Rat Thyroid Epithelial Cell Line Research Articles

Anterograde Trafficking of KCa3.1 by Cytoskeletal Elements and Accessory Proteins in a Polarized Epithelium

We reported that KCa3.1 is trafficked to the basolateral membrane (BLM) of epithelial cells by a Rab1- and Rab8-dependent mechanism (Bertuccio et al. PlosONE 9:e92013, 2014). Here, we examine the role of cytoskeletal elements (microfilaments and microtubules) and SNARE proteins (VAMP3 and syntaxin 4, STX4) in the anterograde trafficking of KCa3.1. We used our Fischer Rat Thyroid epithelial cell line stably expressing KCa3.1-BLAP/Bir-A-KDEL (cells grown on filters), Western blot, and siRNA experiments in this study. Our approach was to streptavidin label channels that arrive at the BLM to determine the effects of cytoskeletal elements or accessory proteins on trafficking of KCa3.1 to the BLM. We examined(10 µM for 0, 3 and 5 hr) the effectsofmicrofilamentinhibitors of actin, cytochalasin D (Cyto-D, n=4) and latrunculin A (LAT-A, n=4) and microtubules: ML-7, an inhibitor of myosin light chain kinase (n=5); and the effects of siRNA knockdown of VAMP3 (n=3) and STX-4 (n=2) on trafficking of KCa3.1 to the BLM. For inhibition of microfilaments, after 5 hr, Cyto-D reduced the trafficking of KCa3.1 to the BLM by 88±4%; while Lat-A decreased the trafficking of KCa3.1 by 70±5%; and ML-7 reduced KCa3.1 by 67±11% when compared to controls. Knockdown of VAMP3 or STX-4 reduced the trafficking of KCa3.1 arriving at the BLM when compared to controls. These data suggest that microfilaments, microtubules and accessory proteins are critical for the trafficking of KCa3.1 to BLM of polarized epithelial cells. This work was supported by the NIH (DCD) and UORG and OSMS Strategic grants from the Univ. of Otago (KLH).

Serotonin activates cell survival and apoptotic death responses in cultured epithelial thyroid cells.

Anatomic and physiological interactions between central serotonergic system and thyroid gland are well established. However, the effects of locally available serotonin on the thyroid functions are poorly known. Here, we first demonstrate the expression of serotonin transporter SERT and 5-HT2A receptor subtype in rat thyroid epithelial cell line FRT both at mRNA and protein levels. In order to investigate the molecular mechanisms of serotonin action, FRT cells were exposed to increasing concentrations of the amine. Low concentrations of serotonin (up to 5μM) enhanced FRT cell growth, and ERK1/2 and SMAD2/3 phosphorylation. Cell exposure to the selective 5-HT2A receptor agonist DOI recapitulated the effects of 5-HT on ERK1/2 phosphorylation. By contrast, administration of M100907, a specific 5-HT2A receptor inhibitor, prevented 5-HT induced ERK1/2 activation. On the other hand, high doses of serotonin (50μM up to 1mM) activated a caspase-3 mediated apoptosis of cells. Overall, our findings demonstrate that low levels of serotonin, interacting with 5-HT2A receptor, are able to activate proliferative signals in the thyroid epithelial cells, while high levels of serotonin cause pro-apoptotic responses, thus suggesting an active role of the amine in the thyroid functions and disorders.

Multiple pathways for cationic amino acid transport in rat thyroid epithelial cell line PC Cl3.

Information regarding cationic amino acid transport systems in thyroid is limited to Northern blot detection of y(+)LAT1 mRNA in the mouse. This study investigated cationic amino acid transport in PC cell line clone 3 (PC Cl3 cells), a thyroid follicular cell line derived from a normal Fisher rat retaining many features of normal differentiated follicular thyroid cells. We provide evidence that in PC Cl3 cells plasmalemmal transport of cationic amino acids is Na+ independent and occurs, besides diffusion, with the contribution of high-affinity, carrier-mediated processes. Carrier-mediated transport is via y+, y(+)L, and b(0,+) systems, as assessed by L-arginine uptake and kinetics, inhibition of L-arginine transport by N-ethylmaleimide and neutral amino acids, and L-cystine transport studies. y(+)L and y(+) systems account for the highest transport rate (with y(+)L > y+) and b(0,+) for a residual fraction of the transport. Uptake data correlate to expression of the genes encoding for CAT-1, CAT-2B, 4F2hc, y(+)LAT1, y(+)LAT2, rBAT, and b(0,+)AT, an expression profile that is also shown by the rat thyroid gland. In PC Cl3 cells cationic amino acid uptake is under TSH and/or cAMP control (with transport increasing with increasing TSH concentration), and upregulation of CAT-1, CAT-2B, 4F2hc/y(+)LAT1, and rBAT/b(0,+)AT occurs at the mRNA level under TSH stimulation. Our results provide the first description of an expression pattern of cationic amino acid transport systems in thyroid cells. Furthermore, we provide evidence that extracellular L-arginine is a crucial requirement for normal PC Cl3 cell growth and that long-term L-arginine deprivation negatively influences CAT-2B expression, as it correlates to reduction of CAT-2B mRNA levels.

Trafficking of lysosomal cathepsin B-green fluorescent protein to the surface of thyroid epithelial cells involves the endosomal/lysosomal compartment.

Cathepsin B, a lysosomal cysteine proteinase, is involved in limited proteolysis of thyroglobulin with thyroxine liberation at the apical surface of thyroid epithelial cells. To analyze the trafficking of lysosomal enzymes to extracellular locations of thyroid epithelial cells, we have expressed a chimeric protein consisting of rat cathepsin B and green fluorescent protein. Heterologous expression in CHO cells validated the integrity of the structural motifs of the chimeric protein for targeting to endocytic compartments. Homologous expression, colocalization and transport experiments with rat thyroid epithelial cell lines FRT or FRTL-5 demonstrated the correct sorting of the chimeric protein into the lumen of the endoplasmic reticulum, and its subsequent transport via the Golgi apparatus and the trans-Golgi network to endosomes and lysosomes. In addition, the chimeras were secreted as active enzymes from FRTL-5 cells in a thyroid-stimulating-hormone-dependent manner. Immunoprecipitation experiments after pulse-chase radiolabeling showed that secreted chimeras lacked the propeptide of cathepsin B. Thus, the results suggest that cathepsin B is first transported to endosomes/lysosomes from where its matured form is retrieved before being secreted, supporting the view that endosome/lysosome-derived cathepsin B contributes to the potential of extracellular proteolysis in the thyroid.

Ischemia-responsive protein (irp94) is up-regulated by endoplasmic reticulum stress.

The expression of the ischemia-responsive protein (irp94) was enhanced by endoplasmic reticulum (ER) stress inducing drugs such as brefeldin A (BFA), calcium ionophor A23187, dithiothreitol (DTT) and tunicamycin in fisher rat thyroid epithelial cell line (FRTL-5 cells). In particular, irp94 mRNA expression was increased dose dependently by tunicamycin, and there was increased irp94 expression when the cells were incubated with the thyroid-stimulating hormone (TSH) together.

Control by the endogenous cannabinoid system of ras oncogene‐dependent tumor growth

We investigated the effect of 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA), a stable analog of the endocannabinoid anandamide, on a rat thyroid epithelial cell line (FRTL-5) transformed by the K-ras oncogene, and on epithelial tumors derived from these cells. Met-F-AEA effect in vivo was evaluated in a nude mouse xenograft model, where K-ras-transformed (KiMol) cells were implanted subcutaneously. Met-F-AEA (0.5 mg/kg/dose) induced a drastic reduction in tumor volume. This effect was inhibited by the CB1 receptor antagonist SR141716A (0.7 mg/kg/dose) and was accompanied by a strong reduction of K-ras activity. Accordingly, KiMol cells and tumors express CB1 receptors. Met-F-AEA inhibited (IC50 ~5 mM) the proliferation in vitro and the transition to the S phase of KiMol cells and it reduced K-ras activity; these effects were antagonized by SR141716A. Met-F-AEA cytostatic action was significantly smaller in nontransformed FRTL-5 cells than in KiMol cells. Met-F-AEA treatment exerted opposite effects on the expression of CB1 receptors in KiMol and FRTL-5 cells, with a strong up-regulation in the former case and a suppression in nontransformed cells. The data suggest that: 1) Met-F-AEA inhibits ras oncogene-dependent tumor growth in vivo through CB1 cannabinoid receptors; and 2) responsiveness of FRTL-5 cells to endocannabinoids depends on whether or not they are transformed by K-ras.

Increase in AP-1 activity is a general event in thyroid cell transformation in vitro and in vivo.

We have recently reported that neoplastic transformation of two rat thyroid epithelial cell lines by retroviruses carrying the v-mos and v-ras Ki oncogenes is associated with a drastic increase of AP-1 activity. The most important effects were represented by the dramatic junB and fra-1 gene induction, which was abolished by the block of the transformation-induced HMGI-C protein synthesis. Here, we have further characterized the transformation-dependent AP-1 activity, by analysing the expression of different jun- and fos-related components, in rat thyroid cell lines transformed by several oncogenes, in human thyroid carcinoma cell lines, and in naturally occurring human thyroid tumours. A significant increase of Fra-1 and JunB protein levels was detected in all oncogene transformed rat thyroid cell lines. Fra-1 gene induction was demonstrated to occur also in human thyroid carcinoma cell lines and tissues. Conversely, c-Jun and JunD proteins, rather than JunB, accumulated in human thyroid carcinoma cell lines. An induction of AP-1 target genes was also detected both in rat and human thyroid transformed cell lines. Therefore, in vivo and in vitro thyroid cell transformation is associated with important compositional changes in the AP-1 complex and an increased transcriptional activity.

From differentiation to proliferation: the secretory amyloid precursor protein as a local mediator of growth in thyroid epithelial cells.

In various species, thyrotropin (TSH) is known to stimulate both differentiation and proliferation of thyroid follicle cells. This cell type has also been shown to express members of the Alzheimer amyloid precursor (APP) protein family and to release the secretory N-terminal domain of APP (sAPP) in a TSH-dependent fashion. In this study on binding to the cell surfaces, exogenously added recombinant sAPP stimulated phosphorylation mediated by mitogen-activated protein kinase and effectively evoked proliferation in the rat thyroid epithelial cell line FRTL-5. To see whether this proliverative effect of sAPP is of physiological relevance, we used antisense techniques to selectively inhibit the expression of APP and the proteolytic release of sAPP by cells grown in the presence of TSH. The antisense-induced inhibition was detected by immunoblot, immunoprecipitation, and immunocytochemical analyses. After the reduced APP expression and sAPP secretion, we observed a strong suppression of the TSH-induced cell proliferation down to 35%. Recombinant sAPP but not TSH was able to overcome this antisense effect and to completely restore cell proliferation, indicating that sAPP acts downstream of TSH, in that it is released from thyroid epithelial cells during TSH-induced differentiation. We propose that sAPP operates as an autocrine growth factor mediating the proliferative effect of TSH on neighboring thyroid epithelial cells.

Identification of a Unique Monocarboxylate Transporter (MCT3) in Retinal Pigment Epithelium

The retinal pigment epithelium transports lactate between two tissue compartments, the interphotoreceptor matrix and the choriocapillaris. In this report we describe a 2.45-kb cDNA isolated from a chick cDNA RPE library that encodes a membrane protein found only in RPE cells. The deduced protein has 542 amino acids with twelve putative membrane spanning domains. The cDNA has been designated MCT3 based on its 45% identity in amino acid sequence and structural similarity with the monocarboxylate transporters MCT1 and MCT2. Stable transfectants (pCl-neo/MCT3), made in a rat thyroid epithelial cell line (FRTL-5), express MCT3 RNA. Transfectants had enhanced pyruvate uptake (used as a measure of lactate uptake) which was proton-dependent and inhibited by α-cyano-4-hydroxycinnamate. In summary, MCT3's unique expression in RPE cells, multiple potential phosphorylation sites, and basolateral distribution suggest that MCT3 may regulate lactate levels in the interphotoreceptor space.

Tolerance of high levels of wild-type p53 in transformed epithelial cells dependent on auto-regulation by mdm-2.

A significant proportion of human cancers express high levels of p53 protein in the absence of an underlying mutation in the gene. Using transformed (Vh1) and non-transformed (FRTL-5) rat thyroid epithelial cell lines as a model, we have examined the mechanisms by which high levels of wild-type p53 may be tolerated. Stable transfection with p53-dependent reporter constructs demonstrated that the 'excess' wild-type p53 in Vh1 cells is not associated with a comparable increase in p53-dependent transcription (though the response to u.v. irradiation is retained). Mdm-2, which binds p53 and inhibits its transactivation activity, is overexpressed in Vh1 cells in the absence of gene amplification and in a p53-dependent manner. Furthermore disruption of p53-mdm-2 complex formation in Vh1 cells by microinjection of an antibody to the p53-binding domain of mdm-2 resulted in a dramatic increase in p53-dependent transcription. Since only a small proportion of the p53 in Vh1 cells was found to be in complex with mdm-2 (the majority of unbound protein being in a latent form), this suggests that mdm-2 selectively binds a pool of p53 that would otherwise be active as a sequence-specific activator of transcription. We suggest that, in some types of tumour, the 'sensitivity' of the p53-driven mdm-2 feedback loop may be sufficient to prevent free, active p53 reaching the level required for growth arrest or apoptosis, making them an ideal target for therapies designed to disrupt p53-mdm-2 interactions.

Norepinephrine and thyrotropin stimulation of [Ca++]i in PC C13 a rat thyroid epithelial cell line: Effect of transformation by E1A gene of adenovirus and polyomavirus middle-T antigen gene

The effect of thyrotropin and norepinephrine on cytosolic calcium levels were evaluated in normal (PC C13) and transformed (PC E1A, PC Py and PC E1APy) rat thyroid epithelial cell lines. A different pattern of response to both norepinephrine and thyrotropin was observed among the distinct cell lines. In PC C13 the cytosolic calcium rise induced by norepinephrine, characterized by an early transient spike followed by a second phase of sustained calcium levels, was greatly enhanced by thyrotropin. The effect of norepinephrine on calcium concentrations was less affected by thyrotropin in PC C13 transformed by the adenovirus E1A oncogene. Conversely, in Polyoma middle-T transformed PC C13 the increase in cytoplasmic calcium was still sensitive to thyrotropin. The most malignant PC E1APy were totally independent of thyrotropin.

The PTC oncogene is able to dedifferentiate a rat thyroid epithelial cell line in culture

We have recently cloned a new oncogene from human papillary thyroid carcinomas, which we have designatedPTC. This oncogene consists of a chimeric gene, the 5′-end of which corresponds to a gene of unknown function, designated H4, while the 3′ portion corresponds to the tyrosine kinase domain of theRET proto-oncogene. We constructed a retroviral vector containing thePTC oncogene under the transcriptional control of the Moloney LTR, and then produced the corresponding defective retrovirus by transfecting the retroviral vector onto Psi-2 cells. We subsequently used this retrovirus to infect a differentiated rat thyroid cell line (PC-C1-3) which is dependent on TSH for growth. Upon infection with thePTC virus the PC-C1-3 cells no longer depended on TSH for growth. Furthermore, the infected cells lost completely their differentiated functions,i.e. thyroglobulin and TSH-receptor genes expression and iodide uptake. However, they retained their non malignant phenotype in that they were not able to grow in semisolid media nor in the athymic mice. The introduction in the PC-C1-3 cell line of both thePTC and the activated viral Ha-ras oncogenes resulted in the appearance of a completely undifferentiated and malignant phenotype. These results demonstrate that:a) ThePTC oncogene, which plays an important role in human thyroid carcinogenesis, is also able to induce the complete dedifferentiation of rat thyroid cellsin vitro;b) Two cytoplasmic oncogenes (Ha-ras andPTC) cooperate in the induction of a fully malignant phenotype of a thyroid differentiated cell line.

Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line.

We examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL-5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electronmicroscopy using the protein A-colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL-5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.

Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes.

Transformation of a rat thyroid epithelial cell line (FRTL5-C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid-soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC-C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v-mos oncogene) and when transformed by transfection with the c-myc proto-oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c-myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI-like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.

Interferon‐Mediated Enhancement of Thyroid Major Histocompatibility Complex Antigen Expression

Epithelial expression of class II antigens encoded by the major histocompatibility complex (MHC) has been proposed as a means by which autoimmune thyroid disease may be initiated and maintained. We studied a rat thyroid epithelial cell line (FRTL-5), which constitutively expresses class I (OX18) but not class II (OX6 or OX17) determinants to quantify in vitro MHC antigen induction using flow cytometry. Recombinant rat gamma interferon (rIFN-gamma) induced dose-dependent expression of OX6 (I-A) antigen at greater than 48 h (maximum 80-90% of cells in culture at 100 U/ml), which was abrogated by DB-1, a monoclonal antibody to rat IFN-gamma. OX17 antigen (I-E) was also induced (86%) and OX18 (class I) markedly increased under these conditions. Other thyroid-active agents including the calcium ionophore A23187, dibutyryl cyclic AMP, thyroid-stimulating autoantibodies from Graves' disease patients (LATS), and TSH, caused no I-A induction. Supernatants from spleen cells stimulated with plant lectins (concanavalin A or phytohaemagglutinin), but not lectin alone, evoked substantial class II induction, which was inhibited by DB-1. These findings suggest that IFN-gamma is the central mediator of thyroid epithelial class II expression. FRTL-5 provides a powerful model for the analysis of thyroid MHC class II dynamics and a potential means of analysing the role of epithelial class II in autoimmune pathogenesis.

Expression of the onc gene of the Kirsten murine sarcoma virus in differentiated rat thyroid epithelial cell lines.

A systematic study has been performed using a series of differentiated rat thyroid epithelial cell lines either uninfected or infected with Kirsten murine sarcoma virus (KiMSV), to determine the levels of the p21 product of the v-ras-Ki oncogene in transformed and normal cell lines. The p21 levels have been assayed by SDS-polyacrylamide gel electrophoresis of immunoprecipitates of 35S-labelled cell extracts and by a GDP binding assay. All cell lines analysed showed a significant increase in the levels of p21 after transformation with KiMSV compared to the p21 levels of uninfected and untransformed differentiated thyroid cells. The results reported here confirm the potential ability of the v-ras-Ki oncogene product to transform epithelial cells. They show, furthermore, that not only is p21 present in some epithelial cells transformed by KiMSV, but also that it is functionally active, as has been shown for fibroblasts transformed by the same virus, and that its functioning is maintained after passaging in vivo of the transformed cells.