Essential role of GATA2 in the negative regulation of thyrotropin beta gene by thyroid hormone and its receptors.

Previously we reported that the negative regulation of the TSHbeta gene by T(3) and its receptor [thyroid hormone receptor (TR)] is observed in CV1 cells when GATA2 and Pit1 are introduced. Using this system, we further studied the mechanism of TSHbeta inhibition. The negative regulatory element (NRE), which had been reported to mediate T(3)-bound TR (T(3)-TR)-dependent inhibition, is dispensable, because deletion or mutation of NRE did not impair suppression. The reporter construct, TSHbeta-D4-chloramphenicol acetyltransferase, which possesses only the binding sites for Pit1 and GATA2, was activated by GATA2 alone, and this transactivation was specifically inhibited by T(3)-TR. The Zn finger region of GATA2 interacts with the DNA-binding domain of TR in a T(3)-independent manner. The suppression by T(3)-TR was impaired by overexpression of a dominant-negative type TR-associated protein (TRAP) 220, an N- and C-terminal deletion construct, indicating the participation of TRAP220. Chromatin immunoprecipitation assays with a thyrotroph cell line, TalphaT1, revealed that T(3) treatment recruited histone deacetylase 3, reduced the acetylation of histone H4, and caused the dissociation of TRAP220 within 15-30 min. The reduction of histone H4 acetylation was transient, whereas the dissociation of TRAP220 persisted for a longer period. In the negative regulation of the TSHbeta gene by T(3)-TR we report that 1) GATA2 is the major transcriptional activator of the TSHbeta gene, 2) the putative NRE previously reported is not required, 3) TR-DNA-binding domain directly interacts with the Zn finger region of GATA2, and 4) histone deacetylation and TRAP220 dissociation are important.

1,25-dihydroxyvitamin D3 and its receptor inhibit the chenodeoxycholic acid-dependent transactivation by farnesoid X receptor.

Farnesoid X receptor (FXR), the receptor for bile acids, including chenodeoxycholic acid (CDCA), is a member of the nuclear receptor superfamily, which also includes the receptors for retinoic acid, vitamin D (D3), thyroid hormone, thiazolidinedione and 22(R)-hydroxycholesterol. Here, we have evaluated the effects of a series of ligands and their receptors on the promoter activity induced by CDCA/FXR. The kidney cell line, CV1, was cotransfected with FXR-expression plasmid and the luciferase-based reporter gene that has a thymidine kinase promoter fused to the canonical FXR-responsive element or the natural promoter for the small heterodimer partner (SHP), bile salt export pump (BSEP), and ileum bile acid (I-BABP) gene. D3 and its receptor (VDR) inhibited the transactivation of all four reporter constructs that are enhanced by CDCA/FXR. The effect of D3 on the expression of the BSEP and SHP genes in HepG2 cells and that of the I-BABP gene in Caco-2 cells were confirmed by reverse transcription (RT)-PCR. Deletion analysis of VDR revealed that its ligand-binding domain (LBD) is responsible for the repression and the DNA-binding domain (DBD) is dispensable. Specific interaction between FXR and VDR was detected with the in vitro pull-down assay using chimeric FXR or VDR fused to glutathione-S-transferase.

[Study on root fracture and alveolar bone absorption of doweled lower premolars].

PURPOSE: The aim of this study was to examine root fractures in the doweled lower premolars and their relationship to alveolar bone absorption on the buccal aspect of the abutment teeth. METHODS: Experiment 1. Fractures in the roots of the abutment teeth and absorption of the surrounding alveolar bone was visualized by Multi-image Micro CT (3DX) in nine lower premolar abutment teeth that had lost both their posts and their. Experiment 2. The stresses in the teeth were analyzed using a three-dimensional finite element model, both models with and without alveolar bone absorption, as determined from Experiment 1. The stress distributions of von Mises equivalent stress and the shear stresses in the XZ, YZ and XY direct ions were examined. RESULTS: Experiment 1. The absorption of the alveolar bone was observed on the buccal side, and the fractures in the roots were observed in the lingual to buccal direction. Experiment 2. In the case without alveolar bone absorption, the XY shear stress in the root for mesial or distal loads was concentrated near the cervix with a positive stress value, and close to the end of the post with a negative stress value. In the alveolar bone absorption model, the XY shear stress in the root for mesial or distal loads was concentrated close to the end of the post on the lingual side with a positive stress value and on the buccal side with a negative stress value. CONCLUSION: In the case without alveolar bone absorption, on stress concentration was observed at the end of the post. Otherwise, based on the results of the finite element model analysis, for mesial or distal loads, the root fracture in the abutment teeth would be considered to be caused by absorption of the alveolar bone and the buccal aspect.

Thyroid-hormone-dependent negative regulation of thyrotropin beta gene by thyroid hormone receptors: study with a new experimental system using CV1 cells.

The molecular mechanism involved in the liganded thyroid hormone receptor suppression of the TSHbeta (thyroid-stimulating hormone beta, or thyrotropin beta) gene transcription is undetermined. One of the main reasons is the limitation of useful cell lines for the experiments. We have developed an assay system using non-pituitary CV1 cells and studied the negative regulation of the TSHbeta gene. In CV1 cells, the TSHbeta-CAT (chloramphenicol acetyltransferase) reporter was stimulated by Pit1 and GATA2 and suppressed by T3 (3,3',5-tri-iodothyronine)-bound thyroid hormone receptor. The suppression was dependent on the amounts of T3 and the receptor. Unliganded receptor did not stimulate TSHbeta activity, suggesting that the receptor itself is not an activator. Analyses using various receptor mutants revealed that the intact DNA-binding domain is crucial to the TSHbeta gene suppression. Co-activators and co-repressors are not necessarily essential, but are required for the full suppression of the TSHbeta gene. Among the three receptor isoforms, beta2 exhibited the strongest inhibition and its protein level was the most predominant in a thyrotroph cell line, TalphaT1, in Western blotting. The dominant-negative effects of various receptor mutants measured on the TSHbeta-CAT reporter were not simple mirror images of those in the positive regulation under physiological T3 concentration.

[A case report of removable partial denture with I-C attachment].

PATIENT: The alveolar bone and teeth for a patient involved in a traffic accident were prepared using removable partial dentures with I-C attachment. DISCUSSION: Because of using no clasp in the front teeth, sufficient supporting force of the dentures and no throbbing pain after insertion of the dentures, the patient was fully satisfied with the appearance and functional results of the treatment. CONCLUSION: In a case of loss of front teeth, the patient was content with the smaller and esthetically excellent attachment instead of a clasp. Moreover, the patient is progressing favorably. It is considered that the I-C attachment was effective for this case.

[Observations on three dimensional images for cracks of doweled teeth--comparison of images from specimen sections and dental tomograms from small three dimensional X-ray CT].

PURPOSE: Intraoral views of teeth with dowel and post hole taken by small three-dimensional X-ray CT (3DX) were compared with three-dimensional images from specimen sections of the same extracted teeth. This comparison shows the usefulness of 3DX for examination of cracked teeth in the oral cavity. MATERIALS AND METHODS: After taking dental tomographic images using 3DX for fractured teeth in the oral cavity, the fractured teeth were extracted and three-dimensional images for them and their cracks were obtained from a set of photographed sections. Then both sets of three-dimensional images for the fractured teeth were compared in terms of the form and region of the cracks. DISCUSSION: The tooth cracks were observed at the root face region in the intraoral view. Also, in the extracted teeth, fracture lines were recognized from the three-dimensional images. Moreover, a discontinuous image was obtained in teeth from the dental tomographic image using 3DX. This discontinuous image in teeth was observed in the same region and direction as the cracks of the three dimensional image from specimen sections of the extracted teeth. CONCLUSIONS: The discontinuous images of teeth in the dental tomographic images from 3DX were observed in the same region and direction as the cracks of teeth in the three-dimensional images from specimen sections of the extracted teeth. It was confirmed that dental tomographic images from 3DX are useful for finding cracks in living teeth. However, dental tomographic images from small three-dimensional X-ray CT are not perfectly reliable because the discontinuous image is not found in some teeth where the cracks are recognized by images from specimen sections after extraction.

Unliganded thyroid hormone receptor-beta1 represses liver X receptor alpha/oxysterol-dependent transactivation.

The thyroid hormone receptor (TR) and liver X receptor (LXR)-alpha are members of the nuclear hormone receptor family and are ligand-dependent transcription factors. Among the promoter target genes, TR and LXR recognize the T3 response element and LXR response element (LXRE), respectively. Because T3 response elements and LXREs have similar configurations, referred to as direct repeat 4, we investigated the possibility of cross-talk between the two ligand-dependent signal transduction pathways. We found that TRbeta1, a major isoform of TR in the liver, binds and transactivates LXREs derived from the mouse mammary tumor virus long-terminal repeat and the promoter of the sterol regulatory element binding protein 1c. Moreover, unliganded TRbeta1 suppresses promoter activity driven by LXRalpha and its ligand, whereas transactivation by T3-bound TRbeta1 is not affected by LXRalpha in the presence or absence of oxysterols. Gel shift, mammalian two-hybrid, and glutathione S-transferase pull-down assays demonstrated the direct binding of TRbeta1 to these LXREs and revealed that the interaction between TRbeta1 and corepressors is important to the unliganded TR-mediated suppression of LXRalpha-transactivation. Our findings suggest that T3 and TR influence lipid metabolism regulated by oxysterol/LXRalpha at the transcriptional level.

[Docetaxel and carboplatin for epithelial ovarian cancer].

We report herein on the efficacy and toxicity of docetaxel and carboplatin in patients with epithelial ovarian cancer. Fifteen patients with FIGO stage I c-IV epithelial ovarian cancer were administered docetaxel (70 mg/m2) and carboplatin (AUC 5) every 3 weeks as 1 course. Eleven patients received this regimen as a first-time chemotherapy, and the other 4 as therapy for recurrence. Seven patients were evaluated for response. Of these, 6 achieved complete response and the other a partial response. CA 125 response was seen in 2 of 8 patients who did not have visible tumors. Our toxicity findings include the following: grade 3 and 4 neutropenia (86.7%), hypersensitive reaction (13.3%), grade 2 alopecia (13.3%), and no edema. Docetaxel and carboplatin are actively used in ovarian cancer, with the major toxicity being bone marrow suppression. But we were able to control myelosuppression with G-CSF. Hypersensitivity reactions were frequent, we thought pre-medication. This chemotherapy combination appears effective for epithelial ovarian cancer.

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.

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.

The role of the amino-terminal domain in the interaction of unliganded peroxisome proliferator-activated receptor gamma-2 with nuclear receptor co-repressor.

Peroxisome proliferator-activated receptor gamma-2 (PPARG2) is a ligand-dependent transcriptional factor involved in the pathogenesis of insulin resistance. In the presence of a ligand, PPARG2 associates with co-activators, while it recruits co-repressors (CoRs) in the absence of a ligand. It has been reported that the interaction of liganded PPARG2 with co-activators is regulated by the amino-terminal A/B domain (NTD) via inter-domain communication. However, the role of the NTD is unknown in the case of the interaction between unliganded PPARG2 and CoRs. To elucidate this, total elimination of the influence of ligands is required, but the endogenous ligands of PPARG2 have not been fully defined. PPARG1-P467L, a naturally occurring mutant of PPARG1, was identified in a patient with severe insulin resistance. Reflecting its very low affinity for various ligands, this mutant does not have transcriptional activity in the PPAR response element, but exhibits dominant negative effects (DNEs) on liganded wild-type PPARG2-mediated transactivation. Using the corresponding PPARG2 mutant, PPARG2-P495L, we evaluated the role of the NTD in the interaction between unliganded PPARG2 and CoRs. Interestingly, the DNE of PPARG2-P495L was increased by the truncation of its NTD. NTD deletion also enhanced the DNE of a chimeric receptor, PT, in which the ligand-binding domain of PPARG2 was replaced with that of thyroid hormone receptor beta-1. Moreover, NTD deletion facilitated the in vitro binding of nuclear receptor CoR with wild-type PPARG2, mutant P495L, and the PT chimera (PPARG2-THRB). Inter-domain communication in PPARG2 regulates not only ligand-dependent transactivation but also ligand-independent silencing.

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.

Beta-catenin/Tcf-1-mediated transactivation of cyclin D1 promoter is negatively regulated by thyroid hormone.

Cyclin D1 is an oncogenic cyclin frequently over-expressed in cancer. To examine the effect of thyroid hormone (T3) and its receptor (TR) on the transcription of cyclin D1 gene, we co-transfected the chloramphenicol acetyl-transferase (CAT) reporter plasmid containing cyclin D1 promoter together with the expression plasmids for TRbeta1 and wild-type or mutant beta-catenin (SA) into 293T cells. In the presence of T3, beta-catenin-dependent transactivation of cyclin D1 promoter was suppressed by co-transfection of TRbeta1. The suppression by T3/TRbeta1 was in a dose-dependent manner. The CAT reporter gene in which Tcf/Lef-1 sites were fused to heterologous promoter was also suppressed by T3/TRbeta1. Furthermore, inhibition of endogenous wild-type beta-catenin by T3/TRbeta1 was observed in SW480 colon carcinoma cells with mutation of the adenomatous polyposis coli gene. These results indicate that the T3-bound TR inhibits the transcription of cyclin D1 through the Tcf/Lef-1 site, which is positively regulated by the Wnt-signaling pathway.