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.

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.

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 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.

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.

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.

Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves' disease.

We performed genetic immunization of outbred NMRI mice, using a cDNA encoding the human thyrotropin receptor (TSHr). All mice produced antibodies capable of recognizing the recombinant receptor expressed at the surface of stably transfected Chinese hamster ovary (CHO) cells, and sera from most of the immunized mice blocked TSH-dependent stimulation of cAMP accumulation in cells expressing the TSHr. Five out of 29 female mice showed sign of hyperthyroidism including elevated total T4 and suppressed TSH levels. The serum of these mice contained thyroid-stimulating activity, as measured in a classic assay using CHO cells expressing recombinant TSHr. In contrast, only 1 male out of 30 had moderately elevated serum total T4 with undetectable TSH values. The hyperthyroid animals had goiters with extensive lymphocytic infiltration, characteristic of a Th2 immune response. In addition, these animals displayed ocular signs reminiscent of Graves' ophthalmopathy, including edema, deposit of amorphous material, and cellular infiltration of their extraocular muscles. Our results demonstrate that genetic immunization of outbred NMRI mice with the human TSHr provides the most convincing murine model of Graves' disease available to date.

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.

Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.

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.

Chimeric proteins can exceed the sum of their parts: implications for evolution and protein design.

Chimeric analogs derived from pairs of homologous proteins routinely exhibit activities found in one or both parents. We describe chimeras of two glycoprotein hormones, human chorionic gonadotropin (hCG) and human follitropin (hFSH), that exhibit activity unique to a third family member, human thyrotropin (hTSH). The results show that biological activity can be separated from hormone-specific amino acid residues. This is consistent with a model for the evolution of homologous ligand-receptor pairs involving gene duplication and the creation of inhibitory determinants that restrict binding. Disruption of these determinants can unmask activities characteristic of other members of a protein family. Combining portions of two ligands to create analogs with properties of a third family member can facilitate identifying key determinants of protein-protein interaction and may be a useful strategy for creating novel therapeutics. In the case of the glycoprotein hormones, this showed that two different hormone regions (i.e., the seat-belt and the intersubunit groove) appear to limit inappropriate contacts with receptors for other members of this family. These observations also have important caveats for chimera-based protein design because an unexpected gain of function may limit the therapeutic usefulness of some chimeras.

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.

Methylation of the thyroid-stimulating hormone receptor gene in epithelial thyroid tumors: a marker of malignancy and a cause of gene silencing.

Thyroid-stimulating hormone receptor (TSHR) expression is frequently silenced in epithelial thyroid cancers associated with decreased or absent TSH-promoted iodine uptake. To study the underlying molecular mechanism of decreased TSHR expression, we examined the methylation status of the TSHR gene promoter by sequencing bisulfite-treated DNA from thyroid tumors. After identification of methylated sites by sequencing bisulfite-treated DNA, we used methylation-specific polymerase chain reaction and found frequent CpG methylation in papillary thyroid cancer (23 of 39 patients; 59%) and follicular thyroid cancers (7 of 15 patients; 47%). In contrast, we saw no methylation in normal thyroid tissues and benign adenomas (0 of 8 patients; 0%). In human thyroid tumor cell lines, we observed that TSHR was normally expressed at the protein and mRNA level in cells where the TSHR gene was unmethylated, whereas it was silenced in cell lines where the TSHR promoter was hypermethylated. Treatment of the latter cells with a demethylating agent partially restored TSHR expression. We thus demonstrate aberrant methylation of human TSHR as a likely molecular pathway responsible for the silencing of this gene in thyroid cancers. We propose that methylation of TSHR may provide a novel diagnostic marker of malignancy and a basis for potential use of demethylating agents in conjunction with TSH-promoted radioiodine therapy for epithelial thyroid cancers.

E-cadherin: a differentiation marker in thyroid malignancies.

Loss of E-cadherin (uvomorulin), a Ca(2+)-dependent cell adhesion molecule required for normal epithelial function, has been attributed a pathogenetic role in tumor invasion. The expression of E-cadherin was studied in normal and neoplastic follicular epithelium of the human thyroid by Northern blot analysis and immunofluorescence on frozen tissue sections. In the normal thyroid (n = 10) and in benign thyroid disorders (n = 21; toxic diffuse goitre; multinodular goitre; follicular adenomas), E-cadherin mRNA levels were equally high and the follicles were generally stained, mainly along the lateral surface of the epithelial cells, by the anti-E-cadherin monoclonal antibody. In anaplastic thyroid carcinomas (n = 6) E-cadherin expression was very low or lacking. In papillary carcinomas (n = 23), E-cadherin mRNA levels varied from nearly normal to highly reduced, which roughly correlated with the overall immunofluorescence intensity. However, the immunostaining also revealed a heterogeneous "all-or-nothing" expression of E-cadherin among adjacent cells in the same tumor. In the follicular carcinomas (n = 9), E-cadherin mRNA levels were in general rather high but the immunostaining varied considerably. A few papillary and follicular tumors lacked immunoreactive E-cadherin in spite of high mRNA levels. In oxyphilic (Hürthle) cell tumors, comprising both adenomas (n = 4) and carcinomas (n = 2), E-cadherin immunoreactivity was reduced and distributed intracellularly rather than at the cell surface. The expression of E-cadherin in relapsing thyroid carcinomas and in tumors with metastatic spreading was, irrespective of the histiotype, low or lacking. Sequential Northern analysis revealed a close correlation between the expression levels of E-cadherin and the thyrotropin receptor. Together, the data suggest that in human thyroid malignancies both gene expression and posttranscriptional control of E-cadherin may be impaired.

Oncogenic potential of a mutant human thyrotropin receptor expressed in FRTL-5 cells.

An abnormal stimulation of the cAMP pathway has been recognized as the primary event in various pathological situations that lead to goitrogenesis or thyroid tumors. Thyroid adenomas are monoclonal neoplasms that become independent of thyroid stimulating hormone (TSH) in their secretory function and growth. Mutated forms of the TSH receptor (TSHR) and the adenylyl cyclase-activating Gs alpha protein, which confer a constitutive activity on these proteins, have been observed in human adenomas. The FRTL-5 rat thyroid cell line is a permanent but untransformed line; the growth of which depends on the presence of TSH, and at least in part, on the stimulation of the cAMP pathway. In order to compare the oncogenic potential of the activated mutant Gs alpha protein and the constitutively activated TSHR, we have transfected FRTL-5 cells with an expression vector bearing either the cDNA of the Gs alpha gene carrying the A201S mutation or the cDNA of the TSH receptor carrying the M453T mutation recently identified in a case of congenital hyperthyroidism. The expression of these two cDNAs was driven by the bovine thyroglobulin gene promoter. We show that, although the expression of both the Gs alpha or TSHR mutant proteins leads to TSH-independent proliferation and to constitutive cAMP accumulation in FRTL-5 cells, only the mutant TSHR is able to induce neoplastic transformation, as demonstrated by growth in semi-solid medium and tumorigenesis in nude mice.

A mutated p53 gene alters thyroid cell differentiation.

p53 is the gene most frequently found mutated in human neoplasias. In the majority of tumors, p53 mutations contribute to the progression towards stages of increasing malignancy with the appearance of an undifferentiated phenotype. Also in thyroid cancerogenesis, p53 mutations correlate with the loss of the differentiated phenotype. The results presented here, suggest a direct involvement of p53 in the molecular mechanisms regulating cellular differentiation in thyroid since a mutated p53 gene markedly affects the growth potential and differentiated functions of the rat thyroid cell line PC Cl 3. Blockage in the expression of the PAX-8 transcription factor seems to be a key event in the loss of thyroid differentiated functions induced by the mutated p53 gene. Thyroid cells carrying a mutated p53 gene did not form colonies in soft agar or tumors in athymic mice, suggesting that a mutation of the p53 gene is not sufficient for the induction of the malignant phenotype and probably a cooperation with other oncogenes is necessary to accomplish full malignancy. No effect on either growth or differentiation of thyroid cells was exerted either by overexpression of the wild-type p53 gene, or by the vector alone.

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.