Second-Generation AUTACs for Targeted Autophagic Degradation.

Targeted protein degradation via the ubiquitin-proteasome system has emerged as one of the most promising drug discovery modalities. Autophagy, another intracellular degradation system, can target a wide range of nonproteinous substrates as well as proteins, but its application to targeted degradation is still in its infancy. Our previous work revealed a relationship between guanine modification of cysteine residues on intracellular proteins and selective autophagy, resulting in the first autophagy-based degraders, autophagy-targeted chimeras (AUTACs). Based on the research background, all the reported AUTACs compounds contain cysteine as a substructure. Here, we examine the importance of this substructure by conducting SAR studies and report significant improvements in the degrader's activity by replacing cysteine with other moieties. Several derivatives showed sub-µM range degrading activity, demonstrating the increased practical value of AUTACs.

Falsely elevated thyroid hormone levels associated with fibrin interference in patients receiving oral anticoagulant therapy.

OBJECTIVE: Unique clinical courses were observed in two asymptomatic patients receiving warfarin who referred to our hospital because of suspected central hyperthyroidism. We eventually diagnosed these patients with falsely elevated thyroid hormone levels caused by macroscopically invisible fibrin. Although false results caused by fibrin interference in vitro have been identified in various immunoassays, especially in blood samples from patients receiving anticoagulant therapy, no studies on thyroid function testing have been reported. The experience in evaluating these cases prompted us to investigate the independent influence of oral anticoagulants via putative fibrin interference on thyroid function testing. METHODS: We retrospectively reviewed known contributing factors that affect thyroid function testing including age, gender, medication history, body mass index, estimated glomerular filtration rate, smoking status, alcohol consumption, and the seasons of hospital visits from participants who presented the Department of Health Checkup between April 2010 and December 2020. RESULTS: A propensity-matched analysis revealed that the median serum free thyroxine levels under oral anticoagulant were significantly higher (17.9 pmol/L, n = 60) than those without anticoagulants (16.0 pmol/L, n = 60; p < 0.001). It was noted that this difference was the largest among contributing factors we analyzed. No significant differences were noted in serum thyroid-stimulating hormone levels. CONCLUSIONS: We report two patients receiving warfarin with falsely elevated thyroid hormone levels caused by fibrin interference resembling central hyperthyroidism for the first time. Our retrospective study suggests that the medication status of oral anticoagulants should be considered when evaluating thyroid function tests.

Clinical outcomes of 34 patients with resistance to thyroid hormone beta: a twenty-year experience in Japan.

Resistance to thyroid hormone beta (RTHß) caused by germline mutations in genes encoding thyroid hormone receptor beta (TRß) is a rare disorder. Little information is available regarding the clinical experience of this syndrome in Japan. We retrospectively reviewed the records of 34 patients with RTHß (21 adult females and 13 adult males) with positive TRß mutations identified at our division between 2000 and 2020. Of the 24 patients with available clinical history, 10 (41.7%) received inappropriate treatments such as antithyroid drugs, thyroidectomy, or radioactive iodine. Diagnostic delay and inappropriate management of RTHß are still present in Japan. Every patient except one demonstrated thyroid hormone profiles indicative of syndrome of inappropriate secretion of thyrotropin (SITSH), characterized by a hormonal profile of hyperthyroxinemia with a non-suppressed TSH concentration. Since the most common forms of hyperthyroidism including Graves' disease feature elevated thyroid hormone levels with suppressed TSH concentrations, early diagnosis of SITSH is critical for preventing inappropriate management. One patient positive for anti-thyroglobulin antibody (Tg-Ab) and anti-thyroperoxidase antibody (TPO-Ab) showed remarkably elevated TSH (>200 µIU/mL) despite thyroid hormone concentrations within the reference ranges. At least one thyroid autoantibody (Tg-Ab, TPO-Ab, or thyrotropin receptor antibodies) was identified in 37.9% (11/29) of the patients tested. One patient developed overt Graves' disease nine years after RTHß diagnosis. These findings suggest that RTHß is frequently comorbid with additional autoimmune thyroid disorders. Further research is required to identify the most appropriate treatments for RTHß patients who develop a second thyroid disorder.

Retroperitoneal Paraganglioma With Asymptomatic Follicular Lymphoma: A Case Report.

Paraganglioma (PGL) is a rare tumor originating from extra-adrenal paraganglionic chromaffin tissues, and most sympathetic PGLs have excessive catecholamine secretion. However, nonfunctional PGLs are sometimes found. Although malignant PGL is defined by metastasis to nonchromaffin tissues, it is difficult to predict malignancies due to the lack of reliable markers of potential malignancies. We report the case of a 69-year-old Japanese woman with an incidental retroperitoneal tumor and multiple enlarged mesenteric lymph nodes simultaneously. The patient had no subjective symptoms and there were no laboratory findings suggesting catecholamine hypersecretion. Both the retroperitoneal tumor and the enlarged mesenteric lymph nodes showed high accumulation of fluorodeoxyglucose (FDG), whereas metaiodobenzylguanidine (MIBG) was accumulated only at the retroperitoneal tumor. Although a retroperitoneal tumor was diagnosed as nonfunctional PGL by examination including MIBG scintigraphy, the cause of enlarged mesenteric lymph nodes could not be diagnosed by imaging and biochemical tests. As a result of retroperitoneal tumor resection and mesenteric lymph nodes sampling, histopathological examination revealed that a retroperitoneal tumor was PGL and enlarged mesenteric lymph nodes were follicular lymphoma. To reveal an underlying genetic factor, we performed whole exome sequencing of genomic DNA, and we identified 2 possible candidate variants in SDHD and DLST, but the pathogenicity of these variants remains uncertain in the present case. This rare case reinforces the importance of histopathological diagnosis of nonchromaffin tissue lesions in patients with PGL for the appropriate treatment strategy.

Impairment of the Hypothalamus-Pituitary-Thyroid Axis Caused by Naturally Occurring GATA2 Mutations In Vitro.

The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus-pituitary-thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone ß (TSHß). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHß promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHß promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro.

Hyperfunctioning Papillary Thyroid Carcinoma with a BRAF Mutation: The First Case Report and a Literature Review.

INTRODUCTION: Hyperfunctioning papillary thyroid carcinoma (PTC) is rare and consequently, little information on its molecular etiology is available. Although BRAF V600E (BRAF c.1799T>A, p.V600E) is a prominent oncogene in PTC, its mutation has not yet been reported in hyperfunctioning PTC. CASE PRESENTATION: Ultrasonography detected a 26-mm nodule in the right lobe of the thyroid gland of a 48-year-old man. Thyroid function tests indicated that he was hyperthyroid with a TSH level of 0.01 mIU/L (reference range: 0.05-5.00) and a free thyroxine level of 23.2 pmol/L (reference range: 11.6-21.9). TSHR autoantibodies were <0.8 IU/L (reference value: <2.0 IU/L). The 99mTc thyroid scintigram revealed a round, right-sided focus of tracer uptake by the nodule with a decreased uptake in the remainder of the gland. The patient underwent total thyroidectomy because fine-needle aspiration cytology revealed a malignancy. The histopathological diagnosis was conventional PTC. Subsequent mutational analysis of BRAF (exon 15), TSHR (exons 1-10), GNAS (exons 7-10), EZH1 (exon 16), KRAS, NRAS, HRAS (codons 12, 13, and 61), and TERT promoter (C250T and C228T) identified a heterozygous point mutation in BRAF V600E in a tumor tissue sample. In addition, we identified a TSHR D727E polymorphism (TSHR c.2181C>G, p.D727E) in both the tumor and the surrounding normal thyroid tissue. DISCUSSION AND CONCLUSIONS: We report a case of hyperfunctioning PTC with a BRAF V600E mutation for the first time. Our literature search yielded 16 cases of hyperfunctioning thyroid carcinoma in which a mutational analysis was conducted. We identified TSHR mutations in 13 of these cases. One case revealed a combination of TSHR and KRAS mutations; the other case revealed a TSHR mutation with a PAX8/PPARG rearrangement. These findings suggest that the concomitant activation of oncogenes (in addition to constitutive activation of the TSHR-cyclic AMP cascade) are associated with the malignant phenotype in hyperfunctioning thyroid nodules.

G ATA2 mediates the negative regulation of the prepro-thyrotropin-releasing hormone gene by liganded T3 receptor ß2 in the rat hypothalamic paraventricular nucleus.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) ß2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin ß subunit (TSHß) gene suggest that there is a tethering mechanism, whereby liganded TRß2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHß gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRß2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRß2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TRß2 interferes with this activation via a tethering mechanism as in the case of the TSHß gene.

CCNB2 and AURKA overexpression may cause atypical mitosis in Japanese cortisol-producing adrenocortical carcinoma with TP53 somatic variant.

BACKGROUND: Many genomic analyses of cortisol-producing adrenocortical carcinoma (ACC) have been reported, but very few have come from East Asia. The first objective of this study is to verify the genetic difference with the previous reports by analyzing targeted deep sequencing of 7 Japanese ACC cases using next-generation sequencing (NGS). The second objective is to compare the somatic variant findings identified by NGS analysis with clinical and pathological findings, aiming to acquire new knowledge about the factors that contribute to the poor prognosis of ACC and to find new targets for the treatment of ACC. METHOD: DNA was extracted from ACC tissue of seven patients and two reference blood samples. Targeted deep sequencing was performed using the MiSeq system for 12 genes, and the obtained results were analyzed using MuTect2. The hypothesis was obtained by integrating the somatic variant findings with clinical and pathological data, and it was further verified using The Cancer Genome Atlas (TCGA) dataset for ACC. RESULTS: Six possible pathogenic and one uncertain significance somatic variants including a novel PRKAR1A (NM_002734.4):c.545C>A (p.T182K) variant were found in five of seven cases. By integrating these data with pathological findings, we hypothesized that cases with TP53 variants were more likely to show atypical mitotic figures. Using TCGA dataset, we found that atypical mitotic figures were associated with TP53 somatic variant, and mRNA expression of CCNB2 and AURKA was significantly high in TP53 mutated cases and atypical mitotic figure cases. CONCLUSION: We believe this is the first report that discusses the relationship between atypical mitotic figures and TP53 somatic variant in ACC. We presumed that overexpression of CCNB2 and AURKA mRNA may cause atypical mitosis in TP53 somatic mutated cases. Because AURKA is highly expressed in atypical mitotic cases, it may be an appropriate indicator for AURKA inhibitors.

Liganded T3 receptor ß2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production.

The serum concentration of thyrotropin (thyroid stimulating hormone, TSH) is drastically reduced by small increase in the levels of thyroid hormones (T3 and its prohormone, T4); however, the mechanism underlying this relationship is unknown. TSH consists of the chorionic gonadotropin α (CGA) and the ß chain (TSHß). The expression of both peptides is induced by the transcription factor GATA2, a determinant of the thyrotroph and gonadotroph differentiation in the pituitary. We previously reported that the liganded T3 receptor (TR) inhibits transactivation activity of GATA2 via a tethering mechanism and proposed that this mechanism, but not binding of TR with a negative T3-responsive element, is the basis for the T3-dependent inhibition of the TSHß and CGA genes. Multiple GATA-responsive elements (GATA-REs) also exist within the GATA2 gene itself and mediate the positive feedback autoregulation of this gene. To elucidate the effect of T3 on this non-linear regulation, we fused the GATA-REs at -3.9 kb or +9.5 kb of the GATA2 gene with the chloramphenicol acetyltransferase reporter gene harbored in its 1S-promoter. These constructs were co-transfected with the expression plasmids for GATA2 and the pituitary specific TR, TRß2, into kidney-derived CV1 cells. We found that liganded TRß2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRß2 functions as a classical transcriptional repressor. Then, we investigated the effect of T3 on the endogenous expression of GATA2 protein and mRNA in the gonadotroph-derived LßT2 cells. In this cell line, T3 reduced GATA2 protein independently of the ubiquitin proteasome system. GATA2 mRNA was drastically suppressed by T3, the concentration of which corresponds to moderate hypothyroidism and euthyroidism. These results suggest that liganded TRß2 inhibits the positive feedback autoregulation of the GATA2 gene; moreover this mechanism plays an important role in the potent reduction of TSH production by T3.

G-protein-coupled receptor 40 agonist GW9508 potentiates glucose-stimulated insulin secretion through activation of protein kinase Cα and ε in INS-1 cells.

OBJECTIVE: The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells. METHODS: Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia. RESULTS: At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 µM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect. CONCLUSION: GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

A case of TSH-secreting pituitary adenoma with cyclic fluctuations in serum TSH levels.

A 29-year-old man was referred to our department due to adrenal insufficiency with the inappropriate secretion of TSH (SITSH). Magnetic resonance imaging revealed a pituitary tumor. A weak TSH response in the TRH test, elevated sex hormone binding globulin (SHBG) levels, and the absence of a family medical history of SITSH or TRß gene mutations supported the diagnosis of TSH-secreting pituitary adenoma (TSHoma). However, complete TSH suppression and a blunted cholesterol response in the T3 suppression test as well as normal glycoprotein α-subunit (α-GSU) levels were not compatible with TSHoma. Since TSH, FT3, and FT4 spontaneously returned to normal ranges after admission, he was discharged. One month after his discharge, thyrotoxicosis with elevated serum TSH levels relapsed. After admission, his serum TSH levels returned to within the normal range. After his discharge from the second admission, his serum TSH levels fluctuated in accordance with serum FT3 and FT4 levels and symptoms, such as palpitations. Ten months after his discharge, he was admitted to our department again due to adrenal insufficiency and thyrotoxicosis with elevated serum TSH levels, suggesting cyclic SITSH. Although resistance to thyroid hormone (RTH) was not completely excluded, the pituitary tumor was removed by transsphenoidal surgery (TSS). A pathological diagnosis confirmed TSHoma. We herein report a case of TSHoma in which serum TSH, FT3, and FT4 levels fluctuated periodically. To the best of our knowledge, this is the first case report of "cyclic TSHoma", which needs to be considered when making a differential diagnosis of SITSH.

The Mechanism of Negative Transcriptional Regulation by Thyroid Hormone: Lessons From the Thyrotropin ß Subunit Gene.

Thyroid hormone (T3) activates (positive regulation) or represses (negative regulation) target genes at the transcriptional level. The molecular mechanism of the former has been elucidated in detail; however, the mechanism for negative regulation has not been established. The best example of the gene that is negatively regulated by T3 is the thyrotropin (thyroid-stimulating hormone) ß subunit (TSHß) gene. Analogous to the T3-responsive element (TRE) in positive regulation, a negative TRE (nTRE) has been postulated in the TSHß gene. However, TSHß promoter analysis, performed in the presence of transcription factors Pit1 and GATA2, which are determinants of thyrotroph differentiation in the pituitary, revealed that the nTRE is dispensable for inhibition by T3. We propose a tethering model in which the T3 receptor is tethered to GATA2 via protein-protein interaction and inhibits GATA2-dependent transactivation of the TSHß gene in a T3-dependent manner.

Discovery of Novel 5-(Piperazine-1-carbonyl)pyridin-2(1H)-one Derivatives as Orally eIF4A3-Selective Inhibitors.

Starting from our previous eIF4A3-selective inhibitor 1a, a novel series of (piperazine-1-carbonyl)pyridin-2(1H)-one derivatives was designed, synthesized, and evaluated for identification of orally bioavailable probe molecules. Compounds 1o and 1q showed improved physicochemical and ADMET profiles, while maintaining potent and subtype-selective eIF4A3 inhibitory potency. In accord with their promising PK profiles and results from initial in vivo PD studies, compounds 1o and 1q showed antitumor efficacy with T/C values of 54% and 29%, respectively, without severe body weight loss. Thus, our novel series of compounds represents promising probe molecules for the in vivo pharmacological study of selective eIF4A3 inhibition.

Crystal Structure of a Human K-Ras G12D Mutant in Complex with GDP and the Cyclic Inhibitory Peptide KRpep-2d.

The Ras proteins play roles in cell differentiation, proliferation, and survival. Aberrant signaling through Ras-mediated pathways in tumor cells occurs as a result of several types of mutational damage, which most frequently affects the amino acids G12, G13, and Q61. Recently, KRpep-2d was identified as a K-Ras(G12D) selective inhibitory peptide against the G12D mutant of K-Ras, which is a key member of the Ras protein family and an attractive cancer therapeutic target. In this study, the crystal structure of the human K-Ras(G12D) mutant was determined in complex with GDP and KRpep-2d at 1.25 Å resolution. This structure revealed that the peptide binds near Switch II and allosterically blocks protein-protein interactions with the guanine nucleotide exchange factor. This discovery of a unique binding pocket provides valuable information that will facilitate the design of direct Ras inhibitors.

Investigation of the structural requirements of K-Ras(G12D) selective inhibitory peptide KRpep-2d using alanine scans and cysteine bridging.

A structure-activity relationship study of a K-Ras(G12D) selective inhibitory cyclic peptide, KRpep-2d was performed. Alanine scanning of KRpep-2d focusing on the cyclic moiety showed that Leu7, Ile9, and Asp12 are the key elements for K-Ras(G12D) selective inhibition of KRpep-2d. The cysteine bridging was also examined to identify the stable analog of KRpep-2d under reductive conditions. As a result, the KRpep-2d analog (12) including mono-methylene bridging showed potent K-Ras(G12D) selective inhibition in both the presence and the absence of dithiothreitol. This means that mono-methylene bridging is an effective strategy to obtain a reduction-resistance analog of parent disulfide cyclic peptides. Peptide 12 inhibited proliferation of K-Ras(G12D)-driven cancer cells significantly. These results gave valuable information for further optimization of KRpep-2d to provide novel anti-cancer drug candidates targeting the K-Ras(G12D) mutant.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

Amino-acid mutations of Gly12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 µM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.

Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor ß2 in Thyrotroph.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the ß subunit of TSH (TSHß). We previously reported that T3-dependent repression of the TSHß gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived TαT1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with TαT1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TRß2. The inhibition by T3-bound TRß2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TRα1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSHß gene.

Non-islet cell tumor-induced hypoglycemia associated with macronodular pulmonary metastases from poorly differentiated thyroid carcinoma.

BACKGROUND: Non-islet cell tumor-induced hypoglycemia (NICTH), a major cause of fasting hypoglycemia, is caused by the overproduction of incompletely processed, high molecular-weight insulin-like growth factor-II (IGF-II), termed "big" IGF-II. To the best of our knowledge, only two cases of thyroid carcinoma associated with NICTH have been documented. PATIENT FINDINGS: We report the case of a 72-year-old woman who was brought to the emergency department with impaired consciousness. The patient had a history of pulmonary metastases from poorly differentiated thyroid carcinoma (PDTC), spanning 12 years since initial treatment. Laboratory tests showed decreased plasma glucose levels even though immunoreactive insulin, IGF-I, and growth hormone (GH) were undetectable. Computed tomography (CT) scan revealed macronodular pulmonary metastases the estimated volume of which was 456 mL. Both the biochemical data and imaging results suggested NICTH. The results of Western blot analysis performed on a fractionated serum sample showed an increased expression of big IGF-II, an important indicator in the diagnosis of NICTH. Because the massive pulmonary metastases were considered inoperable, immunohistochemical analysis of stored formalin-fixed, paraffin-embedded tissues was performed. The analysis revealed that the tumor cells were positive for both IGF-II and thyroglobulin. A whole-body CT excluded extrapulmonary metastatic lesions. A retrospective review revealed a gradual decrease in glycohemoglobin levels accompanied by an increase in the estimated volume of pulmonary metastases. These findings suggested that NICTH had been caused by pulmonary metastases from PDTC. CONCLUSIONS: We describe here the third reported case of NICTH associated with thyroid carcinoma. This is also the first case reporting big IGF-II in the serum of a patient with thyroid carcinoma.

Factors associated with fluorine-18-fluorodeoxyglucose uptake in benign thyroid nodules.

Thyroid nodules that exhibit focal uptake of fluorine-18 ((18)F)-fluorodeoxyglucose (FDG) are relatively frequent. Although the clinical features and associated mechanisms of FDG-avid lesions in both thyroid cancer and cytologically indeterminate nodules have been extensively studied, not much information is available on benign nodules. Therefore, in this retrospective study, the clinical, serological, and sonographic features of 15 benign FDG-avid nodules were compared with those of 17 non-avid lesions. Univariate analysis indicated that the FDG-positive and -negative nodules were similar with regard to age, gender, thyroid stimulating hormone (TSH), anti-thyroglobulin antibodies, tumor size, 4 B-mode sonographic findings (i.e., shape, margin, texture, and echo level), and/or elasticity. The presence of intranodular blood flow and the absence of a cystic component were associated with a greater possibility of positive FDG uptake. Multivariate analysis showed that vascularity was the only independent factor predicting FDG uptake. Across a wide range of tumor types, the extent of FDG uptake is positively correlated with tumor perfusion; this observation is consistent with the results of this study, which shows that FDG uptake in benign thyroid nodules is associated with increased vascularity.