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.

Essential role of TEA domain transcription factors in the negative regulation of the MYH 7 gene by thyroid hormone and its receptors.

MYH7 (also referred to as cardiac myosin heavy chain ß) gene expression is known to be repressed by thyroid hormone (T3). However, the molecular mechanism by which T3 inhibits the transcription of its target genes (negative regulation) remains to be clarified, whereas those of transcriptional activation by T3 (positive regulation) have been elucidated in detail. Two MCAT (muscle C, A, and T) sites and an A/T-rich region in the MYH7 gene have been shown to play a critical role in the expression of this gene and are known to be recognized by the TEAD/TEF family of transcription factors (TEADs). Using a reconstitution system with CV-1 cells, which has been utilized in the analysis of positive as well as negative regulation, we demonstrate that both T3 receptor (TR) ß1 and α1 inhibit TEAD-dependent activation of the MYH7 promoter in a T3 dose-dependent manner. TRß1 bound with GC-1, a TRß-selective T3 analog, also repressed TEAD-induced activity. Although T3-dependent inhibition required the DNA-binding domain (DBD) of TRß1, it remained after the putative negative T3-responsive elements were mutated. A co-immunoprecipitation study demonstrated the in vivo association of TRß1 with TEAD-1, and the interaction surfaces were mapped to the DBD of the TRß1 and TEA domains of TEAD-1, both of which are highly conserved among TRs and TEADs, respectively. The importance of TEADs in MYH7 expression was also validated with RNA interference using rat embryonic cardiomyocyte H9c2 cells. These results indicate that T3-bound TRs interfere with transactivation by TEADs via protein-protein interactions, resulting in the negative regulation of MYH7 promoter activity.

Inappropriate elevation of serum thyrotropin levels in patients treated with axitinib.

BACKGROUND: Although anticancer treatment with the tyrosine kinase inhibitor (TKI) axitinib frequently causes thyroid dysfunction, the associated mechanism and clinical features have not been elucidated. METHODS: Six patients were treated with axitinib for metastatic renal cell carcinoma at the Hamamatsu University School of Medicine between 2008 and 2010. We reviewed their thyroid function results and compared them to those of patients treated with two other TKIs, sunitinib or sorafenib, and to those of subjects with normal hypothalamic-pituitary-thyroid (HPT) function. RESULTS: Axitinib-induced thyroid dysfunction was observed in all patients, and two patterns were observed: increased serum thyrotropin (TSH) levels within one month after administration occurred in five patients and transient thyrotoxicosis due to destructive thyroiditis occurred in five patients within 7 months of treatment. Four patients exhibited both. When the relationship between the serum TSH and thyroid hormones was evaluated using plots of TSH versus both free thyroxine and free triiodothyronine, four patients showed an inappropriate elevation of serum TSH during administration of axitinib. Their values apparently shifted against the regression line compared to data from patients with a normal HPT function. A similar tendency, though weaker, was observed in some patients treated with sunitinib or sorafenib. CONCLUSION: This is the first study to report an inappropriate elevation of serum TSH levels in patients treated with axitinib.

Liganded thyroid hormone receptor inhibits phorbol 12-O-tetradecanoate-13-acetate-induced enhancer activity via firefly luciferase cDNA.

Thyroid hormone receptor (TR) belongs to the nuclear hormone receptor (NHR) superfamily and regulates the transcription of its target genes in a thyroid hormone (T3)-dependent manner. While the detail of transcriptional activation by T3 (positive regulation) has been clarified, the mechanism of T3-dependent repression (negative regulation) remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin ß gene, T3-bound TR (T3/TR) is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1). Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL) gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT)-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA). TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional activity, which is inhibited by T3/TR.

GATA2 mediates thyrotropin-releasing hormone-induced transcriptional activation of the thyrotropin ß gene.

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHß and α-glycoprotein (αGSU) subunit genes. TSHß expression is maintained by two transcription factors, Pit1 and GATA2, and is negatively regulated by thyroid hormone (T3). Our prior studies suggest that the main activator of the TSHß gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHß gene, the involvements of Pit1 and TR have been investigated, but the role of GATA2 has not been clarified. Using kidney-derived CV1 cells and pituitary-derived GH3 and TαT1 cells, we demonstrate here that TRH signaling enhances GATA2-dependent activation of the TSHß promoter and that TRH-induced activity is abolished by amino acid substitution in the GATA2-Zn finger domain or mutation of GATA-responsive element in the TSHß gene. In CV1 cells transfected with TRH receptor expression plasmid, GATA2-dependent transactivation of αGSU and endothelin-1 promoters was enhanced by TRH. In the gel shift assay, TRH signal potentiated the DNA-binding capacity of GATA2. While inhibition by T3 is dominant over TRH-induced activation, unliganded TR or the putative negative T3-responsive element are not required for TRH-induced stimulation. Studies using GH3 cells showed that TRH-induced activity of the TSHß promoter depends on protein kinase C but not the mitogen-activated protein kinase, suggesting that the signaling pathway is different from that in the prolactin gene. These results indicate that GATA2 is the principal mediator of the TRH signaling pathway in TSHß expression.

Syndrome of inappropriate secretion of thyrotropin associated with thymoma-related peripheral nerve hyperexcitability.

Syndrome of inappropriate secretion of thyrotropin (SITSH) is a clinical state of inappropriately elevated secretion of thyrotropin (TSH) in the presence of elevated free thyroid hormones. Peripheral nerve hyperexcitability (PNH) is a rare disorder characterized by muscle twitching at rest. No relation between them is known. A 49-year-old man was referred to our hospital because of elevated serum free thyroxine (2.6 ng/dL; normal range, 0.9-1.7) and normal TSH (2.7 mIU/L; normal range, 0.5-5.0). Genetic analysis revealed no mutations of the thyroid hormone receptor ß gene. Magnetic resonance imaging visualized no pituitary adenoma. He complained of appetite loss, weight loss, myokymia, paraesthesia, hyperhydrosis and insomnia. Chest X ray and computed tomography (CT) scan showed a mediastinal tumor diagnosed as a thymoma by CT-guided biopsy. Electromyography disclosed fasciculations and myokymic discharges. Nerve conduction studies showed prolonged after-discharges following evoked compound muscle action potential. The patient was diagnosed with thymoma-associated PNH based on neurological manifestations and neurophysiological findings, and was treated with pulse therapy with methylprednisolone after thymectomy. Interestingly, the SITSH state became less prominent as his neurological manifestations improved. This is the first case of SITSH possibly caused by thymoma-associated PNH.

High incidence of thyroid cancer in focal thyroid incidentaloma detected by 18F-fluorodeoxyglucose [corrected] positron emission tomography in relatively young healthy subjects: results of 3-year follow-up.

As 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is becoming a common imaging modality, the number of thyroid incidentalomas identified by FDG-PET (PET incidentaloma) is increasing. The purpose of this study was to elucidate the risk of cancer in focal thyroid PET incidentaloma in healthy subjects of relatively younger age as well as the usefulness of repeated FDG-PET. The study was conducted with an observation period of three years. A total of 1,501 healthy volunteers (mean age, 43.5+/-9.7 years) underwent the first FDG-PET from August 2003 to July 2004. When focal thyroid PET incidentaloma was found, further diagnostic examination was conducted. When thyroid cancer was suspected, surgical resection was performed with the patient' s agreement. Patients with PET incidentaloma without surgery were offered annual US and FDG-PET and finally FNAB was performed in the fourth year. Focal thyroid PET incidentaloma was observed in 20 subjects. The final diagnoses in 20 subjects were malignant in 11 (ten papillary thyroid carcinoma (PTC) and one thyroid carcinoma showing thymus-like differentiation), indeterminate in one, and benign in eight subjects. Seven patients not treated surgically at the first examination had annual FDG-PET. One patient with PTC showed increasing SUVmax, but another with a benign nodule exhibited a similar increase. Others (one with PTC, one with an indeterminate nodule, and three with benign nodules) exhibited negligible SUVmax changes. When closely examined, focal thyroid PET incidentaloma in relatively young healthy adults has a high probability of malignancy. Repeated FDG-PET to follow up patients with thyroid nodules is ineffective.

Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin beta promoter.

Thyrotropin (TSH) is a heterodimer consisting of alpha and beta chains, and the beta chain (TSHbeta) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHbeta expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHbeta promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHbeta promoter without PIT1. The deleted region (nt -82/-52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHbeta promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

Inhibition of GATA2-dependent transactivation of the TSHbeta gene by ligand-bound estrogen receptor alpha.

Transcriptional repression of the TSH-specific beta subunit (TSHbeta) gene has been regarded to be specific to thyroid hormone (tri-iodothyronine, T(3)) and its receptors (TRs) in physiological conditions. However, TSHbeta mRNA levels in the pituitary were reported to decrease in the administration of pharmacologic doses of estrogen (17-beta-estradiol, E(2)) and increase in E(2) receptor (ER)-alpha null mice. Here, we investigated the molecular mechanism of inhibition of the TSHbeta gene expression by E(2)-bound E(2)-estrogen receptor 1 (E(2)-ERalpha). In kidney-derived CV1 cells, transcriptional activity of the TSHbeta promoter was stimulated by GATA2 and suppressed by THRBs and ERalpha in a ligand-dependent fashion. Overexpression of PIT1 diminished the E(2)-ERalpha-induced inhibition, suggesting that PIT1 may protect GATA2 from E(2)-ERalpha targeting by forming a stable complex with GATA2. Interacting surfaces between ERalpha and GATA2 were mapped to the DNA-binding domain (DBD) of ERalpha and the Zn finger domain of GATA2. E(2)-dependent inhibition requires the ERalpha amino-terminal domain but not the tertiary structure of the second Zn finger motif in E(2)-ERalpha-DBD. In the thyrotroph cell line, TalphaT1, E(2) treatment reduced TSHbeta mRNA levels measured by the reverse transcription PCR. In the human study, despite similar free thyroxine levels, the serum TSH level was small but significantly higher in post- than premenopausal women who possessed no anti-thyroid antibodies (1.90 microU/ml+/-0.13 S.E.M. vs 1.47 microU/ml+/-0.12 S.E.M., P<0.05). Our findings indicate redundancy between T(3)-TR and E(2)-ERalpha signaling exists in negative regulation of the TSHbeta gene.

Naked eye sensor on polyvinyl chloride platform of chromo-ionophore molecular assemblies: a smart way for the colorimetric sensing of toxic metal ions.

We demonstrate the possibility of fabricating a simple, naked eye colorimetric sensor miniature, using chromo-ionophore molecular assemblies anchored on polyvinyl chloride (PVC) surface. The ion-sensing probe (4-n-dodecyl-6-(2-thiazolylazo)-resorcinol) provides a better efficiency with PVC platform in developing a series of colour transitions, while targeting trace levels of Cd(2+), Pb(2+) and Hg(2+). The physical properties of the film sensor are controlled by measuring the probe isotherm plot. The surface morphology and molecular composition of the solid-state optical sensor are characterized using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The changes in sensor's optical intensity and its response time for the target analytes are followed by absorption spectroscopy. High speed of response (t

Optical nanosensor design with uniform pore geometry and large particle morphology.

Appropriate design of nanosensors for optically selective, sensitive sensing systems is needed for naked-eye detection of pollutants for environmental cleanup of toxic heavy-metal ions. Mesostructured materials with two- or three-dimensional (2D or 3D) geometries and large particle morphologies show promise as probe carriers, and can therefore be used to reproducibly fabricate uniformly packed nanosensors. This is the first report on the effects of significant key properties of the mesostructured carriers, such as morphology, geometry, and pore shape, on the functionality of optical nanosensor designs. Such mesostructured sensors with superior physical characteristics can be used as components in sensing systems with excellent stability and sensitivity, and with rapid detection response. The nanosensor design can enhance the selectivity even at low concentrations of the pollutant target ions (nanomolar level). Among the nanosensors developed here, the large pore-surface grains of highly ordered 3D monoliths (HOM) exhibited a high adsorption capability of the Pyrogallol Red probe and high accessibility to analyte ion transport, leading to possible naked-eye detection of Sb(III) ions at concentrations as low as 10(-9) mol dm(-3) and at a wide detection range of 0.5 ppb to 3 ppm. A key finding in our study was that our mesostructured nanosensor designs retained highly efficient sensitivity without a significant increase in kinetic hindrance, despite the slight decrease of the specific activity of the electron acceptor/donor strength of the probe functional group after several regeneration/reuse cycles. The results, in general, indicate that large-scale reversibility of optical nanosensors is feasible in such metal-ion sensing systems.

Naked-eye cadmium sensor: using chromoionophore arrays of Langmuir-Blodgett molecular assemblies.

This study demonstrates the possibility of a reversible naked-eye detection method for submicromolar levels of cadmium(II) using the Langmuir-Blodgett (L-B) technique. Molecular assemblies of 4-n-dodecyl-6-(2-thiazolylazo)resorcinol are transferred on precleaned microscopic glass slides, to act as a sensing probe. Isotherm (pi-A) measurements were performed to ensure the films' structural rigidity and homogeneity during sensor fabrication. The sensor surface morphology was characterized using atomic force microscopy and scanning electron microscopy. The probe membrane exhibits visual color transition, forming a series of reddish-orange to pinkish-purple complexes with cadmium, over a wide concentration range (0.04-44.5 microM). Cadmium response kinetics and the changes in the sensors' intrinsic optical properties were monitored using absorption spectroscopy and further confirmed using X-ray photoelectron spectroscopy. A hybrid L-B film composite of poly(vinyl stearate) and poly(vinyl-N-octadecylcarbamate) were investigated for enhancing sensor performance. The sensor was tested for its practical approach to prove its cadmium selectivity and sensitivity amid common matrix constituents using synthetic mixtures and real water samples. Using the sensor strips, the respective lower limits of cadmium detection and quantification are 0.039 and 0.050 microM, as estimated from a normalized linear calibration plot.

Supercritical water decomposition of polyethylene samples for the determination of their trace cadmium content.

A novel pretreatment method has been developed for determination of toxic metals in plastic materials by their decomposition under supercritical water conditions. Particularly, quantitative analysis of cadmium in polyethylene has been demonstrated using inductively coupled plasma atomic emission spectrometry combined with supercritical water treatment. All the cadmium in a polyethylene sample was obtained as an aqueous solution by the treatment with supercritical water containing 12.4% of hydrogen peroxide at 673 K. Although a complete recovery of the aqueous solution from the reactors has not yet been attained, we verified that the present method was effective and promising for quantitative analysis of trace amounts of hazardous metals in plastic materials.

A reactivity index study to monitor the role of solvation on the interaction of the chromophores with amino-functional silanol surface for colorimetric sensors.

Amino-functional silanol surface are mostly used for the immobilization of inorganic ions, molecules, organic or biochemical molecules onto the mesopore surface. In analytical chemistry, the metal ion uptake was visualized through colorimetric sensors using chromophore molecules. One needs to know the structure-property correlation between the chromophore and silylating agent while choosing chromophore, which is very important to design the sensors. We have used two chromophores representative of hydrophobic and hydrophilic type. We used density functional calculation on all the interacting molecules in both the unsolvated phase and solvated medium within the domain of hard soft acid base principle (HSAB) to look at the localized activity of the interacting atoms of these reacting molecules to formulate a priori rule to choose of the best chromophore. We have as well postulated the mechanism of interaction between chromophore and the silylating agent. The results were compared with experiment and it is observed that solvation plays a detrimental role in the binding of chromophore with silylating agent. The results also show that, the range of reactivity index can be used as a suitable property to scale activity of chromophore molecules suitable for the sensing process. It is observed that the hydrophobic chromophore binds stronger with both the metal and the silylating agent; whereas for the hydrophilic one, it binds only with the silylating agent when solvated and in all cases the metal ion binding is weaker compared to that of the hydrophobic one.

Naked eye detection of cadmium using inorganic-organic hybrid mesoporous material.

A novel and low-cost optical sensor for the naked eye detection of Cd2+ in aqueous media based on mesoporous silica containing 4-(2-pyridylazo)resorcinol (PAR) as a probe molecule anchored by N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TMAC) was prepared. The effects of various factors such as pH, solvent volume, temperature, reaction time, amount of the material, and the presence of various ions were studied in order to optimize operating conditions. The detection was based on the color change of PAR from orange-yellow to purple as a result of complexation with Cd2+. The intensity of the Cd-PAR complex varies linearly with the Cd2+ concentration, from zero to 1.78x10(-7) mol dm(-3). The detection and quantification limits for the method when determining Cd2+ were 1.75x10(-8) and 5.77x10(-8) mol dm(-3), respectively, with a correlation coefficient of 0.99. Good chemical stability of the material was observed for a period of five months. The developed sensor was applied to the analysis of various industrial effluents and tap water samples.

Fluorometric determination of fluoride ion by reagent tablets containing 3-hydroxy-2'-sulfoflavone and zirconium(IV) ethylenediamine tetraacetate.

A reagent tablet for determination of fluoride ion has been prepared using ethylenediamine-N,N,N',N'-tetraacetate complex of zirconium (Zr-EDTA), 3-hydroxy-2'-flavone (FS) and an appropriate pH buffer. Dissolving of the tablet into water exhibits an intense blue fluorescence (lambda(max)=460 nm) upon excitation at 377 nm and the fluorescence intensity decreases with the presence of fluoride ion. Hence, a simple fluorescent detection procedure for fluoride ion in aqueous media was successfully constructed with this tablet. The principle of this detection system is the ligand exchange reaction of FS bound to Zr-EDTA with fluoride ion. The present system provides an easy, rapid and selective determination method of fluoride ion ranging from 5 x 10(-6) to 1 x 10(-3)mol dm(-3). The measurement of real samples with this tablet showed the similar results as those by the common method with the Alfusone reagent.

Naked-eye detection of fluoride using Zr(IV)-EDTA complex and pyrocatechol violet.

A method for the visual detection of fluoride is presented using an aqueous solution of Pyrocatechol Violet (PV), which changes its color from yellow to blue by the formation of a ternary complex with Zr(H2O)2EDTA; also, the blue color of PV-Zr-EDTA readily shifts to orange red upon the addition of a moderate concentration of fluoride. The mechanism has been interpreted by a ligand-exchange reaction of PV coordinated to Zr(IV) with fluoride. The effect of various factors, such as the pH, ratio of Zr(H2O)2EDTA and PV, and diverse ions were studied to optimize the conditions for the reaction of PV-Zr-EDTA with fluoride. The present naked-eye detection system provides a simple, quick, and sensitive method for the determination of fluoride in the concentration range 1.5 x 10(-5) to 1.5 x 10(-4) mol dm(-3). The detection limit of the method was observed to be 4.5234 x 10(-4) mol dm(-3) with a correlation coefficient of 0.9955. The developed chemosensor was applied to the detection of fluoride in industrial effluents.

Optical sensor for the visual detection of mercury using mesoporous silica anchoring porphyrin moiety.

A low cost, solid optical sensor for the rapid detection of low concentrations of Hg2+ in aqueous media was prepared by the monolayer functionalization of mesoporous silica with 5,10,15,20-tetraphenylporphinetetrasulfonic acid (TPPS), anchored by N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TMAC). The detection is based on the color change of TPPS from orange to green as a result of the formation of a charge-transfer complex with Hg2+. The intensity of the charge-transfer band varies linearly with Hg2+ in the concentration range from zero to 2.5 x 10(-7) mol dm(-3). The lower detection limit observed for Hg2+ concentration is 1.75 x 10(-8) mol dm(-3). The material exhibits good chemical and mechanical stability, and did not show any degradation of TPPS for a period of eight months. The sensor was applied for the analysis of various environmental samples. The effects of pH, sample volume, reaction time, amount of material, and the presence of foreign ions on the detection method are discussed.