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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 β (TSHβ). 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 TSHβ 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 TSHβ 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.

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 β chain (TSHβ). 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 TSHβ 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, TRβ2, into kidney-derived CV1 cells. We found that liganded TRβ2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRβ2 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 LβT2 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 TRβ2 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.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) β2 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 β subunit (TSHβ) gene suggest that there is a tethering mechanism, whereby liganded TRβ2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHβ gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRβ2 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 TRβ2 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 TRβ2 interferes with this activation via a tethering mechanism as in the case of the TSHβ gene.

Background Staphylococcus hominis ( S. hominis ) is an opportunistic pathogen that is often highly resistant to antibiotics and is difficult to treat. In patients diagnosed with an adrenocorticotropic hormone (ACTH)-producing tumor that compromises the immune system due to hypercortisolemia, cancer treatment and infection control should be considered simultaneously. This report presents a case of refractory postoperative S. hominis bacteremia requiring the prolonged administration of several antibiotics in a patient with an ACTH-producing pancreatic neuroendocrine neoplasm (pNEN). Case presentation A 35-year-old man visited a neighboring hospital for a thorough examination after experiencing weight gain and lower limb weakness for several months. Enhanced computed tomography revealed a pancreatic tail tumor and bilateral adrenal enlargement. Elevated plasma ACTH and serum cortisol were noted. Biopsy under endoscopic ultrasonography revealed the tumor as an ACTH-producing pNEN. The patient was transferred to our hospital for further treatment. Pneumocystis pneumonia was noted and treated with sulfamethoxazole and adjunctive glucocorticoids. Hypercortisolism was controlled with metyrapone and trilostane. Somatostatin receptor scintigraphy and ethoxybenzyl magnetic resonance imaging detected other lesions in the pancreatic head. A total pancreatectomy was performed given that the lesions were found in both the pancreatic head and tail. Plasma ACTH and serum cortisol levels decreased immediately after the resection. Pathological examination revealed that the pancreatic tail tumor was NEN G2 and T3N1aM0 Stage IIB and the pancreatic head lesions were SSTR-positive hyperplasia of the islet of Langerhans cells. On postoperative day 11, catheter-associated bacteremia occurred. Initially, meropenem hydrate and vancomycin hydrochloride were administered empirically. S. hominis was identified and appeared sensitive to these antibiotics according to susceptibility testing. However, S. hominis was repeatedly positive in blood cultures for more than one month, despite treatment with several antibiotics. Eventually, with the combined use of three antibiotics (meropenem hydrate, vancomycin hydrochloride, and clindamycin phosphate) for more than 3 weeks, the S. hominis -associated bacteremia improved. He was discharged 79 days after surgery. Conclusions Our patient with an ACTH-producing pNEN was immunocompromised and needed meticulous attention for infectious complications even after successful tumor removal. Specifically, S. hominis bacteremia in such patients demands intensive treatments, such as with combinational antibiotics.

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 β subunit of TSH (TSHβ). We previously reported that T3-dependent repression of the TSHβ 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, TRβ2. The inhibition by T3-bound TRβ2 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 TSHβ gene.

T3 inhibits thyrotropin-releasing hormone (TRH) synthesis in hypothalamic paraventricular nucleus (PVN). Although T3 receptor (TR) β2 is known to mediate the negative regulation of prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene indicate the tethering mechanism, where T3-bound TRβ2 interferes with the function of the transcription factor GATA2, which is essential for TSHβ expression. Interestingly, the transcription factor Sim1, a determinant of PVN differentiation in hypothalamus, is reported to induce the expressions of TRβ2 and GATA2. Indeed, our immunohistochemistry revealed the expression of GATA2 in the TRH neuron of the rat PVN. According to the experimental report with transgenic mice, the DNA sequence from nt. -547 to nt. +84 is sufficient for the expression of the prepro-TRH gene in PVN. Using the CAT reporter gene harboring this region, we found that this promoter is activated by GATA2 approximately 6-fold in CV1 cells. The deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. T3-dependent repression was maintained after the mutation of the putative negative T3 responsive element (site4). Although the melanocortin 4 receptor signaling is known to stimulate the prepro-TRH promoter via protein kinase A pathway in the PVN, inhibition by T3 was dominant over the 8-bromo-cAMP-induced activation. We observed the in vivo recognition of GATA-RE by GATA2 using chromatin immunoprecipitation assay with CA77 cells, which express endogenous TRH. The electrophoretic mobility shift assay also demonstrated that GATA2 bound to oligonucleotide containing the GATA-RE. These results suggest that, as in the case of the TSHβ gene, GATA2 transactivates the prepro-TRH gene and that T3-bound TRβ2 interferes with its function, resulting in the negative regulation of this gene.

T3 negatively regulates thyrotropin-releasing hormone (TRH) secreted from hypothalamus paraventricular nucleus (PVN). As T3 receptor (TR) ₂ is known to mediate the negative regulation of prepro-TRH gene, we previously investigated the T3-dependent negative regulation of the prepro-TRH gene. We reported that T3-bound TR₂ inhibits the transcription of this gene induced by transcription factor GATA2 via tethering mechanism but not negative T3-responsive element (site4), as in the case of thyrotropin (TSH) _(g)ene (Vitam Horm. 106:97-127, 2018). We also confirmed that GATA2 is expressed in the TRH neuron in PVN. Interestingly, it was reported that T3-dependent repression of TSH_(g)ene is blunted in the mice whose steroid receptor coactivator (SRC)-1 and/or 2 were genetically ablated (Endocrinology 143(4) 1554-1557, 2002), suggesting the involvement of SRCs in the T3-dependent negative regulation. Indeed, T3-dependent inhibition of TSH_(s)ecretion is impaired in the knock-in mice, of which TR₁ was substituted with mutant TR₁ E457A, a mutant lacking for the interaction with SRC-1 without defect with T3-binding (J. Clin. Invest. 115(9)2517-2523, 2005). Because similar TR₁ mutants have been reported (Science. 12;280(5370):1747-9, 1998), we attempted here to study their function in the context of the negative regulation of the prepro-TRH promoter in monkey kidney-derived CV-1 cells. Compared with wild-type TR₂, T3-dependent inhibition of the CAT-reporter gene, which was fused with prepro-TRH promoter (nt. -547 to nt. +84bp ), was relieved in all the mutant TR₂s including V337R, K341A, I355R, L507R, L509R, E510K and E510A (corresponding to E457A in TR₁). Unexpectedly, however, over-expression of SRC-1 and SRC-2 did not affect the T3-dependent inhibition mediated by wild-type TR₂. These results indicate that negative regulation of preproTRH gene by T3 may be mediated by unknown factor that is able to interact with the surface of T3-bound TR₂ as in the case of SRC-1 or 2. Alternatively, effect by over-expressed SRC-1 and 2 may be quickly metabolized by ubiquitin-proteasome system or exported to cytosol as previously reported. We are currently investigating the effect of SI-2, bufalin and gossypol, which were reported to degradate the SRC proteins potently (PNAS 113(18) 4970-4975, 2016 and references therein).

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) [beta]2 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 [beta] subunit (TSH[beta]) gene suggest that there is a tethering mechanism, whereby liganded TR[beta]2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSH[beta] gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TR[beta]2 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 TR[beta]2 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 TR[beta]2 interferes with this activation via a tethering mechanism as in the case of the TSH[beta] gene.

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 [beta] subunit of TSH (TSH[beta]). We previously reported that T3-dependent repression of the TSH[beta] 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 T[alpha]T1. 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 T[alpha]T1 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, TR[beta]2. The inhibition by T3-bound TR[beta]2 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 TR[alpha]1. 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 TSH[beta] gene.

Background: Hyperfunctioning papillary thyroid carcinoma (PTC) is a rare tumor and accounts for less than 0.1% of all thyroid tumors. Information about its driver mutations is limited. Our literature search yielded 16 cases wherein a mutational analysis was conducted. Thyrotropin receptor (TSHR) mutations were identified in 11 of these cases. One case revealed a combination of TSHR and KRAS mutations. No mutations were identified in the other four cases. BRAFV600E is a prominent oncogene in PTC; however, hyperfunctioning PTC with this mutation has not yet been reported. Clinical Case: In a 48-year-old man, ultrasonography (US) during an annual medical checkup revealed a nodule at the right lobe of the thyroid gland. He visited the outpatient clinic for further evaluation. Thyroid function tests indicated that he was hyperthyroid with TSH level of 0.01 mIU/L (reference range: 0.05-5.00), free thyroxine level of 1.8 ng/dL (reference range: 0.9-1.7), and free triiodothyronine level of 4.3 pg/mL (reference range: 2.3-4.0). Serum thyroglobulin was 62.1 ng/mL (reference range: <33.7) and TSHR autoantibodies (TRAb) was <0.8 IU/L (reference range: <2.0 IU/L). B-mode US revealed a hypoechoic, heterogeneous nodule with largest diameter of 25 mm, and it had a jagged border and microcalcification. Color Doppler US revealed increased intranodular vascularity. The 99mTc thyroid scintigram revealed a round, right-sided focus of tracer uptake by the nodule with suppression in the remainder of the gland. These findings were consistent with an autonomously-functioning thyroid nodule. The patient underwent total thyroidectomy because fine-needle aspiration cytology revealed a malignant cytological diagnosis. The histopathological diagnosis of the patient was PTC, tall cell variant, pT2, pEx0, pN1b, and M0. Subsequent mutational analysis of BRAF (exon 15), TSHR (exons 9 and 10), GNAS (exons 7-10), KRAS, NRAS, HRAS (codons 12, 13, and 61), and TERT promoter (C250T and C228T) only identified a heterozygous point mutation in BRAFV600E in tissue samples. Conclusion: We report for the first time a case of hyperfunctioning papillary thyroid carcinoma with a BRAF mutation.