Fisher Rat Thyroid Cell Line Research Articles

The exocyst complex is required for the trafficking and delivery of KCa3.1 to the basolateral membrane of polarized epithelia.

Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein, we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. In addition, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, Soluble N-ethylmaleimide-sensitive factor (SNF) Attachment Receptors (SNARE) proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.NEW & NOTEWORTHY Our Ussing chamber and immunoblot experiments demonstrate that when subunits of the exocyst complex were transiently knocked down, this significantly reduced the basolateral population and functional expression of KCa3.1. These data suggest, combined with our protein association experiments, that the exocyst complex regulates the tethering of KCa3.1-containing vesicles to the basolateral membrane prior to the SNARE-dependent insertion of channels into the basolateral membrane of epithelial cells.

The Flavonoid Quercetin Induces AP-1 Activation in FRTL-5 Thyroid Cells.

Previous studies have shown that quercetin inhibits thyroid function both in vitro and in vivo. An attempt to evaluate the effect of quercetin at the promoter level of the thyroid-specific genes led to the observation that this compound induces the basal activity of the reporter vector. Therefore, the action of quercetin has been evaluated on the basal activity of several reporter vectors: The PGL3 basic, promoter and control vectors from Promega, and a pSV-based chloramphenicol acetyltransferase (CAT) reporter vector. In the Fisher Rat Thyroid cell Line FRTL-5 thyroid cells transiently transfected, quercetin 10 μM increased the basal activity of all the reporter vectors evaluated, although the degree of the effect was significantly different among them. The analysis of the difference among the regulatory regions of these vectors identified the activator protein 1 (AP-1) binding site as one of the potential sites involved in the quercetin effect. Electromobility shift assay experiments showed that the treatment with quercetin induced the binding of a protein complex to an oligonucleotide containing the AP-1 consensus binding site. This is the first study showing an effect of quercetin on AP-1 activity in thyroid cells. Further studies are in progress to understand the role of AP-1 activation in the effects of quercetin on thyroid function.

Small Molecule Inhibition of IFN‐γ‐Induced Major Histocompatibility Complex Class II Expression by Thyroid Cells

Major histocompatibility complex (MHC) molecules are cell surface receptors involved in host immune response. Several lines of evidence suggest that increased expression of MHC class I (MHC I) and aberrant expression of MHC class II (MHC II) on non‐immune cells contributes to autoimmune disease, including the autoimmune thyroid disease termed Graves' disease. Graves' disease is often treated, especially in Europe, by methimazole (MMI). Previous work has shown that MMI decreases constitutive MHC I expression on the Fisher rat thyroid cell line 5 (FRTL‐5), a non‐transformed cell line often used to gain insights into human thyroid pathology. Further, MMI decreases interferon‐gamma (IFN‐γ) induction of MHC II in FRTL‐5 cells. Our labs, as well as others, have demonstrated that a phenyl‐derivative of MMI, termed C10, is more potent than MMI in a variety of cell models of disease including the FRTL‐5 model described above. Additionally, we have an ongoing molecular screening program that seeks to identify compounds that are more efficacious than MMI and C10. Thus, we initially sought to screen three novel compounds from our library in the FRTL‐5 model. Two of these compounds (COB‐204, COB‐214) were more effective than C10 in other cell models and the third (COB‐187) appears to be a highly specific inhibitor of glycogen synthase kinase ‐3 (GSK‐3). We first investigated the effect of the compounds on basal expression of MHC I. FRTL‐5 cells were treated with each compound for 24 hours and the level of MHC Class I determined via flow cytometric analysis. In each case, no significant effect on MHC Class I was observed at the levels of compound tested (50 μM for COB‐204 and COB‐214 and 20 μM for COB‐187). We next investigated the effect of the compounds on inhibition of IFN‐γ induction of MHC II. In this case, FRTL‐5 cells were treated for 24 hours with 100 U/ml IFN‐γ in the presence of the compounds or carrier control (DMSO) and MHC II expression determined by flow cytometry. 50 μM COB‐214 caused a slight (19%) inhibition, 50 μM COB‐204 caused a marked (54%) inhibition and 20 μM COB‐187 caused a fairly dramatic (80%) inhibition of IFN‐γ induced MHC II expression. Based on these results, we decided to investigate COB‐187 further and also test the effects of two other known GSK‐3 inhibitors, namely Tideglusib and AR‐A014418. All three compounds appeared to inhibit IFN‐γ induction of MHC II albeit with varying potencies. The IC50 for AR‐A014418 was 4.8 μM, for COB‐187 9.6 μM and for Tideglusib > 80 μM. The MHC II inhibition did not appear to be due to non‐specific toxicity since an MTS assay revealed the 24 hour TC50 to be 50 μM for AR‐A014418, and > 80 μM for COB‐187 and Tideglusib. Combined, these results suggest a connection between GSK‐3 and IFN‐γ induced aberrant expression of MHC II on FRTL‐5 cells and provides a possible avenue for the development of molecular therapeutics for Graves' disease.Support or Funding InformationNIH R15GM110602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium

In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.

CFTR and SLC5A8 Associate in Thyroid Epithelial Cells

The cystic fibrosis transmembrane conductance regulator, CFTR, is expressed at the apical membranes of many epithelial cells, where it mediates cAMP‐stimulated anion secretion. In thyroid, CFTR may indirectly and/or directly furnish iodide for hormone synthesis. CFTR functionally interacts with many membrane transporters via PSD‐Discs large homologous‐ZO‐1 (PDZ) domain‐containing scaffolding proteins, thereby forming macromolecular transport complexes. SLC5A8 is an electrogenic, sodium‐coupled monocarboxylate transporter that also is expressed at thyrocyte apical membranes. CFTR and SLC5A8 contain the identical PDZ domain interaction motif; preliminary studies utilizing overexpression systems indicate a role for PDZ adaptor proteins in linking CFTR and SLC5A8. The present studies use the Fisher rat thyroid cell line, FRTL‐5, to test the hypothesis that PDZ proteins facilitate CFTR/SLC5A8 interactions in an endogenously expressing system. Expression of CFTR (~150 kDa), SLC5A8 (~68 kDa) and NHERF‐1 (~50 kDa) in total FRTL‐5 lysates was confirmed by immunoblot analysis using anti‐CFTR (M3A7 and Cystic Fibrosis Foundation Therapeutics (CFFT) 660), anti‐SLC5A8 (Abcam) and anti‐NHERF‐1 (Alomone) antibodies. Anti‐SLC5A8‐immunoprecipitated (IPd) fractions of FRTL‐5 lysates reacted with both CFFT 660 and anti‐NHERF‐1. Fractions IPd using anti‐CFTR (M3A7) were probed with anti‐SLC5A8 and anti‐NHERF‐1. M3A7‐IPd fractions showed positive reactivity with anti‐SLC5A8, but NHERF‐1 was not detected. These data suggest that although CFTR and SLC5A8 associate in FRTL‐5 cells, the participation of NHERF‐1 is not required.
 Support: NIH/NIGMS Award 1R15GM101674‐01A1 (to PF); Office of the Director, NIH Award Number T35OD010979.

Functional coupling of TRPC2 cation channels and the calcium‐activated anion channels in rat thyroid cells: Implications for iodide homeostasis

The initial step in a synthesis of thyroid hormones is the uptake of iodide from the circulation. Iodide (I(-)) is transported into thyroid cells via a Na(+)/I(-) symporter (NIS), which is electrogenic and thus sensitive to alterations in membrane potential (V(m)). I(-) is then released to the lumen of thyroid follicles where the hormones are synthesised and stored. The mechanisms of I(-) release to follicle lumen are poorly characterised. Our whole-cell voltage clamp recordings revealed the presence of a Ca(2+) activated Cl(-) current (CaCC) in Fisher rat thyroid cell line 5 (FRTL-5). Transcripts of anoctamin 1 (ANO1) and anoctamin 10 (ANO10), putative molecular constituents of CaCC, were detected. The anion channels underlying CaCC are highly permeable to I(-). Both niflumic acid (NFA) and 2-aminoethyl diphenylborinate (2-APB), antagonists of CaCC and transient receptor potential channels, respectively, inhibited CaCC. Canonical transient receptor potential channel 2 (TRPC2) is the only TRPC member present in FRTL-5 cells. The activation rate of CaCC was markedly slower in shTRPC2 knock-down cells, indicating that Ca(2+) entry via TRPC2 contributes to CaCC activation. The uptake of iodide was enhanced and the resting V(m) was more depolarised in TRPC2 knock-down cells. We suggest that the interplay between TRPC2 and ANO1 may have dual effects on iodide transport, modulating I(-) release via ANO channels and I(-) uptake via the V(m) sensitive NIS.

Evaluation of [18F]-tetrafluoroborate as a potential PET imaging agent for the human sodium/iodide symporter in a new colon carcinoma cell line, HCT116, expressing hNIS

Accumulation of iodide and other substrates via the human sodium/iodide symporter (hNIS) is fundamental to imaging and therapy of thyroid disease, hNIS reporter gene imaging and hNIS-mediated gene therapy. There is no readily available positron emission tomography (PET) tracer for hNIS. Our aim was to develop a colon carcinoma cell line stably expressing hNIS, and use it to evaluate a novel hNIS PET tracer, [18F]-tetrafluoroborate. Colon carcinoma cell line, HCT116, was stably transfected with hNIS, thus producing a cell line, HCT116-C19, with high hNIS expression. A Fisher rat thyroid cell line, FRTL5, which expresses rat sodium/iodide symporter when stimulated with thyroid-stimulating hormone, was used for comparison. Accumulation of [188Re]-perrhenate, [99mTc]-pertechnetate and [18F]-tetrafluoroborate was evaluated with and without perchlorate inhibition using an automated radioimmune assay system, LigandTracer. The affinity of [18F]-tetrafluoroborate for hNIS, and its half-maximal inhibitory concentration (IC50) for the inhibition of [99mTc]-pertechnetate transport were determined from the plateau accumulation of [18F]-tetrafluoroborate and [99mTc]-pertechnetate, respectively, as a function of tetrafluoroborate concentration. [18F]-tetrafluoroborate accumulated effectively in both FRTL5 and HCT116-C19 cells. The accumulation in HCT116-C19 cells (plateau accumulation 31%) was comparable to that of [188Re]-perrhenate (41%) and [99mTc]-pertechnetate (46%). Its affinity for hNIS and half-maximal inhibitory concentration (IC50) for the inhibition of pertechnetate uptake was approximately micromolar. We have produced a human colon cell line with a stable constitutive expression of functional hNIS (HCT116-hNIS-C19). [18F]-tetrafluoroborate accumulates in cells expressing hNIS or rat sodium/iodide symporter and is a potential PET imaging agent in thyroid disease and hNIS reporter gene imaging.

NF-κB p65 Subunit Mediates Lipopolysaccharide-Induced Na+/I−Symporter Gene Expression by Involving Functional Interaction with the Paired Domain Transcription Factor Pax8

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) elicits a variety of biological responses. Na(+)/I(-) symporter (NIS)-mediated iodide uptake is the main rate-limiting step in thyroid hormonogenesis. We have recently reported that LPS stimulates TSH-induced iodide uptake. Here, we further analyzed the molecular mechanism involved in the LPS-induced NIS expression in Fisher rat thyroid cell line 5 (FRTL-5) thyroid cells. We observed an increase in TSH-induced NIS mRNA expression in a dose-dependent manner upon LPS treatment. LPS enhanced the TSH-stimulated NIS promoter activity denoting the NIS-upstream enhancer region (NUE) as responsible for the stimulatory effects. We characterized a novel putative conserved kappaB site for the transcription factor nuclear factor-kappaB (NF-kappaB) within the NUE region. NUE contains two binding sites for the transcription factor paired box 8 (Pax8), main regulator of NIS transcription. A physical interaction was observed between the NF-kappaB p65 subunit and paired box 8 (Pax8), which appears to be responsible for the synergic effect displayed by these transcription factors on NIS gene transcription. Moreover, functional blockage of NF-kappaB signaling and site-directed mutagenesis of the kappaB cis-acting element abrogated LPS stimulation. Silencing expression of p65 confirmed its participation as an effector of LPS-induced NIS stimulation. Furthermore, chromatin immunoprecipitation corroborated that NIS is a novel target gene for p65 transactivation in response to LPS. Moreover, we were able to corroborate the LPS-stimulatory effect on thyroid cells in vivo in LPS-treated rats, supporting that thyrocytes are capable of responding to systemic infections. In conclusion, our results reveal a new mechanism involving p65 in the LPS-induced NIS expression, denoting a novel aspect in thyroid cell differentiation.

Effects of iodine excess on mitochondrial superoxide production and mitochondrial membrane potential in rat thyroid cell line cells

Objective To investigate the effects of iodine excess on mitochondrial superoxide production and mitoehondrial membrane potential(△ψ)changes in Fisher rat thyroid cell line(FRTL)cells.Methods FRTL cells were treated with 10-4mol/L potassium iodine(KI),10 U/L thyrotropin(TSH),10-4 mol/L KI+10 U/L TSH respectively for 24 h.Effects on cell proliferation were assayed by methyl thiazolyl tetrazolium(MTT)colorimetric method.Changes of mitochondrial superoxide production and △ψ were measured by live cell imaging and spectrofluorometer using MitoSOX and rhodamine 123(rh123)respectively.Results Absorbance(A)in the KI group (0.794±0.144)showed a significant decline compared to the control group(1.000 ±0.183,P 0.05),but the former was marked higher than the KI group(P 0.05). Conclusion Iodine excess (10-4 mol/L KI) may lead to peroxide damage on the mitochondria of FRTL cells, and cell proliferation is inhibited. Combining treatment with 10 U/L TSH may attenuate mitochondrial peroxide damage and inhibition of cell proliferation caused by iodine excess. Key words: Iodine; Thyroid gland; Superoxides; Membrane potential, mitochondrial; Thyrotropin

Regulation of major histocompatibility complex gene expression in thyroid epithelial cells by methimazole and phenylmethimazole

Increased expression of major histocompatibility complex (MHC) class-I genes and aberrant expression of MHC class-II genes in thyroid epithelial cells (TECs) are associated with autoimmune thyroid diseases. Previous studies have shown that methimazole (MMI) reduces MHC class-I expression and inhibits interferon-gamma (IFN-gamma or IFNG as listed in the MGI Database)-induced expression of the MHC class-II genes in TECs. The action of MMI on the MHC class-I genes is transcriptional, but its mechanism has not been investigated previously. In the present study, we show that in Fisher rat thyroid cell line 5 cells, the ability of MMI and its novel derivative phenylmethimazole (C10) to decrease MHC class-I promoter activity is similar to TSH/cAMP suppression of MHC class-I and TSH receptor genes, and involves a 39 bp silencer containing a cAMP response element (CRE)-like site. Furthermore, we show that C10 decreases MHC class-I gene expression to a greater extent than MMI and at 10- to 50-fold lower concentrations. C10 also reduces the IFN-gamma-induced increase in the expression of MHC class-I and MHC class-II genes more effectively than MMI. Finally, we show that in comparison to MMI, C10 is a better inhibitor of specific protein-DNA complexes that are formed with a CRE-like element on the MHC class-II promoter. These data support the conclusion that the immunosuppressive mechanism by which MMI and C10 inhibit MHC gene expression mimics 'normal' hormonal suppression by TSH/cAMP.

Bacterial Lipopolysaccharide Stimulates the Thyrotropin-Dependent Thyroglobulin Gene Expression at the Transcriptional Level by Involving the Transcription Factors Thyroid Transcription Factor-1 and Paired Box Domain Transcription Factor 8

The bacterial lipopolysaccharide (LPS) is a biological activator that induces expression of multiple genes in several cell types. LPS has been proposed as an etiopathogenic agent in autoimmune diseases. However, whether LPS affects the expression of autoantigens has not been explored. Thyroglobulin (TG) is a key protein in thyroid hormonogenesis and one of the major thyroid autoantigens. This study aimed to analyze the action of LPS on TG gene expression in Fisher rat thyroid cell line FRTL-5 thyroid cells. We demonstrate that LPS increases the TSH-induced TG protein and mRNA level. Evidence that the effect of LPS is exerted at the transcriptional level was obtained by transfecting the minimal TG promoter. The C element of the TG promoter, which contains sequences for paired box domain transcription factor 8 (Pax8) and thyroid transcription factor (TTF)-1 binding, is essential for full TG promoter expression under TSH stimulation. The transcriptional activity of a construct containing five tandem repeats of the C site is increased by LPS, indicating a possible involvement of the C site in the LPS-induced TG gene transcription. We demonstrate that the TG promoter mutated at the Pax8 or TTF-1 binding element in the C site does not respond to LPS. In band shift assays, binding of Pax8 and TTF-1 to the C site is increased by LPS. The Pax8 and TTF-1 mRNA and protein levels are augmented by LPS. The half-lives of TG, Pax8, and TTF-1 are increased in endotoxin-treated cells. Our results reveal the ability of LPS to stimulate the expression of TG, a finding of potential pathophysiological implication.

P2X7 Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X(7) receptor activated a reversible, Ca(2+)-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (C(m)) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5'-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in C(m) without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2',3'-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2'4'-disulfonic acid, adenosine 5'-triphosphate-2'3'-dialdehyde, and Cu(2+). The capacitance decrease persisted in Na(+)-, Ca(2+)- and Cl(-)-free external saline or with Ca(2+)-free pipette solution. It is concluded that ATP activation of the inotropic P2X(7) receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca(2+) or influx of Na(+) or Ca(2+) through the receptor-gated channel.

Binding of rat thyroglobulin to heparan sulfate proteoglycans.

We previously showed that rat thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits Tg binding to megalin (gp330), an endocytic Tg receptor found on the apical surface of thyrocytes. Cooperation between cell surface receptors and heparin-like molecules, namely heparan sulfate proteoglycans (HSPGs), can facilitate cell surface binding of some heparin-binding proteins. Based on our previous findings indicating that heparin and megalin-binding sites of rat Tg are functionally related, here we investigated whether rat Tg binds to HSPGs, which are expressed by thyroid cells. We showed in solid phase assays that unlabeled rat Tg binds to a heparan sulfate (HS) preparation in a dose-dependent, saturable manner, with moderately high affinity (Kd approximately 19 nM, Ki approximately 25 nM). Binding was inhibited by heparin and by HS itself. We then studied the role of HSPGs in Tg binding to FRTL-5 cells, a differentiated Fisher rat thyroid cell line. As previously reported, after incubation of FRTL-5 cells with unlabeled rat Tg at 4 degrees C, heparin released virtually all the cell-bound Tg. Co-incubation of Tg with HS or with a preparation of HSPGs resulted in a reduction of binding by 35%-40%. When FRTL-5 cells were preincubated with heparitinase or heparinase I, which released 20%-30% of cell surface HSPGs, Tg binding was reduced to a similar extent. An antibody against a Tg heparin-binding site functionally related to a major megalin-binding site virtually abolished Tg binding to HS and to FRTL-5 cells, supporting the hypothesis that combined interactions of Tg with HSPGs and with megalin are involved in Tg binding to rat thyroid cells.

The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity.

Iodide concentration by the thyroid gland, an essential step for thyroid hormone synthesis, is mediated by the Na+/I- symporter (NIS). To identify factors that may regulate this process, we have studied NIS gene expression in the Fisher rat thyroid cell line (FRTL-5) by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. Increasing concentrations of bovine TSH (0.1, 1, 10, 50 and 100 mU/l), with or without tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) or interleukin-1 alpha (IL-1 alpha) were added to FRTL-5 cells previously deprived of TSH for a minimum of 5 days. RNA was extracted and samples were studied for NIS expression. TSH enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.1 mU/l, reaching a peak at 50 mU/l, an effect detected after 6 h of stimulation, but not in the first 2 h. Both TNF alpha and, to a lesser extent, IL-1 alpha inhibited basal and TSH-induced NIS expression. High concentrations of IFN gamma also downregulated TSH-stimulated NIS mRNA expression. Using the same technique, we also investigated NIS mRNA tissue distribution in two male and one female Wistar rats. High levels of NIS expression were detected in the thyroid, stomach, and mammary gland, lower levels were found in the intestine, adipose tissue and liver, borderline levels were expressed in the salivary gland, and no expression was detected in the kidneys. In summary, we have shown that TSH upregulates rat NIS gene expression in vitro, and this induction can be modulated by cytokines. Analysis of the distribution of rat NIS mRNA ex vivo demonstrated variable levels of NIS transcription in different tissue samples.

TGF-beta1 actions on FRTL-5 cells provide a model for the physiological regulation of thyroid growth.

Little is known about the TGF-beta1 mechanism that promotes thyroid cell growth arrest. We assessed TGF-beta1 effects on Fisher rat thyroid cell line (FRTL-5). This allowed us to study TGF-beta1 action on thyroid cells in various physiological situations such as actively proliferating cells, resting cells stimulated to proliferate by the action of various mitogens, and resting cells. TGF-beta1 arrested proliferating FRTL-5 cells, increasing c-myc mRNA levels and reducing p27-free cyclin D1 protein levels, without affecting either the cellular content of p27 or the cyclin D1-p27 complexes. Moreover, TGF-beta1 treatment reduced the activity of cyclin E-CDK2 complexes and, consequently, pRB was found to be hypophosphorylated. TGF-beta1 prevented resting cells to enter in the cell cycle when stimulated with growing medium (newborn calf serum plus a mixture of five hormones) but not when TSH (thyroid stimulating hormone) plus IGF-1 (Insulin-like growth factor I) were used as mitogens. Both stimuli increased the levels of cyclins D1, D3 and E but TGF-beta1 had a greater effect in decreasing these cyclin levels in growing-medium stimulated cells than in TSH + IGF-1. This suggests that for FRTL-5 cells, the content of these cyclins must exceed a threshold to progress through the cell cycle. TGF-beta1 induced apoptosis in quiescent cells, accompanied by a reduction in p27 protein levels and an increase in c-myc expression. Interestingly, TGF-beta1-induced variations in prothymosin alpha and c-myc mRNA levels were not correlated. TGF-beta1 always promoted an increase of p15 mRNA levels. In summary, our results point to the fact that TGF-beta1 could play a physiological role in the control of thyroid growth through the modification of cell cycle regulatory proteins.

Thyroid-Stimulating Antibody Assay Using a Human Thyrotropin Receptor Transfected Cell Line: Relationship to Clinical Features of Graves’ Disease

To evaluate a Chinese hamster ovary cell line transfected with the human thyrotropin (thyroid-stimulating hormone or TSH) receptor (CHO-TSHR) for its use in routine clinical testing and compare it with the currently used Fisher rat thyroid cell line (FRTL-5) relative to the cyclic adenosine monophosphate (cAMP) responses to thyroid-stimulating antibodies (TSAb). We analyzed 112 serum samples prospectively. TSAb were measured concurrently by both CHO-TSHR cells and FRTL-5 cells. In addition, thyrotropin-binding inhibitory immunoglobulin (TBII) was measured by a radioreceptor assay. Among the study subjects, 51 had active Graves' disease, 40 had other thyroid disease, and 21 had no thyroid disease. Of 51 patients with Graves' disease, 38 (74%) had positive results in both cell assays, 4 (8%) had negative results in both assays, and 9 (18%) had positive responses with CHO-TSHR cells but negative results with FRTL-5 cells. No patient had negative findings with CHO-TSHR cells and positive results by FRTL-5 cells. All 61 patients in the other two groups had negative results by both assays (diagnostic specificity = 100%). The diagnostic sensitivity and accuracy of TSAb activity by the two cell lines (CHO-TSHR versus FRTL-5) were 92.2% versus 74.5% and 96.4% versus 88.4%, respectively. A significant correlation was noted between the TSAb activities in active Graves' sera by the two assays (r = 0.58; P<0.0001); however, values by the CHO-TSHR method were significantly higher (P<0.003). TSAb activities determined with both cell lines were positively correlated with the 24-hour thyroidal 131 I uptake (r = 0.62 and P<0.0005 in CHO-TSHR cells; r = 0.41 and P<0.05 in FRTL-5 cells). The difference in TSAb activity between patients with ophthalmopathy and those without ophthalmopathy was statistically significant with both FRTL-5 (P<0.01) and CHO-TSHR (P = 0.05) assays. The correlation between the severity of eye disease and TSAb activities was significant only with the FRTL-5 assay (P = 0.009) but not with the CHO-TSHR assay (P = 0.16). TSAb activity showed a significant positive correlation with serum thyroxine, triiodothyronine, and TBII by both cell assays. Neither cell line showed a significant correlation between TSAb activity and antimicrosomal antibody titer. The CHO-TSHR cell line is superior to the standard FRTL-5 cell line in the cAMP bioassay for TSAb with reference to diagnostic sensitivity and accuracy, correlation with disease activity, simplicity, and cost.

Characterization of ganglioside associated with the thyrotrophin receptor.

The receptor protein for thyrotrophin (thyroid-stimulating hormone; TSH) is associated with a glycosphingolipid moiety. The protein belongs to the family of receptors that couple to guanine nucleotide binding proteins; the glycosphingolipid contains sialic acid and belongs to the family of gangliosides. This report defines the structure of the receptor ganglioside in the Fisher rat thyroid cell line (FRTL-5). Receptor protein was purified by TSH affinity chromatography from FRTL-5 cells, biosynthetically labelled with [3H]galactose and [3H]glucosamine, and resolved by SDS-PAGE. A single radiolabelled band of Mr approximately 80 kDa, corresponding to the predicted size of the cloned receptor, contained ganglioside. Gangliosides were extracted from unlabelled receptor protein after SDS-PAGE and probed on TLC plates with 125I-labelled Limax flavus agglutinin or the B subunit of cholera toxin, before and after digestion with Vibrio cholerae sialidase or beta-galactosidase. The TSH receptor (TSH-R) ganglioside belongs to the gangliotetraose family, having sialic acid attached to both galactose molecules. Its sialic acid is devoid of negative charge because of the formation of internal esterlactones. Its structure is lactonized N-acetylneuraminyl-(alpha 2-->3)galactosyl(beta 1-->3)-N-acetylgalactosaminyl(beta 1-->4)-[N-acetylneuraminyl(alpha 2-->3)]galactosyl(beta 1-->4)glucosyl(beta 1-->1)ceramide (GDla-lactone). Ganglioside lactones have not been previously described as components of thyroid cells. They are highly rigid and are more likely than their parent structures to serve as molecular recognition sites and elicit immunoreactivity. Identification of this unique ganglioside intimately associated with the TSH receptor implies that it has an integral role in receptor structure and function.

Polarized distribution of ? interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells)

The characteristics of a polarized epithelial cell line and dynamics of an endogenous polarized plasma membrane constituent were studied by use of an subclone, FRT-9, from the Fisher rat thyroid cell line, FRT. Transmission electron microscopy (conventional, freeze-fracture), determination of transepithelial electrical parameters and immuno-fluorescence study, were used to establish polarity and demonstrated the basolateral distribution of transferrin receptors and the major histocompatibility complex antigens (constitutive Class I or gamma interferon-induced Class II).

Strain-Specific Determination of the Degree of Thyroid Cell MHC Class II Antigen Expression: Evaluation of Established Wistar and Fisher Rat Thyroid Cell Lines*

The major histocompatibility complex (MHC) genes, primarily of the MHC class II region, are linked to increased susceptibility to autoimmune thyroid disease in animals and humans. The quantity of MHC class II antigens that are expressed on the appropriate cell surface, as well as their allotype, are of vital importance to immune function. We have, therefore, compared MHC class II (RT1.D) gene activation in a newly available Wistar rat thyroid (WRT) cell line (a thyroiditis-susceptible strain) with a cloned cell (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) (a thyroiditis-resistant strain) to determine differences in their degree of MHC class II gene activation and antigen expression. There was no detectable constitutive MHC class II antigen or RT1.D alpha-chain messenger RNA expression in either WRT or 1B-6 cells. Cultures of both cells expressed MHC class II gene induction in response to recombinant rat gamma-interferon (gamma IF). After 72 h of exposure to 25 U/ml gamma IF, over 80% of WRT cells expressed class II antigen, detected by flow cytometric assessment, as compared to only 15% of 1B-6 cells. This earlier and greater expression of MHC class II antigen was reflected in the RT1.D alpha-chain mRNA responses with peak levels in WRT cells after only 24 h of exposure to gamma IF, a period in which 1B-6 cells showed only minor increases in mRNA. To examine whether these differences in class II MHC expression were thyroid-cell specific, primary pulmonary fibroblast lines from Wistar and Fisher rats were developed and a similar, although less marked, variation in susceptibility to gamma IF was observed. Hence, those rats strains that are resistant to autoimmune thyroid disease exhibit a fundamental difference in their MHC class II gene responsiveness to cytokines, which may contribute to their disease susceptibility profiles.

Activation of MHC-restricted rat T cells by cloned syngeneic thyrocytes.

We have previously demonstrated that rat thyrocytes express MHC class II Ag (RT1.B&D) in response to IFN-gamma. To determine whether MHC class II-positive thyrocytes can be recognized by MHC-restricted T cells, we used our clone of rat thyroid cells (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) and known to express MHC class II Ag in response to recombinant rat IFN-gamma. CD4+ and CD8+ normal syngeneic Fisher rat spleen T cells were selected by flow cytometry and averaged greater than 96% purity. We demonstrated that irradiated MHC class II-positive but not class II-negative 1B-6 thyrocytes stimulated CD4+ T cells in a primary sensitization reaction over 4 days. In contrast, CD8+ T cells had no response in similar experiments. This stimulation of CD4+ T cells was dose dependent for 1B-6 thyrocytes and was abrogated by anti-rat MHC class II mAb (MRC OX-6). Autoreactive (Fisher) and alloreactive (Buffalo) T cell lines and isolated CD4+ T cells derived from these lines, which were developed against Fisher rat spleen cells, similarly recognized MHC class II Ag expressed on 1B-6 cells but had no detectable response to 1B-6 MHC class II-negative thyrocytes or MHC class II-positive human thyroid cells. The CD4+ T cell recognition of 1B-6 cells via MHC class II Ag supports our previous data with autologous human thyroid T cell co-cultures and is indicative of an autospecific role for thyrocytes in the development of autoimmune thyroiditis.