Growth factor expression in cold and hot thyroid nodules.

Hot thyroid nodules (HTNs) are predominantly caused by constitutively activating thyrotropin receptor (TSHR) mutations leading to an activation of the cyclic adenosine monophosphate (cAMP)-cascade that stimulates growth and function of thyroid epithelial cells and confers growth advantage. In contrast to HTNs, the molecular etiology of szintigraphically cold thyroid nodules (CTNs) is largely unknown. An increased prevalence of toxic multinodular goiters in iodine-deficient regions has been reported. Growth factors increase during early stages of iodine deficiency in rats. These growth factors could modulate the proliferation of thyrocytes. In order to determine if and which growth factors could modulate the increase in thyroid epithelial cell proliferation in late stages of CTNs and HTNs we investigated epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and TGF-beta1 concentrations by enzyme-linked immunosorbant assay (ELISA) in CTNs (n = 7), HTNs (n = 9), and their normal surrounding tissue (ST). Insulin-like growth factor-1 (IGF-1) was determined in CTNs (n = 5) and HTNs (n = 10) and their surrounding tissues by radioimmunoassay (RIA). We found lower concentrations of all investigated growth factors and iodine in CTNs compared to surrounding normal tissues (ST). Only iodine showed a significant difference. Furthermore, we found significantly lower concentrations of EGF and TGF-beta1 concentration in HTNs compared to their STs. Differences of TGF-alpha and IGF-1 were not significant. In conclusion, low EGF, TGF-alpha, and IGF-1 concentrations in most CTNs in spite of low iodine concentrations argue against a pathophysiologic role of EGF, TGF-alpha, or IGF-1 in late stages of CTNs. The low EGF, TGF-alpha, and IGF-1 concentrations in HTNs irrespective of their clonal origin or the presence or absence of activating mutations argue for increased cAMP as the primary cause for thyroid epithelial cell proliferation in established HTNs. However, the pathophysiologic significance of low TGF-beta1 concentrations in CTNs and HTNs remains to be elucidated. It might be possible that growth factors like EGF, TGF-alpha, TGF-beta1, and IGF-1 play a more prominent role during early clonal expansion and that aberrant intrinsic signaling through a somatic mutation (e.g., TSHR for HTNs) confers the predominant selective growth advantage in later stages of HTNs or CTNs.     

Meta- and reanalysis of gene expression profiles of hot and cold thyroid nodules and papillary thyroid carcinoma for gene groups

CONTEXT: There are an increasing number of studies analyzing gene expression profiles in various benign and malignant thyroid tumors. This creates the opportunity to validate results obtained from one microarray study with those from other data sets. This process requires rigorous methods for accurate comparison. OBJECTIVE: The ability to compare data sets derived from different Affymetrix GeneChip generations and the influence of intra- and interindividual comparisons of gene expression data were evaluated to build multigene classifiers of benign thyroid nodules to verify a previously proposed papillary thyroid carcinoma (PTC) classifier and to look for molecular pathways essential for PTC oncogenesis. METHODS: Gene expression profile data sets from autonomously functioning and cold thyroid nodules and from PTC were analyzed by support vector machines. GenMAPP analysis was used for PTC data analysis to examine the expression patterns of biologically relevant gene sets. RESULTS: Only intraindividual reference samples allowed the identification of subtle changes in the expression patterns of relevant signaling cascades, such as the MAPK pathway in PTC. Using an artificial intelligence approach, the autonomously functioning and cold thyroid nodule multigene classifiers were derived and evaluated by cross-comparisons. CONCLUSION: We recommend defining classifiers within one generation of gene chips and subsequently checking them across different array generations. Using this approach, we have demonstrated the specificity of a previously reported PTC classifier on an independent collection of benign tumors. Moreover, we propose multigene classifiers for different types of benign thyroid nodules.     

PI3K/Akt信号转导通路与甲状腺结节

近年来研究发现,磷脂酰肌醇3激酶(PDK)/蛋白激酶B(Akt)信号转导通路在细胞的增殖、分化、生存和凋亡过程中起重要调节作用.结节性甲状腺肿、甲状腺腺瘤、甲状腺癌及桥本甲状腺炎病变中存在着PI3K/Akt信号转导通路的过度激活.PI3K/Akt信号转导通路的活化使甲状腺细胞增殖指数和生长指数明显升高,刺激甲状腺滤泡持续生长,促进甲状腺结节的发生、发展.研究此通路中相关分子的表达,对探索甲状腺结节的病因、发病机制和防治策略具有重要意义.     

Kinetic analysis of iodine and thyroxine metabolism in “hot” thyroid nodules

Iodine kinetic studies were performed in seven patients with hot thyroid nodules, three diffuse toxic goiters and one normal control, using a double label technique (¹³¹I and ¹²⁵I-thyroxine). The investigation was complemented by partial kinetic studies in 29 further subjects, including normal control subjects and euthyroid or hyperthyroid patients with diffuse or nodular goiter. Thyroid iodide uptake and hormonal secretion parameters were significantly increased, up to twice control values, in hot nodules without clinical signs of thyrotoxicosis. Values similar to those of Graves' disease were found in toxic nodular goiter. The exchangeable iodine pool was markedly reduced in those glands where the nodule was adenomatous and radioiodide uptake of normal parenchyma was completely suppressed. In two cases of nontoxic hot nodule with histologic diagnosis of multinodular goiter, and incomplete suppression of the “extranodular” thyroid, the exchangeable iodine pool was comparable to that of normals. The T₄ disposal rate was increased, mainly due to a significantly higher metabolic deiodination which seemed to keep pace with the increased T₄ production rate in nontoxic nodules. This could be the reason why the mean peripheral T₄ pool of clinically nontoxic patients with hot nodules is comparable to that of normal persons, and there are no evident signs of hypermetabolism in spite of the greater thyroid hormone production. A significant “non-T4-iodine” release, probably mainly iodide, was found in every subject. The amount of iodine lost by the thyroid through this “iodide leak” did not relate to the T₄ secretion rate but seems to be directly related to the thyroid exchangeable iodine pool size.     

Decreased expression of G-protein coupled receptor kinase 2 in cold thyroid nodules.

G-protein coupled receptor kinases (GRKs) have been shown to regulate the homologous desensitization of different G-protein coupled receptors. We have previously demonstrated that the expression of GRK 3 and 4 is increased in hyperfunctioning thyroid nodules (HTNs) and that GRKs 2, 3, 5 and 6 are able to desensitize the TSHR in vitro. Since cold thyroid nodules (CTNs) and HTNs show different molecular and functional properties, different expression patterns of GRKs in these nodules can be expected. The comparison of GRK expression between CTNs and HTNs could give additional insight into the regulation mechanisms of these nodules. We therefore examined the expression of GRKs in CTNs and analyzed the differences to HTNs. The expression of the different GRKs in CTNs was measured by Western blot followed by chemiluminescence imaging. We found a decreased expression of GRK 2 in CTNs compared to their surrounding tissues and an increased expression of GRK 3 and 4 in CTNs, which is similar to HTNs. The decreased GRK 2 expression most likely results from reduced cAMP stimulation in CTNs. However, the increased GRK 3 and 4 expression in CTNs remains unclear and requires further investigations.     

Thyroglobulin biosynthesis in "cold" and "hot" nodules in the human thyroid gland.

Studies on "cold" and "hot" nodules from human thyroid gland have been carried out to investigate thyroglobulin biosynthesis and to correlate carbohydrates incorporation and thyroid hormone formation in thyroglobulin. The aim of the investigation was to ascertain the role of carbohydrates of the protein molecule in its migration to the iodinating site of the cell. Thyroid slices from two cases with "cold" and two with "hot" nodules were incubated for 30 to 120 min with [3H]leucine, [3H]carbohydrates (ManNAc, Gal, Man, GlcNAc, GalNAc) and 125I. Soluble and particulate iodoproteins, solubilized by digitonin, were identified by density gradient centrifugation and immunoprecipitation; following hydrolysis carbohydrates and iodothyronines were identified by paper chromatography and chemically determined. While the rate of leucine incorporation into TG increased with time in both "cold" and "hot" nodules, the "cold" nodule, with respect to the "hot" tissue, showed: much lower efficiency incorporating carbohydrates and iodine into TG; lower thyroid/medium ratio of radioiodine; less total soluble protein and soluble TG; more particulate protein and solubilized TG; 1/3 of the carbohydrate and iodine chemical content; only minute amounts of labeled iodothyronines in TG compared to normal levels in "hot" tissue. The formation of thyroid hormone, in enzymatically iodinated TG, and the iodinating activity of the 105,000 X g pellet, incubated with low iodine TG, were similar in both "cold" and "hot" tissues. These results demonstrate that the "cold" nodule is able to synthesize thyroglobulin but the protein is defective in its carbohydrate content. The deficient iodine transport into "cold" tissue and impaired hormonal synthesis are confirmed. The incomplete incorporation of carbohydrates into thyroglobulin in the "cold" nodule could be important for the maturation and migration of the molecule to the iodinating site of the cell     

Gene expression analysis reveals evidence for increased expression of cell cycle-associated genes and Gq-protein-protein kinase C signaling in cold thyroid nodules

In contrast to the molecular etiology of autonomously functioning thyroid nodules, the molecular cause of cold thyroid nodules (CTNs), their benign, functional inactive counterparts, are so far largely unknown. Because of the partially dedifferentiated phenotype of CTNs, alterations in signaling cascades that favor proliferation, but not differentiation, are likely candidates for tumor induction and progression. The importance of RAS mutations for the development of benign nodules with follicular histology is still in question. However, differentially expressed genes in the context of their signaling cascades could define aberrant signaling in CTNs. Therefore, we investigated gene expression in 22 CTNs and their normal surrounding tissue using Affymetrix GeneChips. Most prominently, data analysis revealed an increased expression of cell cycle-associated genes and a special relevance of protein kinase C signaling, whereas no evidence of RAS-MAPK signaling in CTNs was found. Moreover, we determined 31 differentially regulated genes in CTNs, including several histone mRNAs. Taken together, these results explain recent findings showing an increased proliferation in CTNs and draw attention to protein kinase C signaling, but away from RAS-MAPK signaling, as being involved in the etiology of CTNs.     

Coincidence of hot thyroid nodules and primary hyperparathyroidism.

Hyperthyroidism is frequently associated with hypercalcemia, which usually subsides after successful treatment of hyperthyroidism. Moreover, thyroid nodules are frequently detected by preoperative thyroid ultrasound in patients with primary hyperparathyroidism. Sensitised by the observation of a patient with coexisting hyperthyroidism and hyperparathyroidism we prospectively evaluated thyroid nodules in euthyroid patients with hyperparathyroidism by thyroid scintigraphy. Whereas the first patient with hyperparathyroidism was hyperthyroid the subsequent four patients with hyperparathyroidism and thyroid nodules had normal fT3 and fT4. Two patients had hypercalcemia and nephroureterolithiasis. Three patients suffered from hypercalcemia and bone pain due to osteoporosis. In the hyperthyroid patient hypercalcemia persisted after euthyroidism was achieved intact parathyroid hormone was found to be elevated. Subsequently, thyroid nodules, detected by preoperative ultrasound in four euthyroid patients with primary hyperparathyroidism, were identified as compensated hot nodules by thyroid scintigraphy. All patients underwent combined subtotal thyroidectomy and parathyroid resection. Histology showed hyperplastic parathyroid glands in one patient and a single parathyroid adenoma in four cases. Postoperatively calcium and PTH levels returned to normal and TSH levels increased in all patients. Persistence of hypercalcemia after successful treatment of hyperthyroidism should be reason for the determination of parathyroid hormone. Thyroid nodules detected by preoperative ultrasound in patients with hyperparathyroidism living in areas of iodine deficiency should be further evaluated by scintigraphy even if TSH is normal. In the case of hot thyroid nodules both parathyroid and partial thyroid resection should be performed.     

Inborn errors of signal transduction: Mutations in G proteins and G protein-coupled receptors as a cause of disease

A vast array of neurotransmitters, polypeptide hormones and other extracellular signalling molecules utilize G protein-coupled pathways for transmembrane signalling. In recent years, mutations in G protein-coupled receptors and in G protein subunits have been identified as the cause of a variety of human diseases. Both loss and gain of function mutations have been described in disorders such as Albright hereditary osteodystrophy, nephrogenic diabetes insipidus, McCune-Albright syndrome, and familial male precocious puberty. Identification of mutations in G protein-coupled receptors and in G proteins in human diseases has provided unique insights into G protein-coupled signal transduction, has important implications for diagnosis and potentially for treatment, and should stimulate the search for additional defects in G protein-coupled signal transduction in other diseases.     

Complementary DNA microarray analysis of chemokines and their receptors in allergic rhinitis.

OBJECTIVE: To analyze the roles of chemokines and their receptors in the pathogenesis of allergic rhinitis by observing the complementary DNA (cDNA) expression of the chemokines and their receptors in the nasal mucosa of patients with and without allergic rhinitis, using gene chips. METHODS: The total RNAs were isolated from the nasal mucosa of 20 allergic rhinitis patients and purified to messenger RNAs, and then reversely transcribed to cDNAs and incorporated with samples of fluorescence-labeled with Cy5-dUPT (rhinitis patient samples) or Cy3-dUTP (control samples of nonallergic nasal mucosa). Thirty-nine cDNAs of chemokines and their receptors were latticed into expression profile chips, which were hybridized with probes and then scanned with the computer to study gene expression according to the different fluorescence intensities. RESULTS: The cDNAs of the following chemokines were upregulated: CCL1, CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL17, CCL18, CCL19, CCL24, and CX3CL1 in most of the allergic rhinitis sample chips. CCR2, CCR3, CCR4, CCR5, CCR8 and CX3CR1 were the highly expressed receptor genes. Low expression of CXCL4 was found in these tissues. CONCLUSION: The T helper cell (T(H)) immune system is not well regulated in allergic rhinitis. Most of the upregulated genes we identified are of chemokines and their receptors that play important roles in T(H)2 response, and some are involved in the induction of allergic reaction, accumulation of inflammatory cells, and degranulation of sensitized cells. These findings can point to new strategies for allergic rhinitis immunotherapy.     

The natural history of thyroid autonomy and hot nodules.

Solitary hyperfunctioning thyroid adenomas are benign monoclonal tumors characterized by their capacity to grow and produce thyroxine (T4) and triiodothyronine (T3) autonomously, i.e. in the absence of thyrotropin (TSH). Mutations of the TSH receptor (TSH-R) have been found in the majority of solitary hyperfunctioning thyroid adenomas. On radioisotope scanning they generally appear as hot nodules because they concentrate radioiodide or 99mTc pertechnate, whereas the normal surrounding and contralateral tissue concentrate little isotopes. A toxic adenoma probably evolves gradually from a small autonomously hyperfunctioning adenoma that initially is only slightly more active than the extranodular tissue. This has been referred to as a "warm" nodule or a "compensated" adenoma. The diagnostic criterion for this designation is the persistence of detectable serum TSH maintaining some radioiodine uptake by the extranodular tissue. This "compensated" adenoma persists as long as the autonomous hormone output is not sufficient to suppress thyrotropin, i.e. to cause hyperthyroidism. The rate of development of thyrotoxicosis in patients with hyperfunctioning adenomas who are euthyroid initially is about 4% per year and depends on the size of the adenoma, iodine intake and age of the patient. No clear relationship can be establish between the nature of the TSH receptor mutations and the phenotype of the tumor.     

The interaction of signal transduction pathways in FRTL5 thyroid follicular cells: studies with stable expression of beta 2-adrenergic receptors.

Multiple signal transduction pathways interact in FRTL5 cells to promote thyroid follicular cell differentiated function and cell proliferation. In these cells, TSH is a tissue-specific mitogen that promotes DNA synthesis primarily through activation of adenylate cyclase. To further test the role of adenylate cyclase in regulating cell growth and differentiated function we have introduced into FRTL5 the human beta 2-adrenergic receptor (BAR) complementary DNA and have studied the ability of isoproterenol, alone and in combination with insulin-like growth factor I (IGF-I), to stimulate cAMP accumulation, iodide transport, [3H]thymidine incorporation into DNA, and cell growth. Wild-type FRTL5 were infected with a PLJ retroviral construct containing the BAR in either a sense (FRTL BAR) or antisense (FRTL RBAR) orientation, and cell populations were selected on the basis of resistance to the antibiotic geneticin. FRTL BAR expressed approximately 1.3 x 10(5) high affinity binding sites per cell for the beta 2-specific ligand, CGP-12177, while neither FRTL5 wild-type nor RBAR cells demonstrated any specific binding. FRTL BAR had significantly higher levels of intracellular cAMP, [3H]thymidine incorporation, and iodide uptake in the absence of added isoproterenol than FRTL RBAR or wild-type cells. In FRTL BAR, but not RBAR cells, isoproterenol stimulated a dose-dependent accumulation of cAMP, iodide uptake, [3H]thymidine incorporation, and cell growth. FRTL BAR and RBAR cells were equally responsive to TSH and to IGF-I. Isoproterenol enhanced the ability of IGF-I to stimulate [3H]thymidine incorporation in BAR but not RBAR cells. Isoproterenol partially inhibited the ability of TSH to stimulate cAMP generation and DNA synthesis. These studies demonstrate that activation of adenylate cyclase through the BAR introduced into FRTL5 cells by retroviral infection reproduces the range of biological effects in these cells stimulated by TSH and suggest that activation of adenylate cyclase is sufficient to stimulate thyroid differentiated function and cell growth. FRTL BAR cells will provide an interesting model system with which to study the heterologous regulation of both TSH and BARs through activation of a common signal transduction pathway, adenylate cyclase.     

Sequence analysis, endocrine regulation, and signal transduction of GnRH receptors in teleost fish

Three gonadotropin-releasing hormones (GnRHs) and three cognate receptors have been identified in vertebrates, with distinct distributions and functions. According to their sequences, the receptors can be grouped into distinct classes: types I, II, and III. One branch contains all type-I GnRH receptors (GnRH-R-I) from mammals and fish; another branch clusters mainly amphibian and human type-II GnRH receptors; and a third branch includes evolved fish, mainly perciform species, type-III GnRH receptors. Taken tilapia GnRH receptors as a model, the present study summarizes the information regarding the amino-acid residues assumed to be involved in the receptors' structure, binding, activation, and intracellular signal transduction, including arrangement of the disulfide bonds, glycosylation sites, coupling to G proteins, and protein kinase A or protein kinase C phosphorylation sites.     

Ras mutations are rare in solitary cold and toxic thyroid nodules

OBJECTIVE: Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. DESIGN: To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. MEASUREMENTS: Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. RESULTS: Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. CONCLUSION: Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.     

Mutations in the thyrotropin receptor signal transduction pathway in the hyperfunctioning thyroid nodules from multinodular goiters: a study in the Turkish population

Many studies have been carried out to determine G(s) alpha and TSHR mutations in autonomously functioning thyroid nodules. Variable prevalences for somatic constitutively activating TSHR mutations in hot nodules have been reported. Moreover, the increased prevalence of toxic multinodular goiters in iodine-deficient regions is well known. In Turkey, a country with high incidence rates of goiter due to iodine deficiency, the frequency of mutations in the thyrotropin receptor signal transduction pathway has not been evaluated up to now. In the present study, a part of the genes of the TSHR, G(s)alpha and the catalytic subunit of the PKA were checked for activating mutations. Thirty-five patients who underwent thyroidectomy for multinodular goiters were examined. Genomic DNAs were extracted from 58 hyperactive nodular specimens and surrounding normal thyroid tissues. Mutation screening was done by single-strand conformational polymorphism (SSCP) analysis. In those cases where a mutation was detected, the localization of the mutation was determined by automatic DNA sequencing. No G(s)alpha or PKA mutations were detected, whereas ten mutations (17%) were identified in the TSHR gene. All mutations were somatic and heterozygotic. In conclusion, the frequency of mutations in the cAMP signal transduction pathway was found to be lower than expected in the Turkish population most likely because of the use of SSCP as a screening method and sequencing only a part of TSHR exon 10.