scholarly journals A Novel Thyroid Transcription Factor Is Essential for Thyrotropin-Induced Up-Regulation of Na+/I− Symporter Gene Expression

1998 ◽  
Vol 12 (5) ◽  
pp. 727-736 ◽  
Author(s):  
Masayuki Ohmori ◽  
Toyoshi Endo ◽  
Norikazu Harii ◽  
Toshimasa Onaya
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Mobility Shift ◽  
Thyroid Cells ◽  
Thyroid Transcription Factor 1 ◽  
Nuclear Extracts ◽  
Thyroid Transcription Factor ◽  
Nis Gene ◽  
Responsive Element ◽  
Gel Mobility Shift

Abstract The stimulation of iodide (I−) transport by TSH in FRTL-5 thyroid cells is partly due to an increase in Na+/I− symporter (NIS) gene expression. The identification of a TSH-responsive element (TRE) in the NIS promoter and its relationship to the action of thyroid transcription factor-1 (TTF-1) on the promoter are the subjects of this report. By transfecting NIS promoter-luciferase chimeric plasmids into FRTL-5 cells in the presence or absence of TSH, we identify a TRE between −420 and −370 bp of the NIS 5′-flanking region. Nuclear extracts from FRTL-5 cells cultured in the absence of TSH form two groups of protein-DNA complexes, A and B, in gel mobility shift assays using an oligonucleotide having the sequence from −420 to −385 bp. Only the A complex is increased by exposure of FRTL-5 cells to TSH or forskolin. The addition of TSH to FRTL-5 cells can increase the A complex at 3–6 h, reaching a maximum at 12 h. FRTL-5, but not nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cell nuclear extracts, form the A complex. The TSH-increased nuclear factor in FRTL-5 cells interacting with the NIS TRE is distinct from TTF-1, thyroid transcription factor-2, or Pax-8, as evidenced by the absence of competition using oligonucleotides specific for these factors in gel shift assays. Neither is it the nuclear protein interacting with cAMP response element. The TRE is in the upstream of a TTF-1-binding site,− 245 to −230 bp. Mutation of the TRE causing a loss of TSH responsiveness also decreases TTF-1-induced promoter activity in a transfection experiment. The formation of the A complex between FRTL-5 nuclear extracts and the NIS TRE is redox-regulated. In sum, TSH/cAMP-induced up-regulation of the NIS requires a novel thyroid transcription factor, which also appears to be involved in TTF-1-mediated thyroid-specific NIS gene expression.

scholarly journals Thyroglobulin Repression of Thyroid Transcription Factor 1 (TTF-1) Gene Expression Is Mediated by Decreased DNA Binding of Nuclear Factor I Proteins Which Control Constitutive TTF-1 Expression

2000 ◽  
Vol 20 (22) ◽  
pp. 8499-8512 ◽  
Author(s):  
Minoru Nakazato ◽  
Hyun-Kyung Chung ◽  
Luca Ulianich ◽  
Antonino Grassadonia ◽  
Koichi Suzuki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Nuclear Factor ◽  
Factor I ◽  
Thyroid Transcription Factor 1 ◽  
Nuclear Extracts ◽  
Thyroid Transcription Factor ◽  
Nuclear Factor I ◽  
Transcription Factor 1

ABSTRACT Follicular thyroglobulin (TG) selectively suppresses the expression of thyroid-restricted transcription factors, thereby altering the expression of thyroid-specific proteins. In this study, we investigated the molecular mechanism by which TG suppresses the prototypic thyroid-restricted transcription factor, thyroid transcription factor 1 (TTF-1), in rat FRTL-5 thyrocytes. We show that the region between bp −264 and −153 on the TTF-1 promoter contains two nuclear factor I (NFI) elements whose function is involved in TG-mediated suppression. Thus, NFI binding to these elements is critical for constitutive expression of TTF-1; TG decreases NFI binding to the NFI elements in association with TG repression. NFI is a family of transcription factors that is ubiquitously expressed and contributes to constitutive and cell-specific gene expression. In contrast to the contribution of NFI proteins to constitutive gene expression in other systems, we demonstrate that follicular TG transcriptionally represses all NFI RNAs (NFI-A, -B, -C, and -X) in association with decreased NFI binding and that the RNA levels decrease as early as 4 h after TG treatment. Although TG treatment for 48 h results in a decrease in NFI protein-DNA complexes measured in DNA mobility shift assays, NFI proteins are still detectable by Western analysis. We show, however, that the binding of all NFI proteins is redox regulated. Thus, diamide treatment of nuclear extracts strongly reduces the binding of NFI proteins, and the addition of higher concentrations of dithiothreitol to nuclear extracts from TG-treated cells restores NFI-DNA binding to levels in extracts from untreated cells. We conclude that NFI binding to two NFI elements, at bp −264 to −153, positively regulates TTF-1 expression and controls constitutive TTF-1 levels. TG mediates the repression of TTF-1 gene expression by decreasing NFI RNA and protein levels, as well as by altering the binding activity of NFI, which is redox controlled.

scholarly journals Modulation of Thyroid-Specific Gene Expression in Normal and Nodular Human Thyroid Tissues from Adults: An in Vivo Effect of Thyrotropin

2005 ◽  
Vol 90 (10) ◽  
pp. 5692-5697 ◽  
Author(s):  
Rocco Bruno ◽  
Elisabetta Ferretti ◽  
Emanuele Tosi ◽  
Franco Arturi ◽  
Paolo Giannasio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Human Thyroid ◽  
Normal Tissues ◽  
Thyroid Transcription Factor 1 ◽  
Thyroid Transcription Factor ◽  
Group 2 ◽  
Transcription Factor 1 ◽  
Tsh Levels

Context: Evidence from in vitro studies or animal models has shown that TSH affects thyrocytes by thyroid-specific expression modulation. Objective: The objective of our study was to analyze the role of TSH in human thyroid gene expression in vivo. Design/Setting: Thirty-nine normal thyroid tissues were collected at the same center. Study Subjects: Patients were divided into two groups based on serum TSH levels: 17 with normal TSH levels (1–4 mU/liter; group 1) and 22 with TSH levels below 0.5 mU/liter (group 2). Intervention: Group 2 underwent thyroidectomy after suppressive l-T4 therapy. Main Outcome Measures: mRNA levels of thyroid genes such as sodium/iodide symporter (NIS), apical iodide transporter, pendrin, thyroglobulin, thyroperoxidase, TSH receptor, paired box transcription factor 8, and thyroid transcription factor-1 were evaluated by quantitative PCR. Results: The reduction of TSH stimulation causes decreases in NIS and apical iodide transporter gene expression in normal tissues and more limited reductions in thyroglobulin, thyroperoxidase, and paired box transcription factor 8, but it has no significant effect on TSH receptor, pendrin, or thyroid transcription factor-1. Comparison of NIS levels in normal and nodular tissues from the same patient confirmed that it is differentially expressed in nodules only in the presence of normal TSH (P < 0.01). In patients with suppressed TSH, nodular NIS levels were similar to those in normal tissues. Conclusions: Our data represent the first demonstration in human thyroid tissues that TSH contributes to the regulation of thyrocyte differentiation by modulating thyroid gene levels. It exerts a particularly important effect on the transcription of NIS, which becomes very low after prolonged TSH suppression.

Disrupted co-ordination of Pax-8 and thyroid transcription factor-1 gene expression in a dedifferentiated rat thyroid tumour cell line derived from FRTL-5

1996 ◽  
Vol 150 (3) ◽  
pp. 377-382 ◽  
Author(s):  
C J H van der Kallen ◽  
D C J Spierings ◽  
J H H Thijssen ◽  
M A Blankenstein ◽  
T W A de Bruin
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Cell Line ◽  
Thyroid Peroxidase ◽  
Wild Type ◽  
Specific Expression ◽  
Thyroid Transcription Factor 1 ◽  
Thyroid Transcription Factor ◽  
Rat Thyroid ◽  
Transcription Factor 1

Abstract The mutant rat thyroid cell line FRTL-5/TA, isolated from a non-functional tumour which originated spontaneously from wild-type FRTL-5 cells, shows autonomous TSH-independent growth and loss of the thyroid-specific phenotype, lacking thyroid-specific expression of thyroglobulin (Tg) and thyroid peroxidase (TPO) genes. To investigate the role of the transcription factors Pax-8 and thyroid transcription factor-1 (TTF-1) in rat thyroid tumorigenesis, RNA expression of these two thyroid-specific nuclear factors was measured in FRTL-5/TA tumour cells and compared with the expression in wild-type FRTL-5 cells. TTF-1 gene expression was similar to that in wild-type FRTL-5, and showed a similar down-regulation after stimulation with TSH. The finding suggested normal TTF-1 mRNA and protein expression in both cell lines. By contrast, Pax-8 mRNA transcript signal was markedly reduced in FRTL-5/TA cells, reaching levels as low as 8% of the normal, basal level in FRTL-5 cells. These data indicated that the loss of thyroid-specific expression of Tg and TPO genes in FRTL-5/TA cells was not related to changes in TTF-1 gene expression but rather to reduced Pax-8 gene expression. It was concluded that a disruption of the co-ordinated expression of TTF-1 and Pax-8 is implicated in the loss of thyroid phenotype of FRTL-5/TA cells in terms of reduced Tg and TPO expression. Journal of Endocrinology (1996) 150, 377–382

Association of SMAD3/4, COUP-Tf, HNF4α, C/EBPα, GATA2 and STAT5 to the Hamp1 Promoter

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3837-3837
Author(s):  
Pauline Lee ◽  
Jaroslav Truksa ◽  
Ernest Beutler
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Nuclear Extract ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Bzip Transcription Factor ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Genes Encoding ◽  
Responsive Element

Abstract There are two regions of the murine Hamp1 promoter that have been shown to be critical for Hamp1 expression. The 260 bp proximal region and the distal −1.6 to −1.8 Kb regions appear to be required for responsiveness to IL-6, BMPs and iron. Analyses of 160 bp proximal promoter for consensus transcription factor motifs by MatInspector identified a STAT5 site at the location identified previously by Wrighting et al., Blood 2006, as a functional STAT3 site and by Courselaud et al., J Biol Chem 2002, as a C/EBPα site. Although a SMAD responsive site was not predicted in this region, we (in press), and Verga-Falzacappa et al., J Mol Med 2008, have demonstrated that there is a functional BMP responsive element (GGCGCC) in this region. A probe encompassing the putative BMP-RE1, STAT, C/EBPα, and AP1 motifs were used in electrophoretic mobility shift assays (EMSA). We found that the addition of cold competitor DNA corresponding to STAT3, C/EBPα and AP1 consensus motifs did not block the binding of transcription factors from liver nuclear extracts to the BMP-RE1/STAT/C/EBPα/AP1 probe. In contrast, the addition of cold competitor DNA corresponding the SMAD3/4 or STAT5 completely blocked essentially all binding of liver nuclear transcription factors to the BMP-RE1/STAT/C/EBPα/AP1 probe. Analyses of the −161 to −260 bp proximal promoter for consensus transcription factor motifs identified a GATA2 binding site and a SMAD responsive site (TGTCTGCCC). Two long probes encompassing the to −161 to −260 bp region were used in EMSAs. Binding of liver nuclear extracts to a probe encompassing the GATA motif was blocked by the addition of a GATA consensus DNA. Similarly, binding to a long probe encompassing the SMAD responsive site was blocked by the addition of a SMAD3/4 consensus DNA. Analyses of the 1.6 to 1.7 Kb region of the distal murine Hamp1 promoter identified several transcription factor motifs: bZIP transcription factor that acts on nuclear genes encoding mitochondrial proteins, COUP-Tf/HNF4α, and MEL1 (MDS1/EVI1-like gene1) to be both in human and mouse Hamp genes. Although a SMAD responsive site was not identified in this region, we have demonstrated that there is a functional BMP responsive element (GGCGCC) in this region. Using EMSA with probes corresponding to the −1.6 to −1.7 bp region of the hepcidin promoter, we examined the binding of transcription factors from liver nuclear extracts derived from mice. Binding of liver nuclear extract to a probe corresponding to the BMP-RE2, bZIP, HNF4α, COUP motifs was blocked by cold competitor probes corresponding to SMAD3/4, HNF4α, COUP-Tf, and Stat5. Whereas competitor probes to Smad3/4 and HNF4α competed for the binding of specific bands to the radiolabelled probe, total binding was blocked with cold competitor probes to the consensus COUP-Tf and Stat5 motifs. Supershift analyses using antibodies to HNF4α, COUP, SMAD4 demonstrated the binding of these transcription factors to the radiolabeled BMP-RE2/bZIP/HNF4α/COUP probe. Binding to a probe encompassing a MEL motif was blocked by the addition of cold competitor to C/EBPα and could be supershifted with antibodies against C/EBPα. In conclusion, SMAD 3/4, COUP-Tf, HNF4α, C/EBPα, GATA2 and STAT5 appear to be important in the regulation of Hamp1 expression.

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