Akio Matsushita

Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β 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.

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.

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

Thyrotropin-releasing hormone (TRH) activates not only the secretion of thyrotropin (TSH) but also the transcription of TSHβ and α-glycoprotein (αGSU) subunit genes. TSHβ 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 TSHβ gene is GATA2, not Pit1 or unliganded T3 receptor (TR). In previous studies on the mechanism of TRH-induced activation of the TSHβ 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 TSHβ 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 TSHβ 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 TSHβ 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 TSHβ expression.

Inhibition of GATA2-dependent transactivation of the TSHβ gene by ligand-bound estrogen receptor α

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.

Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor β2 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 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.

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 β) 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) β1 and α1 inhibit TEAD-dependent activation of the MYH7 promoter in a T3 dose-dependent manner. TRβ1 bound with GC-1, a TRβ-selective T3 analog, also repressed TEAD-induced activity. Although T3-dependent inhibition required the DNA-binding domain (DBD) of TRβ1, it remained after the putative negative T3-responsive elements were mutated. A co-immunoprecipitation study demonstrated the in vivo association of TRβ1 with TEAD-1, and the interaction surfaces were mapped to the DBD of the TRβ1 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.

The Mechanism of Negative Transcriptional Regulation by Thyroid Hormone: Lessons From the Thyrotropin β 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) β subunit (TSHβ) gene. Analogous to the T3-responsive element (TRE) in positive regulation, a negative TRE (nTRE) has been postulated in the TSHβ gene. However, TSHβ 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 TSHβ gene in a T3-dependent manner.

A sudden onset and the spontaneous remission of severe hypo-high-density lipoprotein cholesterolemia without serious underlying disease: a case report.

BACKGROUND Severe hypo-high-density lipoprotein (HDL) cholesterolemia is defined by serum values less than 20mg/dl. Few acquired cases, without serious underlying disease, have been reported. CASE An asymptomatic 75-y-old man was admitted for evaluation of low serum HDL-cholesterol (HDL-C) levels (2-8 mg/dl). The record of periodic medical examinations revealed that a sudden decrease had occurred 5 y ago. Mild anemia and proteinuria were noted but the liver and thyroid function tests were normal. β-Quantification revealed a relatively low HDL-C (10.8 mg/dl) and the serum lecithin cholesterol acyltransferase (LCAT) activity was low (29.4 nmol/ml/h). Unexpectedly, serum HDL-C levels recovered 2 y after hospital discharge. In addition, the serum LCAT activity, hemoglobin concentrations, and urine protein tests all returned to within the reference interval. Subsequent examinations could not clarify the cause of the sudden onset and spontaneous recovery of the extremely low HDL-C. CONCLUSIONS We describe an unusual case of acquired HDL-C deficiency in a 75-y-old man that did not have serious pre-existing disease. Recently, extremely low HDL-C levels in patients with the nephrotic syndrome, associated with acquired LCAT deficiency, have been reported. The present case might illustrate a milder form of this disorder, because the clinical findings show many similarities.

The importance of imaging procedures in evaluating painful neck masses: two patients with a painful internal jugular vein thrombosis.

Internal jugular vein thrombosis (IJVT) is a rare but potentially life-threatening status, as it can lead to fatal pulmonary embolism. One of its clinical manifestations is a painful anterior neck mass (1). Here, we report two patients with IJVT in whom a painful anterior neck mass initially suggested subacute thyroiditis (SAT). The first patient was a 68-year-old woman with Graves’ disease, in remission after antithyroid drug treatment, who consulted our hospital for right anterior neck pain. Palpation revealed a painful firm 6.5 cm · 4.0 cm nodule on the right side of her neck. It extended from the level of the laryngeal prominence to the supraclavicular region. Since the mass was painful and appeared consistent with an enlarged right thyroid lobe, SAT was considered. Unexpectedly, thyroid function tests indicated she was euthyroid and the serum thyroglobulin (Tg) was normal but the serum C-reactive protein level was high (4.5 mg/dL). Thyroid ultrasonography (US) revealed a diffuse hypoechoic and moderately heterogeneous thyroid gland, consistent with treated Graves’ disease, but no features of SAT. US also showed a hypoechoic spindle-shaped mass of 6.3 cm · 2.0 cm in the noncompressible right internal jugular vein (IJV) that was laterally adjacent to the thyroid lobe (see Supplementary Data, available online at www.liebertonline.com/ thy). D-dimer (normal range < 1.0lg/mL) was 6.3lg/mL and fibrinogen (normal range 150–400 mg/dL) was 449 mg/dL. She was hospitalized and diagnosed with IJVT associated with stomach cancer after examinations that included contrast-enhanced computed tomography (CT) and endoscopic biopsy. The second patient was a 51-year-old woman referred to our hospital because of suspected SAT. She complained of a oneweek history of right anterior neck pain and vague right shoulder and brachial discomfort. A painful, firm, 8.0 cm · 4.0 cm nodule was palpable in the region of the right thyroid lobe. Although SAT was suspected, thyroid function tests and serum Tg were normal. As US indicated a hypoechoic thrombus of 7.4 cm · 3.2 cm in the right IJV (see Supplementary Data), and since both D-dimer and fibrinogen were high (6.0 lg/mL and 429 mg/dL, respectively), she was admitted to our hospital. Thrombosis of the IJV and subclavian vein caused by a Pancoast tumor was diagnosed using US, contrastenhanced CT, and subsequent transbronchial lung biopsy. Deep vein thrombosis (DVT) occurs most commonly in the lower extremities or pelvis. IJVT is uncommon in the whole DVT because only about 10% of DVT develops in the internal jugular, axillary, and subclavian veins in the upper torso. Pulmonary embolism, a life-threatening complication, occurs with similar frequency in patients with DVT in the lower and upper extremities, at rates of 3%–36% (2). Since the mortality rate of pulmonary embolism is as high as 10%–30%, the early identification and treatment of DVT are mandatory. The location of an upper torso DVT is not important in this regard, as the mortality is similar among thromboses of the subclavian, axillary, and IJV (3). Central venous catheters and malignant neoplasms are two leading causes of IJVT. Two mechanisms have been suggested for the positive relationship between thrombogenesis and malignant neoplasms. One is the occurrence of a hypercoagulable state in malignancy and the other is venous stasis due to compression or direct tumor invasion of veins (1). Patients 1 and 2 appeared to have the former and latter factors, respectively. Painful anterior neck masses are usually associated with thyroid-related lesions, such as SAT, acute infectious thyroiditis, or hemorrhage in a thyroid nodule. Other very rare painful neck lesions include cervical lymphadenitis and infected cysts (4). Our two cases were confused with SAT because the palpation findings closely resembled those of SAT, and because initial imaging procedures were not performed. US is an easily available and useful noninvasive method for evaluating such conditions. It is widely accepted that noncompressibility of a normally compressible vein with or without a visible thrombus is definitive proof of IJVT. The sensitivity and specificity of US for IJVT is 78%–100% and 82%–100%, respectively. CT has also been useful for the assessment of IJVT, the main advantage of which is the detection of pulmonary embolism, together with IJVT (2). In conclusion, we encountered two patients with IJVT whose clinical findings resembled SAT. Imaging modalities such as US and CT are important for the close evaluation of painful neck masses.

Negative Regulation of the Thyrotropin β Gene by Thyroid Hormone

Thyroid hormone (T3 and T4) is secreted from the thyroid gland, and is known to reduce the level of serum thyrotropin (thyroid-stimulating hormone, TSH) in the pituitary gland (Sarapura et al., 2002; Shupnik et al., 1989) (Fig. 1A). This is a typical example of negative feedback between the pituitary and endocrine organs, and is a key component of thyroid hormone homeostasis. TSH is one of the peptide hormones generated in the anterior pituitary, and is a heterodimer composed of an ┙ chain (┙-glycoprotein subunit, ┙GSU) and a ┚ chain (TSH┚) (Shupnik et al., 1989). While ┙GSU is common to follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG), TSH┚ is specific to TSH alone. Although the concentration of serum T4 is much higher than that of T3, T4 is converted to T3 by deiodinase (Dio) in the TSH-producing cells (thyrotrophs) of the pituitary (Christoffolete et al., 2006), and T3 exhibits biological activity as a thyroid hormone (Gereben et al., 2008). T3 inhibits expression of both TSH┚ and ┙GSU at the transcriptional level (Shupnik et al., 1989). The magnitude of T3-induced repression of the TSH┚ gene is greater than that of ┙GSU. Here, we provide an overview of the molecular mechanisms involved in T3-induced negative regulation of the TSH┚ gene and its related genes.