1b is essential for endoplasmic reticulum-Golgi transport. Although it is ubiquitously expressed, its mRNA levels vary among different tissues. This work aims to characterize the role of the high Rab1b levels detected in some secretory tissues. We report that, in HeLa cells, an increase in Rab1b levels induces changes in Golgi size and gene expression. Significantly, analyses applied to selected genes, KDELR3, GM130 (involved in membrane transport), and the proto-oncogene JUN, indicate that the Rab1b increase acts as a molecular switch to control the expression of these genes at the transcriptional level, resulting in changes at the protein level. These Rab1b-dependent changes require the activity of p38 mitogen-activated protein kinase and the cAMP-responsive element-binding protein consensus binding site in those target promoter regions. Moreover, our results reveal that, in a secretory thyroid cell line (FRTL5), Rab1b expression increases in response to thyroid-stimulating hormone (TSH). Additionally, changes in Rab1b expression in FRTL5 cells modify the specific TSH response. Our results show, for the first time, that changes in Rab1b levels modulate gene transcription and strongly suggest that a Rab1b increase is required to elicit a secretory response.

Cholesteryl Phosphocholine forms Fluid Bilayer Membranes with Saturated or Monounsaturated Ceramides

Some ceramides have been shown to displace cholesterol from liquid ordered domains rich in sphingomyelin or saturated phosphatidylcholine in model membranes. The addition of ceramide will stabilize the formed ceramide/sphingomyelin domains and the subsequent partitioning of cholesterol to the disordered phase will also increase order and stabilization in this phase. The displacement of cholesterol may be due to more favorable interactions of ceramide with the ordered phase lipids, mainly sphingomyelin, compared to cholesterol. The ceramide could also interact less favorably with the disordered phase lipids compared to cholesterol, driving it into the ordered domains. It would also be of interest to better understand how cholesterol and ceramide can interact with each other in bilayer membranes. To study these questions, we have prepared a cholesterol analog with a phosphocholine head group on the 3-oxygen. We have studied the distribution and behavior of chol-PC and ceramides in bilayer systems on their own, and together with saturated and monounsaturated phospholipids. Chol-PC together with ceramide was able to form stable bilayer phases (extruded LUVs, electroformed GUVs). Phosphatidylcholine acyl chain order measurements showed that chol-PC was less efficient in inducing acyl chain order compared to cholesterol. Fluorescence quenching measurements in POPC/ceramide/sterol mixtures further showed a decrease in ordered domain stability of saturated ceramide in the presence of cholesterol or chol-PC. We have also examined the possibility to use chol-PC:ceramide mixtures to load ceramide into cultured cells. Proliferation experiments on HeLa and FRTL-5 cells indicates that chol-PC:ceramide bilayer mixtures are more efficient in inducing apoptosis than DMSO:ceramide mixtures. Our results suggest that chol-PC and ceramide interact with each other under specific conditions, and that the binary bilayers could constitute an interesting formulation for providing cells and tissues with solvent-free ceramides.

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This study investigates the in vitro effect of eleven thyroid-active compounds known to affect pituitary and/or thyroid weights in vivo, using the proliferation of GH3 rat pituitary cells in the so-called "T-screen," and of FRTL-5 rat thyroid cells in a newly developed test denoted "TSH-screen" to gain insight into the relative value of these in vitro proliferation tests for an integrated testing strategy (ITS) for thyroid activity. Pituitary cell proliferation in the T-screen was stimulated by three out of eleven tested compounds, namely thyrotropin releasing hormone (TRH), triiodothyronine (T3) and thyroxine (T4). Of these three compounds, only T4 causes an increase in relative pituitary weight, and thus T4 was the only compound for which the effect in the in vitro assay correlated with a reported in vivo effect. As to the newly developed TSH-screen, two compounds had an effect, namely, thyroid-stimulating hormone (TSH) induced and T4 antagonized FRTL-5 cell proliferation. These effects correlated with in vivo changes induced by these compounds on thyroid weight. Altogether, the results indicate that most of the selected compounds affect pituitary and thyroid weights by modes of action different from a direct thyroid hormone receptor (THR) or TSH receptor (TSHR)-mediated effect, and point to the need for additional in vitro tests for an ITS. Additional analysis of the T-screen revealed a positive correlation between the THR-mediated effects of the tested compounds in vitro and their effects on relative heart weight in vivo, suggesting that the T-screen may directly predict this THR-mediated in vivo adverse effect.

Canonical Transient Receptor Potential Channel 2 (TRPC2) as a Major Regulator of Calcium Homeostasis in Rat Thyroid FRTL-5 Cells: IMPORTANCE OF PROTEIN KINASE C δ (PKCδ) AND STROMAL INTERACTION MOLECULE 2 (STIM2)

Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCβ1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca(2+)-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca(2+)-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.

FUNCTIONAL EXPRESSION OF TSH RECEPTOR IN THE VENTRICLE OF RATS AND MICE: INVOLVEMENT A REGULATION OF HMGCR BY TSH VIA THE CAMP/PKA/PCREB SIGNALLING PATHWAY

ObjectivesElevated thyroid-stimulating hormone (TSH) and hypercholesterolemia commonly coexist, as typically seen in subclinical hypothyroid, which can lead to and aggravate heart disease if it gets serious. However, there is no...

Communication Between the Calcium and cAMP Pathways Regulate the Expression of the TSH Receptor: TRPC2 in the Center of Action

Transient receptor potential (TRP) cation channels are widely expressed and function in many physiologically important processes. Perturbations in the expression or mutations of the channels have implications for diseases. Many thyroid disorders, as excessive growth or disturbed thyroid hormone production, can be a result of dysregulated TSH signaling. In the present study, we found that of TRP canonicals (TRPCs), only TRPC2 was expressed in Fischer rat thyroid low-serum 5% cells (FRTL-5 cells). To investigate the physiological importance of the channel, we developed stable TRPC2 knockdown cells using short hairpin RNA (shTRPC2 cells). In these cells, the ATP-evoked entry of calcium was significantly decreased. This led to increased cAMP production, because inhibitory signals from calcium to adenylate cyclase 5/6 were decreased. Enhanced cAMP signaling projected to Ras-related protein 1-MAPK kinase 1 (MAPK/ERK kinase 1) pathway leading to phosphorylation of ERK1/2. The activated ERK1/2 pathway increased the expression of the TSH receptor. In contrast, secretion of thyroglobulin was decreased in shTRPC2 cells, due to improper folding and glycosylation of the protein. We show here a novel role for TRPC2 in regulating thyroid cell function.

The Effect of Tanespimycin (17-AAG) on Radioiodine Accumulation in Sodium-Iodide Symporter Expressing Cells.

The heat shock protein 90 inhibitor, tanespimycin, is an anticancer agent known to increase iodine accumulation in normal and cancerous thyroid cells. Iodine accumulation is regulated by membrane proteins such as sodium iodide symporter (NIS) and pendrin (PDS), and thus we attempted to characterize the effects of tanespimycin on those genes. Cells were incubated with tanespimycin in order to evaluate (125)I accumulation and efflux ability. Radioiodine uptake and efflux were measured by a gamma counter and normalized by protein amount. RT-PCR were performed to measure the level of gene expression. After tanespimycin treatment, (125)I uptake was increased by ∼2.5-fold in FRTL-5, hNIS-ARO, and hNIS-MDA-MB-231 cells, but no changes were detected in the hNIS-HeLa cells. Tanespimycin significantly reduced the radioiodine efflux rate only in the FRTL-5 cells. In the FRTL-5 and hNIS-ARO cells, PDS mRNA levels were markedly reduced; the only other observed alteration in the levels of NIS mRNA after tanespimycin treatment was an observed increase in the hNIS-ARO cells. These results indicate that cellular responses against tanespimycin treatment differed between the normal rat thyroid cells and human cancer cells, and the reduction in the (125)I efflux rate by tanespimycin in the normal rat thyroid cells might be attributable to reduced PDS gene expression.

Regulation of Dual Oxidase Expression and H2O2Production by Thyroglobulin

Thyroglobulin (Tg) is a macromolecular precursor in thyroid hormone synthesis to which iodine is stably bound. Tg, which is stored in the follicular space, is also a potent negative feedback regulator of follicular function, and this is achieved by suppressing mRNA levels of thyroid-specific genes such as the sodium/iodide symporter (Slc5a5), Tg, and thyroid peroxidase. Dual oxidase 1 (DUOX1) and DUOX2, originally identified in the thyroid, are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are necessary to produce the H2O2 required for thyroid hormone biosynthesis. Since follicular Tg regulates the expression of genes that are essential for thyroid hormone synthesis, we hypothesized that Tg might also regulate DUOX expression and H2O2 production. Rat thyroid FRTL-5 cells were treated with Tg, and the mRNA expression of Duox1 and Duox2 and their corresponding maturation factors Duoxa1 and Duoxa2 were evaluated by DNA microarray and real-time PCR. Duox2 promoter activity was examined by luciferase reporter gene assay. Protein levels of DUOX2 were also examined by Western blot analysis. Intracellular H2O2 generation was quantified by a fluorescent dye, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, and acetyl ester (CM-H2DCFDA). mRNA levels of Duox2 and its activation factor Duoxa2 (but not Duox1 or Duoxa1) were significantly suppressed by Tg in a dose-dependent manner and a time-dependent fashion in rat thyroid FRTL-5 cells. DUOX2 promoter activity was significantly suppressed by Tg in a dose-dependent manner. Protein levels of DUOX2 and H2O2 generation in cells were also reduced by Tg treatment. We show that physiological concentrations of Tg suppressed the expression and function of DUOX2 in thyroid cells. These results suggest that Tg is a strong suppressor of the expression and the activity of DUOX2/DUOXA2, thereby regulating iodide organification and hormone synthesis in the thyroid. The evidence supports a reported model in which accumulated Tg in thyroid follicles plays important roles in autoregulating the function of individual follicles, which produces the basis of follicular heterogeneity.

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.

Propylthiouracil Increases Sodium/Iodide Symporter Gene Expression and Iodide Uptake in Rat Thyroid Cells in the Absence of TSH

Propylthiouracil (PTU) and methimazole (MMI) are drugs that are widely used to treat Graves' disease. Although both exert an antithyroid effect primarily by blocking thyroid peroxidase activity, their molecular structure and other actions are different. We hypothesized that PTU and MMI may have differential effects on thyroid-specific gene expression and function. The effects of PTU and MMI on thyroid-specific gene expression and function were examined in rat thyroid FRTL-5 cells using DNA microarray, reverse transcriptase (RT)-polymerase chain reaction (PCR), real-time PCR, Western blot, immunohistochemistry, and radioiodine uptake studies. DNA microarray analysis showed a marked increase in sodium/iodide symporter (NIS) gene expression after PTU treatment, whereas MMI had no effect. RT-PCR and real-time PCR analysis revealed that PTU-induced NIS mRNA levels were comparable to those elicited by thyroid-stimulating hormone (TSH). PTU increased 5'-1880-bp and 5'-1052-bp activity of the rat NIS promoter. While PTU treatment also increased NIS protein levels, the size of the induced protein was smaller than that induced by TSH, and the protein localized predominantly in the cytoplasm rather than the plasma membrane. Accumulation of (125)I in FRTL-5 cells was increased by PTU stimulation, but this effect was weaker than that produced by TSH. We found that PTU induces NIS expression and iodide uptake in rat thyroid FRTL-5 cells in the absence of TSH. Although PTU and MMI share similar antithyroid activity, their effects on other thyroid functions appear to be quite different, which could affect their therapeutic effectiveness.