Expression and Purification of the Human Thyroid-Stimulating Hormone Receptor.

The thyroid-stimulating hormone receptor (TSHR) is a Class A G protein-coupled receptor (GPCR) that mediates signalling through the hypothalamic-pituitary-thyroid axis. Inappropriate activation of TSHR by autoantibodies or mutations, results in human disease such as Grave's disease and Hashimito's thyroiditis. Therefore, there is a need to develop novel therapeutics targeting the TSHR. Understanding the structure and mechanism of activation of this receptor would help elucidate the pathogenesis of disease and aid drug development. Here, we describe a method for the expression of the human TSHR in a mammalian cell line generated through a lentiviral expression system. The receptor is then purified by affinity chromatography in the ligand-free state and is suitable for structure determination by single-particle electron cryo-microscopy (cryo-EM).

Regulation of TSHR Expression in the Thyroid and Thymus May Contribute to TSHR Tolerance Failure in Graves' Disease Patients via Two Distinct Mechanisms.

Graves' disease (GD) involves the presence of agonistic auto-antibodies against the thyrotropin receptor (TSHR), which are responsible for the clinical symptoms. While failure of TSHR tolerance is central to GD pathogenesis, the process leading to this failure remains poorly understood. Two mechanisms intimately linked to tolerance have been proposed to explain the association of SNPs located in TSHR intron 1 to GD: (1) differential alternative splicing in the thyroid; and (2) modulation of expression in the thymus. To elucidate the relative contribution to these two mechanisms to GD pathogenesis, we analyzed the level of full-length and ST4 and ST5 isoform expression in the thyroid (n = 49) and thymus (n = 39) glands, and the influence of intron 1-associated SNPs on such expression. The results show that: (1) the level of flTSHR and ST4 expression in the thymus was unexpectedly high (20% that of the thyroid); (2) while flTSHR is the predominant isoform, the levels are similar to ST4 (ratio flTSHR/ST4 = 1.34 in the thyroid and ratio flTSHR/ST4 in the thymus = 1.93); (3) next-generation sequencing confirmed the effect of the TSHR intron 1 polymorphism on TSHR expression in the thymus with a bias of 1.5 ± 0.2 overexpression of the protective allele in the thymus compared to the thyroid; (4) GD-associated intron 1 SNPs did not influence TSHR alternative splicing of ST4 and ST5 in the thyroid and thymus; and (5) three-color confocal imaging showed that TSHR is associated with both thymocytes, macrophages, and dendritic cells in the thymus. Our findings confirm the effect of intron 1 polymorphisms on thymic TSHR expression and we present evidence against an effect on the relative expression of isoforms. The high level of ST4 expression in the thymus and its distribution within the tissue suggest that this would most likely be the isoform that induces central tolerance to TSHR thus omitting most of the hinge and transmembrane portion. The lack of central tolerance to a large portion of TSHR may explain the relatively high frequency of autoimmunity related to TSHR and its clinical consequence, GD.

Thyroid-Stimulating Hormone Receptor Expression on Primary Cultured Human Extraocular Muscle Myoblasts.

PURPOSE: To isolate and culture human extraocular muscle (EOM) myoblasts and facilitate their differentiation to myotubes in vitro, and to determine whether these myoblasts express thyroid-stimulating hormone receptor (TSHR). MATERIALS AND METHODS: Human EOM myoblasts were isolated from EOM samples, and identified by immunostaining for PAX7 and MYOD1 (markers of human skeletal myoblasts), and western blot for desmin (muscle marker). In addition, we investigated the expressions of SHOX2 (a genetic marker of EOM myoblasts) and HOXC10 (an exclusive marker of hind-limb muscle-derived myoblasts) by RT-PCR. Fusion index and myotube area were measured to quantify myotube differentiation. TSHR immunostaining and western blot were used to determine the presence of TSHR on human EOM myoblasts and investigate its expression during myogenesis. RESULTS: Human EOM myoblasts were immunopositive for PAX7 and MYOD1 staining, and had desmin expression during myogenesis. The EOM-specific gene SHOX2 was detected by RT-PCR, but HOXC10 was not detected. The significant change in both fusion index and myotubes were shown at 8 days after induction of differentiation myotubes. Immunostaining revealed TSHR was expressed on human EOM myoblasts and western blot demonstrated the presence of TSHR protein and highest TSHR protein expression was shown at 10 days after myogenic differentiation. CONCLUSIONS: Human EOM myoblasts were cultured and underwent myogenic differentiation in vitro. TSHR protein was detected on human EOM myoblasts and increasing TSHR expression during myogenic differentiation.

Identification of Functional Thyroid Stimulating Hormone Receptor and TSHR Gene Mutations in Hepatocellular Carcinoma.

BACKGROUND/AIM: Extra-thyroid expression of thyroid stimulating hormone (TSH) receptor (TSHR) has been reported in normal liver tissues, but never assessed in hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Paired cancerous and non-cancerous HCC tissues were analyzed with TSHR expression assays. TSHR functional assessments and sequence analysis for the TSHR exon-10 were performed. RESULTS: TSHR overexpression was found in 150/197 (76.1%) HCCs. Higher TSHR expression was associated with unfavorable postoperative outcomes. Immunohistochemical analysis revealed predominantly nuclei/peri-nuclei localization of TSHR in cancerous tissues but cell membrane localization in non-cancerous parts. TSH stimulation on hepatoma cells resulted in increased cyclic adenosine monophosphate levels with altered cell sensitivity to cisplatin. Gene mutations leading to TSHR truncation were detected in 8/81 (9.9%) HCC tissues. CONCLUSION: Overexpression of TSHR was found in a great majority of HCC tissues and associated with unfavorable prognosis. Cell-based experiments and gene mutation analysis suggested that TSHR in HCCs was functional.

Cloning, characterization, expression, and feeding response of thyrotropin receptor in largemouth bass (Micropterus salmoides).

Thyrotropin receptor (TSHR) is a G-protein-coupled receptor that regulates the synthesis, storage, and secretion of thyroid hormones in the thyroid tissue. The aims of the present study were to characterize the full-length TSHR cDNA in largemouth bass (Micropterus salmoides), and to determine the TSHR gene transcription levels in different tissues. In addition, the response of TSHR transcription levels to daily feeding in thyroid tissue was investigated. The results showed that the full-length cDNA sequence was 2743 bp with an open reading frame of 2340 bp encoding a 779-amino acid peptide. BLAST analysis indicated that the amino acid sequence displayed 58.4-90.2% identity and 5.6-125.8 divergence, compared with other known fish species. The most abundant TSHR transcription levels were found in the spleen, head kidney, and kidney. Feeding did not affect the transcription level of TSHR in thyroid tissue over the course of the day. Thus, the current study suggests that there was no relationship between daily nutritional status and TSHR transcription level in the thyroid tissue of largemouth bass. The spleen, head kidney, and kidney exhibited the most abundant TSHR transcription levels.

[Construction of stable expression of human thyrotropin receptor alpha-subunits on HEK 293T cells].

The aim of this study was to establish stable expression of human thyroid stimulating hormone receptor (TSHR) alpha-subunit (hTSHRA) on human embryonic kidney 293T (HEK 293T). HEK 293T cell lines with stable expression of hTSHRA could be used for detecting affinity between hTSHRA and potential TSHR blocking-peptide. We firstly constructed hTSHRA gene into lentiviral vectors GV218. The sequence comparison indicated that we had constructed GV218-hTSHRAA. Western blot demonstrated the 52 kD aim band of hTHSRA on over-expressed HEK 293T cells. GV218-hTSHRA constructions and pHelper were then co-transfected into HEK 293T cells to form packaging plasmid. The HEK 293T cells that stably expressed hTSHRA could also express green fluorescent pro tein. The titer of lentiviral packaging vector is 2 x 10(8) TU/mL with qPCR. The lentiviral packaging vector thereafter was transfected into HEK 293T cells again. The hTSHRA expressed on the HEK 293T cells. Human TSHRA stab ly expressed on HEK 293T upon continuously passaging. Therefore, we established hTSHRA stable expression on HEK 293T cells by constructing GV218-hTHSR lentiviral packaging vector. It is a useful tool for studying TSHR affinity with anti-thyroid peptide.

Thyrotropin acts as a T-cell developmental factor in mice and humans.

Using gene expression profiling, we detected differential thyrotropin receptor (TSH-R) expression during human T-cell development in the thymus. This expression pattern indicated a potential role for the TSH-R within the thymus, independent of its function in the thyroid gland. Here, we demonstrate that TSH-R expression is thymus-specific within the immune system. TSH was able to bind and activate the TSH-R present on thymocytes, thereby activating calcium signaling and cyclic adenosine monophosphate signaling pathways. Mice lacking functional TSH-R expression (hyt/hyt mice) were shown to have lower frequencies of DP and SP thymocytes compared to their heterozygous littermates. Moreover, addition of TSH to co-cultures of human thymocytes enhanced T-cell development. Thus, TSH acts as a previously unrecognized growth factor for developing T cells, with potential clinical use to enhance thymic output and thereby the functional T-cell repertoire in the periphery. The direct effects of TSH on thymocytes may also explain the thus far enigmatic thymic hyperplasia in Graves' disease.

Falso incremento de la hormona estimulante del tiroides asociado a la presencia de macro-TSH / Falsely elevated levels of thyrotropin caused by macro-TSH

Mujer que presentó un importante incremento de la hormona estimulante del tiroides (TSH) (62,2 mU/L) con hormonas tiroideas dentro de los intervalos de referencia. La paciente se encontraba eutiroidea y no presentaba bocio. Se realizó un estudio inicial para determinar la posible causa del incremento en la concentración de TSH. La recuperación de TSH tras precipitación con polietilenglicol fue del 1%, sugiriendo la presencia de alguna molécula de elevado peso molecular que podría interferir en la determinación. Mediante cromatografía de exclusión, se confirmó la presencia de macro-TSH, un complejo autoinmune formado por TSH unido a una Inmunoglobulina G que es inmunorreactivo pero biológicamente inactivo, por lo que, si no se detecta, induce a una interpretación errónea de la concentración de TSH (AU)

Woman showing an important increase of serum TSH (62.2 mU/L) with thyroid hormones within the reference interval. The patient was clinically euthyroid and without goitre. Investigations were carried out to determine the origin of the unexpected high TSH. Polyethylene glycol precipitation test showed low TSH recovery (1%), indicating the presence of large molecules that could interfere with the measurement. The serum sample was fractionated by gel filtration chromatography and the presence of a macro-TSH form was confirmed, an immunoreactive but biologically inactive TSH-Immunoglobulin G autoantibody complex. Its detection is important to avoid a misleading interpretation of the TSH concentration (AU)

Effective expression of human proteins on bacterial magnetic particles in an anchor gene deletion mutant of Magnetospirillum magneticum AMB-1.

Biologically synthesized magnetic particles by magnetotactic bacteria (BacMPs) have promising potential in the area of functional protein display technology for various biotechnological and biomedical applications. Functional proteins fused with an anchor protein, Mms13, have been demonstrated to be an effective and stable method for the display of functional proteins on BacMPs. However, the expression of some human proteins is relatively low. Useful host strains of Escherichia coli have been developed for the enhanced expression of recombinant proteins using a genetic engineering approach. To improve human protein expression level on BacMPs in Magnetospirillummagneticum AMB-1, a mutant strain with a deleted native mms13 gene (Δmms13 strain) was established and evaluated for effective functional protein display technology. As a result, the Δmms13 strain synthesized BacMPs with significantly improved expression of two human proteins, thyroid-stimulating hormone receptor (TSHR) and the class II major histocompatibility complex (MHC II) molecules. The Δmms13 strain could therefore be an effective strain for the display of other important human proteins on BacMPs and may be useful for further applications.

Expression of functional TSH receptor in white adipose tissues of hyt/hyt mice induces lipolysis in vivo.

To determine the relative importance of TSH in white adipose tissue, we compared the adipose phenotypes of two distinct mouse models of hypothyroidism. These models differed in that the normal reciprocal relationship between thyroid hormone and TSH was intact in one and disrupted in the other. One model, thyroidectomized (THYx) mice, had a 100-fold increase in TSH and a normal TSH receptor (TSHR); in contrast, the other model, hyt/hyt mice, had a 120-fold elevation of TSH but a nonfunctional TSHR. Although both THYx and hyt/hyt mice were in a severe hypothyroid state, the epididymal fat (mg)/body wt (g) (F/B) ratio of THYx mice was much smaller than that of hyt/hyt mice (8.2 ± 0.43 vs. 14.4 ± 0.40, respectively, P < 0.001). The fat cell diameter in THYx mice was also smaller than that in hyt/hyt mice (79 ± 2.8 vs. 105 ± 2.2 µm, respectively, P < 0.001), suggesting that TSH induced lipolysis in adipose tissues. When we transferred a functional mouse TSHR gene and a control plasmid into opposite sides of epididymal fat of hyt/hyt mice by plasmid injection combined with electroporation, fat weight of the TSHR side was decreased to 60% of that of the control side. Messenger RNA levels of hormone-sensitive lipase in epididymal fat containing the transferred TSHR gene were twofold higher than those in tissue from the control side. These results indicated that TSH worked as a lipolytic factor in white adipose tissues, especially in mice in a hypothyroid state.

Expression of thyroid-specific transcription factors in thyroid carcinoma, contralateral thyroid lobe and healthy thyroid gland in dogs.

Thyrotropin receptor (TSH-R), thyroglobulin (Tg), thyroperoxidase (TPO), thyroid specific transcription factor-1 (TTF-1), paired box 8 transcription factor (PAX-8), insulin like growth factor-1 (IGF-1) and estrogen receptor alpha (ERα) transcripts were determined by real-time PCR in follicular carcinoma and contralateral (CL) lobes, and healthy thyroid canine glands. Concentrations of TSH-R, PAX-8, and ERα mRNA were not different among groups; the carcinoma group had lower Tg and TPO mRNA than healthy and CL groups, while no differences were found between the two latter groups, suggesting that the carcinoma tissue presents an altered capacity to synthesize thyroid hormones. The transcription factor that promotes thyrocytes proliferation, TTF-1 as well as IGF-1, presented a greater mRNA expression in the CL group, suggesting that the CL lobe may function in a compensatory state.

A new small-molecule antagonist inhibits Graves' disease antibody activation of the TSH receptor.

CONTEXT: Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. OBJECTIVE: Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. DESIGN: We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. RESULTS: We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. CONCLUSION: NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

A fast method to detect cell surface expression of thyrotropin receptor (TSHr): the microchip flow cytometry analysis.

Loss-of-function mutations of the thyrotropin receptor (TSHr) may be responsible for congenital hypothyroidism or isolated hyperthyreotropinemia. To study cell surface expression of inactivating TSHr mutations detected in patients with isolated hyperthyreotropinemia (L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T), we used the Agilent 2100 bioanalyzer to perform microchip flow cytometry analysis. The previously described TSHr inactivating mutation T477I was used as control. The level of receptor expression in COS-7 cells transfected with the T477I measured by binding assay was four times lower with respect to the wild-type TSHr. The very low expression of T477I was confirmed by fluorescence-activated cell sorting (FACS) analysis and by microchip flow cytometry analysis, suggesting that this method can be a reliable system to measure receptor cell surface expression. Other inactivating TSHr mutations were expressed in COS-7 cells for binding studies, FACS analysis, and microchip flow cytometry analysis. Binding studies showed that L252P, Q8fsX62, P27T, E34K, R46P, D403N, W488R, and M527T mutants had a low expression at the cell surface, as demonstrated by Bmax values. Data obtained by binding studies were in good agreement with data obtained by FACS analysis and microchip flow cytometry analysis. In conclusion, the low number of cells required for analysis and the ease of use make the microchip flow cytometry analysis a very reliable and favorable system to study cell surface expression of TSHr mutations.

The expression of TRH, its receptors and degrading enzyme is differentially modulated in the rat limbic system during training in the Morris water maze.

TRH administration induces arousal, improves cognition, and modulates glutamatergic and cholinergic transmission in hippocampal neurons. To study the possible involvement of TRH neurons in learning and memory processes, gene expression of TRH, its receptors, and pyroglutamyl peptidase (PPII), were measured in limbic regions of water-maze trained rats. Hypothalamus and amygdala showed changes related to the task but not specific to spatial learning while in hippocampus, pro-TRH and TRH-R1 mRNA levels were specifically increased in those animals trained to find a hidden platform. Variation of TRH content and mRNA levels of pro-TRH, TRH-R1, TRH-R2 and PPII are observed in conditions known to activate TRH hypophysiotropic neurons. Changes in some of these parameters could indicate the activation of TRHergic neurons and their possible involvement in some memory related process. Male Wistar rats were immersed (10 times) for 1, 3 or 5 days in a Morris water-maze containing, or not (yoked control) a platform and sacrificed 5, 30 and 60 min after last trial. TRH content and TSH serum levels were determined by radioimmunoassay; mRNA levels of pro-TRH, TRH-R1, TRH-R2, and PPII, by RT-PCR. Exclusive changes due to spatial training were observed in posterior hippocampus of rats trained for 5 days sacrificed after 60min: decreased TRH content and increased mRNA levels of pro-TRH and TRH-R1, particularly in CA3 region (measured by in situ hybridization). The hypothalamus-pituitary axis responded in both yoked and trained animals (increasing serum TSH levels and pro-TRH expression, due to swim-stress); in the amygdala of both groups, pro-TRH expression increased while diminished that of both receptors and PPII. Differential expression of these parameters suggests involvement of TRH hippocampal neurons in memory formation processes while changes in amygdala could relate to TRH anxiolytic role. The differential modulation in anterior and posterior portions of the hippocampus is discussed.

Probing the genetic basis for thyrotropin receptor antibodies and hyperthyroidism in immunized CXB recombinant inbred mice.

Immunization with adenovirus encoding the TSH receptor (TSHR) or its A-subunit induces Graves' hyperthyroidism in BALB/c and BALB/c x C57BL/6 offspring but not C57BL/6 mice. High-resolution genetic maps are available for 13 recombinant inbred CXB strains generated from BALB/c x C57BL/6 progeny by repeated brother x sister matings to establish fully inbred lines. CXB strains were studied before and after A-subunit adenovirus immunization for TSHR antibodies (TBI, inhibition of TSH binding), serum T4, and thyroid histology. All strains developed TBI activity (at variable levels), six strains became hyperthyroid, and one was overtly thyrotoxic. No low TBI responders became hyperthyroid, but high TBI did not predict hyperthyroidism. Preimmunization T4 levels varied in different CXB strains and was unrelated to subsequent T4 elevation. Linkage analysis indicated that different chromosomes were involved in generating TSHR antibodies and serum T4 before and after immunization. TBI activity was linked in part with major histocompatibility (MHC) genes on chromosome 17 (Chr 17) but induced Graves' disease involved non-MHC genes (Chr 19 and 10). The Chr 10 locus is close to the Trhde gene that encodes TSH-releasing hormone degrading enzyme. Expression of Trhde is controlled by thyroid hormones and linkage with a thyroid function-related gene is intriguing. Our data, the first genome scan in murine Graves' disease, provides insight into the role of MHC and non-MHC genes in human and murine Graves' disease. Finally, our study demonstrates the potential of recombinant inbred mice for discriminating between immune-response genes and thyroid function susceptibility genes in Graves' disease.

Targeted expression of the human thyrotropin receptor A-subunit to the mouse thyroid: insight into overcoming the lack of response to A-subunit adenovirus immunization.

The thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves' hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, "non-self" regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.

Modulation of thyroid-specific gene expression in normal and nodular human thyroid tissues from adults: an in vivo effect of thyrotropin.

CONTEXT: Evidence from in vitro studies or animal models has shown that TSH affects thyrocytes by thyroid-specific expression modulation. OBJECTIVE: The objective of our study was to analyze the role of TSH in human thyroid gene expression in vivo. DESIGN/SETTING: Thirty-nine normal thyroid tissues were collected at the same center. STUDY SUBJECTS: Patients were divided into two groups based on serum TSH levels: 17 with normal TSH levels (1-4 mU/liter; group 1) and 22 with TSH levels below 0.5 mU/liter (group 2). INTERVENTION: Group 2 underwent thyroidectomy after suppressive L-T4 therapy. MAIN OUTCOME MEASURES: mRNA levels of thyroid genes such as sodium/iodide symporter (NIS), apical iodide transporter, pendrin, thyroglobulin, thyroperoxidase, TSH receptor, paired box transcription factor 8, and thyroid transcription factor-1 were evaluated by quantitative PCR. RESULTS: The reduction of TSH stimulation causes decreases in NIS and apical iodide transporter gene expression in normal tissues and more limited reductions in thyroglobulin, thyroperoxidase, and paired box transcription factor 8, but it has no significant effect on TSH receptor, pendrin, or thyroid transcription factor-1. Comparison of NIS levels in normal and nodular tissues from the same patient confirmed that it is differentially expressed in nodules only in the presence of normal TSH (P < 0.01). In patients with suppressed TSH, nodular NIS levels were similar to those in normal tissues. CONCLUSIONS: Our data represent the first demonstration in human thyroid tissues that TSH contributes to the regulation of thyrocyte differentiation by modulating thyroid gene levels. It exerts a particularly important effect on the transcription of NIS, which becomes very low after prolonged TSH suppression.

Reverse transcriptase inhibitors down-regulate cell proliferation in vitro and in vivo and restore thyrotropin signaling and iodine uptake in human thyroid anaplastic carcinoma.

CONTEXT: Two classes of repeated genomic elements, retrotransposons and endogenous retroviruses, encode for endogenous nontelomeric reverse transcriptase (RT), a gene that is down-regulated in differentiated cells but is highly expressed in embryonic and transformed tissues. Two nonnucleosidic RT inhibitors, efavirenz and nevirapine, currently used in HIV treatment, reversibly down-regulate tumor growth and induce differentiation in several human tumor cell models. OBJECTIVES: Aggressive biological behavior and loss of specific thyroid cell functions, such as thyroglobulin, thyroid peroxidase, TSH receptor, Na/I symporter expression, and iodine uptake are features of anaplastic thyroid cancer. Thus, we evaluated the use of RT inhibitors as a potentially differentiating and molecular-targeted treatment of this neoplasm. RESULTS: Our findings showed that nevirapine and efavirenz reversibly inhibit cell proliferation without triggering cell death in the undifferentiated thyroid carcinoma ARO and FRO cells, which exhibited high levels of endogenous RT activity. Inhibition of cell growth was correlated with accumulation of cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the S phase. Moreover, treated cells demonstrated a differentiated phenotype and a significant reprogramming of gene expression characterized by up-regulation of the TSH receptor, thyroglobulin, thyroid peroxidase, and Na/I symporter genes. Interestingly, RT inhibition reestablished the ability to uptake iodine in response to TSH either in vitro or in vivo and reversibly down-regulated tumor growth in mice xenografts of ARO cells. CONCLUSIONS: These findings support the need for clinical trials to clarify whether RT inhibitors may restore the sensitivity to radiometabolic therapy in anaplastic thyroid tumors.

Thyroid transcription factor-1 in orbital adipose tissues: potential role in orbital thyrotropin receptor expression.

Thyroid transcription factor-1 (TTF-1) is required for maximal expression of thyrotropin receptor (TSHR) in the thyroid. Extrathyroidal TSHR expression is detectable in normal orbital adipose tissues, with increased levels found in orbital tissues from patients with Graves' ophthalmopathy (GO), and in orbital preadipocyte cultures following differentiation. In order to determine whether TTF-1 might be involved in orbital TSHR expression, we used quantitative real-time polymerase chain reaction (PCR) to assess relative expression of this and other thyroid-associated transcription factors (TTF-2 and Pax-8) in GO orbital tissue specimens (n = 28) and cultures (n = 3), and in normal orbital tissues (n = 19) and cultures (n = 3). We detected TTF-1 and TTF-2 mRNA in GO and normal orbital tissue samples, with no difference in levels noted between the tissues. In the GO orbital cultures, TTF-1 mRNA was higher in differentiated than in control (undifferentiated) cultures (p < 0.05), while TTF-2 was unchanged. In the normal cultures, neither TTF-1 nor TTF-2 mRNA levels increased in differentiated cultures. Pax8 was undetectable in all orbital tissues and cell cultures. The presence of mRNA encoding TTF-1 in orbital tissues and cultures suggest that this transcription factor may play an important role in extrathyroidal, as it does in thyroidal, TSHR expression.