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

Masayuki Ohmori, Toyoshi Endo, Norikazu Harii and Toshimasa Onaya

Third Department of Internal Medicine Yamanashi Medical University Tamaho, Yamanashi 409–38, Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TSH controls the function and growth of the thyroid in a cell cycle-dependent manner (1, 2, 3). When TSH is added to FRTL-5 thyroid cells that are maintained in medium with no TSH, the cAMP signal induces an increase in genes with products important for the function of the thyroid during the first 24 h: thyroid peroxidase (TPO), thyroglobulin (TG), and the iodide (I-) transporter (3).

I- transport into the thyroid is the first and main rate-limiting step in the biosynthesis of the thyroid hormones, T3 and T4 (4). The regulation of I- transport by TSH has been widely reported (4, 5, 6, 7). Halmi et al. (5) observed that a single injection of TSH into rats caused a 50–100% increase in thyroid I- accumulation after 8 h, as measured by the thyroid/serum radioiodide gradient. Similar experiment in isolated bovine thyroid cells also showed 50–100% stimulation of I- accumulation after the addition of TSH or (Bu)2cAMP (6). Weiss et al. (7) examined the effect of TSH on I- transport using FRTL-5 cells instead of isolated cells. In their experiments, FRTL-5 cells were subjected to TSH withdrawal. Readdition of (Bu)2cAMP or agents that increase intracellular levels of cAMP as well as TSH to the medium restored I- accumulation after a latency of 12–24 h (7). At present it is widely believed that I- is cotransported with Na+ by a membrane carrier, Na+/I- symporter (NIS), into the thyroid cells (4, 8). Dai et al. (9) succeeded in the cloning of the rat NIS cDNA (9). This opened the way to analyze the mechanisms of TSH regulation of I- transport at the molecular level, and we reported that the stimulation of I- transport activity by TSH in FRTL-5 cells is partly due to an increase in NIS gene expression (10).

We recently sequenced 1.9 kb of 5'-flanking region of the rat NIS gene and showed it exhibits the cell type-selective expression in FRTL-5 thyroid cells (11). One regulatory element defined therein, -245 to -230 bp, binds thyroid transcription factor-1 (TTF-1), a homeodomain-containing, DNA-binding protein essential for tissue-specific expression of the TG, TPO, and TSH receptor (TSHR) (12, 13, 14, 15, 16, 17). The TTF-1 element contributes to cell type-selective NIS gene expression (11).

The present study identifies a TSH-responsive element (TRE) upstream of the TTF-1 site in the NIS promoter and establishes a relationship between the promoter element important for TSH regulation and that for FRTL-5-specific expression of the NIS gene. A nuclear factor interacting with the NIS TRE is FRTL-5 cell-specific, but distinct from TTF-1, thyroid transcription factor-2 (TTF-2) (18), or Pax-8 (19), all of which are involved in thyroid-specific regulation of the TG and TPO genes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization of a TSH-Responsive Element between -420 and -370 bp in the Rat NIS Promoter
In our initial study, we showed that TSH up-regulates NIS gene expression in FRTL-5 cells (10). Thus, the addition of TSH to FRTL-5 cells causes an increase in NIS mRNA levels within 6–12 h. The ability of TSH to increase NIS mRNA levels is transcriptional (A. Kawaguchi and T. Onaya, unpublished observations). In contrast, withdrawal of TSH from FRTL-5 cells results in a gradual return of NIS mRNA levels over a 6- to 7-day period. Since transient transfection into FRTL-5 cells by electroporation requires the presence of TSH in the medium for optimal growth and viability (16), the promoter activity assessed in cells that are cultured in the absence of TSH for the last 48 h after initial 12-h incubation with TSH is assumed to be enhanced almost maximally. We cannot demonstrate the effect of TSH on the NIS promoter activity in a transient transfection system. To estimate TSH effect on the 1.9-kb promoter and the deletions thereof in FRTL-5 cells, therefore, we established stable transfectants of the NIS promoter-luciferase chimeric plasmids. For each construct, more than 20 G418-resistant colonies were pooled to minimize possible position effects. These stable transfectants were cultured in the presence or absence of 10 mU/ml TSH for 6–7 days. Since we did not assess the copy number of the chimeric plasmid integrated into the FRTL-5 genome, we could not directly compare the luciferase activity expressed in one stable transfectant to the others. It is reasonable, however, to compare the luciferase activity expressed in the presence of TSH to that in the absence of TSH within the same stable transfectant. Figure 1BGo shows the fold increase of the luciferase activity in the presence vs. in the absence of TSH.



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Figure 1. Effect of TSH on the Luciferase Activity of Chimeric Plasmids Containing 5'-Deletions of the 5'-Flanking Region of NIS Gene

A, Regulatory elements of the NIS promoter (11) are diagrammatically presented. The putative TRE between -420 and -385 bp (dark bar) is approximately 170 bp upstream of the TTF-1-binding site. B, The deletions and their chimeric luciferase plasmids are as follows: -1968 to -1128 [p(-1128)], to -590 [p(-590)], to -420 [p(-420)], to -370 [p(-370)], to -320 (p(-320)), to -210 [p(-210)]. FRTL-5 cells stably transfected with the noted plasmids were maintained either in 6H or in 5H (6H minus TSH) medium for the last 6 to 7 days of culture. Luciferase activities of the plasmids are shown as the ratio of activity in cells with or without TSH, [TSH (+)/TSH (-)], and presented as the mean ± SE for three separate experiments. A statistically significant increase induced by TSH is noted by an asterisk.

 
TSH significantly stimulates the luciferase activity of a 1968-bp rat NIS promoter chimera, p(-1968) (Fig. 1BGo). 5'-Deletion mutants between -1968 and -420 bp retain TSH responsiveness, approximately 2- to 3-fold higher than in cells without TSH (Fig. 1BGo). The TSH response is lost with p(-370), p(-320), and p(-210), approximately 0.5- to 0.7-fold (Fig. 1BGo). These results suggest a TRE exists between -420 and -370 bp of the rat NIS promoter (Fig. 1AGo).

A TSH-Increased Protein-DNA Complex Interacts with the NIS TRE
Gel mobility shift experiments were performed using the double-stranded, synthetic oligonucleotides, -420 to -385 bp and -395 to -360 bp of the rat NIS, containing the putative TRE sequence, as radiolabeled probes. The oligonucleotide, -420 to -385 bp, formed multiple protein-DNA complexes with nuclear extracts from FRTL-5 cells cultured with no TSH (Fig. 2AGo). The protein-DNA complexes could be divided into two groups based on the influence of TSH; group A was increased in nuclear extracts from FRTL-5 cells exposed to TSH for 6 days, while group B was not (Fig. 2AGo). The oligonucleotide, -395 to -360 bp, did not form a specific protein-DNA complex with nuclear extracts from FRTL-5 cells maintained in the absence or presence of TSH (Fig. 2AGo). The TSH effect is cAMP mediated, since it is duplicated by forskolin (Fig. 2BGo), as well as cholera toxin or (Bu)2cAMP (data not shown). The TSH/cAMP-induced increase in the formation of the A complex in FRTL-5 cells mimics the TSH/cAMP-induced increase in NIS gene expression (10). Thus, the formation of the protein-DNA complex was increased after 3 h of TSH stimulation, slowly growing, and reaching a maximum after 12 h (Fig. 2CGo). In sum, the NIS TRE is likely to be between -420 and -385 bp, and TSH/cAMP increases the formation of one of the protein-DNA complexes when an oligonucleotide encompassing the TRE is used in gel mobility shift assays with nuclear extracts from FRTL-5 cells.



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Figure 2. Ability of an Oligonucleotide Containing the NIS TRE to Form Protein-DNA Complexes with Nuclear Extracts from FRTL-5 Cells as Assayed in Gel Mobility Shift Analyses

A, A radiolabeled oligonucleotide spanning -420 to -385 bp or -395 to -360 bp of NIS was incubated with nuclear extracts from FRTL-5 cells that had been maintained in medium with (+) or without (-) TSH for the last 6 days of culture. B, The radiolabeled oligonucleotide, -420 to -385 bp of NIS, was incubated with the noted nuclear extracts. Nuclear extracts were prepared from FRTL-5 cells cultured in 5H medium (None), 5H medium plus 10 mU/ml TSH (+TSH), or 5H medium plus 10 µM forskolin (+FSK) for 6 days. C, The radiolabeled oligonucleotide, -420 to -385 bp of NIS, was incubated with the nuclear extracts from FRTL-5 cells. Nuclear extracts were prepared from cells that were cultured in 5H medium for 7 days and then treated with 10 mU/ml TSH for the noted hours.

 
The Nuclear Factor Interacting with NIS TRE Is FRTL-5-Specific and Distinct from TTF-1, TTF-2, or Pax-8
To characterize the nuclear factor in FRTL-5 thyroid cells whose DNA- binding activity was increased by TSH, we performed gel mobility shift experiments using synthetic oligonucleotide spanning -420 to -385 bp, oligo NIS-TRE, and nuclear extracts from FRTL-5 cells cultured with (6H) or without (5H) TSH, from nonfunctioning FRT thyroid cells, and from Buffalo rat liver (BRL) cells (Fig. 3AGo). The protein-DNA complex A formed with nuclear extracts from FRTL-5 cells was specific, as evidenced by self-competition with the homologous unlabeled oligonucleotide (Fig. 3Go, B and C). The TSH-induced protein-DNA complex A was FRTL-5 cell-specific (Fig. 3AGo, solid arrow). Thus, nuclear extracts from FRTL-5 cells, but not from FRT or BRL cells, formed a protein-DNA complex A (Fig. 3AGo). We tentatively termed the protein, NIS TSH-responsive factor-1 or NTF-1, in accord with its proposed characteristics. Unlike the protein-DNA complex A, B complexes appear to be more ubiquitously expressed, since they are seen in nuclear extracts from FRT and BRL cells (Fig. 3AGo, lanes 3 and 4 vs. 1 and 2). The formation of the B complexes is not similarly prevented, as a function of concentration, by the homologous unlabeled oligonucleotide (data not shown). The nature of the A complexes are, therefore, characterized as follows.



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Figure 3. Analysis by EMSA of the Nuclear Factor in FRTL-5 Cells Whose DNA- Binding Activity Is Increased by TSH

A, The oligonucleotide spanning -420 to -385 bp of NIS, oligo NIS TRE, was incubated with nuclear extracts from FRTL-5 cells maintained with (6H) or without (5H) TSH, from FRT cells, or from BRL cells. The position of the TSH-increased protein/NIS DNA complex A, NTF-1 complex, is indicated by a solid arrow. The small arrows depict the other B complexes, which exist in FRT and BRL as well as FRTL-5 cells and are not increased by TSH. B, The TSH-increased protein/DNA complex A, NTF-1 complex, formed by the oligo-NIS TRE and nuclear extracts from FRTL-5 cells cultured in 6H medium, was competed by increasing amounts of the unlabeled oligo-NIS TRE, C, DS, or K. The amount of each unlabeled oligonucleotide present in each assay, in fold excess over probe, is noted. C, NTF-1 complex between the oligo-NIS TRE and nuclear extracts from FRTL-5 cells cultured in 6H medium was competed by increasing amounts of the unlabeled oligonucleotides NIS TRE and CRE at the noted fold excess over probe.

 
The NTF-1/NIS DNA complex (complex A) formed between the FRTL-5 cell extracts and radiolabeled oligo NIS TRE is competed by the homologous unlabeled oligonucleotide (Fig. 3BGo, lanes 2 and 3 vs. 1), but not at all by a 250-fold excess of the oligonucleotides containing the TTF-1-/Pax-8-binding site on the TG promoter, the TTF-1-binding site on the TSHR promoter, or the TTF-2-binding site on the TG promoter: oligo C (Fig. 3BGo, lanes 4 and 5 vs. 1), oligo DS (Fig. 3BGo, lanes 6 and 7 vs. 1), and oligo K (Fig. 3BGo, lanes 8 and 9 vs. 1), respectively (12, 13, 14, 15, 16, 17, 18, 19). Additionally, formation of the NTF-1/NIS DNA complex was not prevented by the oligonucleotide CRE (cAMP response element) (Promega, Madison, WI), which contains the CRE from the somatostatin gene (Fig. 3CGo, lanes 4 and 5 vs. 1, 2, and 3). These results indicate that the NTF-1 complex does not involve TTF-1, TTF-2, Pax-8, or CRE-binding protein (CREB)/ATF family.

Methylation Interference Analyses Identify the Nucleotides on the NIS Promoter Interacting with NTF-1
To define the binding site of NTF-1, the contacts with purines on both strands of the oligonucleotide spanning -420 to -385 bp, oligo-NIS TRE, were determined by methylation interference (Fig. 4AGo). The dimethyl sulfate-modified, double-stranded oligonucleotide spanning -420 to -385 bp labeled in either the coding or noncoding strand was incubated with nuclear extracts from FRTL-5 cells maintained for 7 days with TSH. NTF-1-DNA complex and free DNA were eluted from the gel, cleaved at the modified residues, and resolved by electrophoresis (Fig. 4AGo). As summarized in Fig. 4BGo, NTF-1-binding was inhibited by methylation of nucleotides at -410, -406, -405, -404, and -402 bp on the coding strand (Fig. 4BGo, top line, open circles) and by methylation of nucleotide at -407 bp on the noncoding strand (Fig. 4BGo, bottom line, open circle). Methylation of nucleotides at -413, -412, -411, -403, and -396 bp on the coding strand, and at -409, -408, and between -401 through -397 bp on the noncoding strand, did not alter the NTF-1-binding. Thus, methylation interference identified the nucleotides at -410, -402, and between -407 through -404 bp as contact points.



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Figure 4. Methylation Interference Analyses of the Complex Formed between NTF-1 in FRTL-5 Cell Extracts and the Oligonucleotide Spanning -420 to -385 bp

A, The dimethyl sulfate-modified, double-stranded oligonucleotide spanning -420 to -385 bp 32P-labeled in either the coding (left) or noncoding (right) strand was incubated with 20 µg nuclear extract from FRTL-5 cells maintained for 7 days with TSH. Protein-DNA complexes were resolved on a preparative native gel as described in Materials and Methods. The bands corresponding to the NTF-1 complex and free DNA were eluted from the gel and cleaved at the modified residues. The cleavage products were resolved by electrophoresis through an 8% denaturing gel and visualized by a Bas 2000 Image Analyzer (Fuji Film Co., Tokyo, Japan). The sequence of the bases is indicated at the right; numbering of nucleotides is relative to the ATG start codon which is defined as +1. Open circles define the bases whose modification reduces the proportion of DNA in the bound fraction; X defines bases whose methylation does not alter the protein binding. B, The sequence of both strands are summarized, together with those bases whose methylation interferes with the binding of NTF-1. Open circles define the bases whose modification reduces the proportion of DNA in the bound fraction; X defines bases whose methylation does not alter protein binding.

 
Mutation Data Indicate the TSH-Increased NTF-1 Complex Is Related to TSH-Increased NIS Promoter Activity
To determine the functional relevance of the NTF-1 element, we mutated both in the p(-420) chimeric construct and in the oligonucleotide used for electrophoretic mobility shift assays (EMSAs) (Fig. 5AGo). The mutation (MT) changes four of six residues of the NTF-1-binding site that were identified as contact points by methylation interference (Fig. 5AGo, open circles, vs. Fig. 4Go). The NTF-1/NIS DNA complex formed by the radiolabeled wild-type (WT) oligonucleotide spanning -420 to -385 bp and FRTL-5 cell extracts is increased by TSH treatment of FRTL-5 cells, as previously shown (Fig. 5BGo, lanes 1 and 2, vs. Figs. 2AGo and 3AGo). The complex is no longer evident when the radiolabeled mutant oligonucleotide, MT, is a probe (Fig. 5BGo, lanes 3 vs. 2). The NTF-1/NIS DNA complex formed between the radiolabeled WT oligonucleotide and nuclear extracts from FRTL-5 cells maintained with TSH is competed by the homologous unlabeled oligonucleotide WT (Fig. 5CGo, lanes 2 and 3 vs. 1), but not at all by a 100-fold excess of the mutant oligonucleotide MT (Fig. 5CGo, lanes 4 and 5 vs. 1). Mutation in p(-420) that matched the MT sequence between -420 and -385 bp, p(-420MT), was stably transfected into FRTL-5 cells, as in the case of p(-420). p(-420MT) lost TSH- (data not shown) or forskolin-responsiveness (Fig. 5DGo), whereas p(-420) exhibited both TSH (Fig. 1BGo) and forskolin responsiveness (Fig. 5DGo). These results clearly link the TSH/forskolin-increased NTF-1 complex formation and TSH/forskolin responsiveness of the NIS promoter.



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Figure 5. Effect of Mutations in the NTF-1-Binding Site on the Promoter Activity of NIS Promoter-Luciferase Chimeras Stably Expressed in FRTL-5 Cells

A, Mutations are denoted by comparison to the sequence of WT promoter and by comparison to bases at which methylation interferes with the binding of NTF-1 on both strands (open circles). Mutations are noted as coding strand changes. B, The oligonucleotide spanning -420 to -385 bp (NIS TRE WT), or its mutated counterpart (MT), were, respectively, used as the radiolabeled probe. Each was incubated with nuclear extracts from FRTL-5 cells maintained for 7 days with (6H) or without (5H) TSH. C, The radiolabeled WT oligonucleotide spanning -420 to -385 bp of NIS (WT) was used as a probe and incubated with nuclear extracts from FRTL-5 cells cultured for 7 days with TSH (6H). Increasing amounts of the unlabeled homologous oligonucleotide (WT) or its mutated counterpart (MT) were used as a competitor at the noted fold excess over probe. D, FRTL-5 cells were stably transfected with p(-420) or p(-420MT) containing mutations in the NTF-1 site. Luciferase activities are presented as the ratio of activity in the presence or absence of 10 µM forskolin in the medium for 7 days [FSK (+)/FSK (-)]. Values are presented as the mean ± SE from three separate experiments. One asterisk (*) denotes a statistically significant increase relative to the activity of p(-420MT) or pGL2-Basic.

 
NTF-1 Is Redox-Regulated
Recent investigation (20) suggested that thyroid transcription factors, TTF-1 and Pax-8, could be redox regulated and that redox regulation increased the binding of these transcription factors to their response elements in the rat TG gene. We therefore asked whether redox regulation of NTF-1 would increase the NTF-1 interaction with the NIS TRE. The effect of redox regulation of NTF-1 on its binding to the NIS TRE was evaluated in EMSA using the synthetic oligonucleotide spanning -420 to -385 bp (Fig. 6Go). The NTF-1/NIS DNA complex was not seen or was negligible when the nuclear extracts from FRTL-5 cells cultured in medium with (6H, Fig. 6Go, lanes 1 and 2) or without TSH (5H, data not shown) were incubated in an EMSA reaction buffer without dithiothreitol (DTT) or in the reaction buffer containing 0.1 mM DTT. The addition of 0.5 mM or 1 mM DTT into the reaction buffer increased NTF-1/NIS DNA complex formation (Fig. 6Go, lanes 3 and 4 vs. 1 and 2). Subsequent treatment with 1 mM diamide, which reversibly oxidizes free sulfhydryls (21), totally abolished the complex formation (Fig. 6Go, lanes 5 and 6 vs. 3 and 4). The effect of DTT and/or diamide on the DNA-binding activity was NTF-1-specific, since the addition of DTT and/or diamide did not alter the formation of the B complexes (data not shown). These results suggest that free sulfhydryl(s) are important for NTF-1 binding in vitro.



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Figure 6. Redox Regulation of NTF-1-Binding Activity

A radiolabeled oligonucleotide spanning -420 to -385 bp of NIS gene was incubated with nuclear extracts from FRTL-5 cells. Nuclear extracts from cells cultured with TSH in the medium for 7 days were incubated with the probe in the assay buffer containing the noted amounts of dithiothreitol (DTT) and/or 1 mM diamide.

 
TTF-1 Activity in the NIS Promoter Is Related to the Activity of the TRE
To determine whether the TTF-1 site downstream of the TRE (11) was functionally related to the NIS TRE, we transfected a TTF-1 expression vector, RcCMV-THA (13), or its control plasmid, pRc/CMV, into FRTL-5 cells stably expressing NIS promoter-luciferase chimeras containing the intact TRE sequence or mutations of the TRE, p(-420) and p(-420MT), respectively (Fig. 7Go). p(-420) has both the intact TRE sequence and the intact TTF-1 site; p(-420MT) has the mutated TRE sequence and the intact TTF-1 element. Transfection into FRTL-5 cells by electroporation was performed in the condition of 6H medium, and the endogeneous TTF-1 in FRTL-5 cells was maximally suppressed (16, 17). Cotransfection of the TTF-1 expression vector significantly (P < 0.05) stimulated the activity of p(-420MT) compared with that in the pGL2-Basic (Fig. 7Go). More importantly, however, cotransfection of TTF-1 increased the activity of p(-420), which has both the intact TRE and TTF-1 site, significantly (P < 0.05) more (2.0-fold) than the activity of p(-420MT), which has only the TTF-1 site and was increased only 1.4-fold (Fig. 7Go). A functional TRE in the NIS is, therefore, required for the full activation by TTF-1.



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Figure 7. Effect of Mutations in the NTF-1-Binding Site on the Ability of TTF-1 to Stimulate NIS Promoter Activity in FRTL-5 Cells

Mutations are identical to those in Fig. 5Go. FRTL-5 cells stably expressing p(-420), p(-420MT), or pGL2-Basic were transfected with a TTF-1 expression vector, RcCMV-THA (13), or its control plasmid, pRc/CMV. The cells were also cotransfected with pCH110-ß-gal, and luciferase activities were normalized to ß-gal levels. Luciferase activities of the plasmids are shown as the ratio of activity in cells transfected with RcCMV-THA against those with pRc/CMV, [TTF-1 (+)/TTF-1 (-)], and presented as the mean ± SE for three separate experiments. One asterisk (*) denotes a statistically significant increase relative to the activity of pGL2-Basic but a significant decreased level of activity by comparison to that of p(-420); two asterisks (**) denotes a statistically increase relative to the activity of p(-420MT).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
I- transport is a highly specialized process in the thyroid gland (4). Since iodine is an essential constituent of the thyroid hormones, T4 and T3, the I- concentrating mechanism of the thyroid is of considerable physiological importance: it serves as a highly specific and efficient supply route of I- into the gland (4). The active transport of I- at the basolateral plasma membrane against an electrochemical gradient is the initial and rate-limiting step in the biosynthesis of the thyroid hormones (4). These hormones, in turn, are of major significance for the intermediary metabolism of virtually all tissues, and they play an essential role in the growth and maturation of the nervous system of the developing fetus and the newborn (22, 23). The Na+/I- symporter (NIS) model, in which I- is cotransported with Na+ by a membrane carrier into the cell, was proposed and confirmed by several studies carried out in the intact thyroid gland, thyroid slices, primary cell culture systems, and FRTL-5 cells (4, 5, 6, 7, 8, 24, 25, 26).

The cDNA encoding the NIS was recently cloned as a result of functional screening of a FRTL-5 cDNA library in Xenopus laevis oocytes (9). We have reported that autoantibody against NIS frequently exists in the sera of patients with autoimmune thyroid disease and that the autoantibody in the sera from Hashimoto’s thyroiditis possesses I- transport-inhibitory activity (27, 28). Thus, autoantibodies to the NIS could contribute to the pathogenesis of autoimmune thyroid disease by inhibiting one of the primary functions of the gland (28). We have separately shown that TSH increases NIS mRNA and protein levels in FRTL-5 cells (10). To clarify the molecular mechanisms in TSH regulation of NIS gene expression, we recently sequenced 1.9 kb of the 5'-flanking region of the rat NIS (11). In the present report, we performed deletion analyses of the 5'-flanking region of the NIS gene and identified an element, -420 to -385 bp of the NIS 5'-flanking region, which confers the positive stimulatory effect of TSH on the NIS promoter. This site has properties consistent with its being the functionally relevant TRE in the NIS promoter. Thus, the TSH-induced increase in protein/DNA complex between the TRE oligonucleotide and FRTL-5 cell nuclear extract is similar to the TSH-induced increase in NIS mRNA levels (10). The TRE is upstream of the minimal NIS promoter, -291 to -36 bp, which was tentatively defined and exhibits the cell type-selective expression of NIS gene (11). We show that mutation of the TRE sequence results in the decrease in the TTF-1-induced transactivation as well as in the loss of TSH responsiveness of the NIS promoter. We would, therefore, suggest that the minimal NIS promoter, which has properties of cell type-selective expression and TSH responsiveness of the NIS gene in FRTL-5 thyroid cells, is between -420 and -36 bp of 5'-flanking region.

In the present study, TSH stimulates the promoter activity of a 1968-bp rat NIS promoter chimera and 5'-deletion mutants between -1968 to -420 bp approximately 2- to 3-fold higher than in cells without TSH. We previously showed that TSH increases NIS mRNA approximately 5- to 6-fold over basal levels (10). One possible explanation for this discrepancy between promoter and mRNA is the upstream region of the 1968-bp 5'-flanking region. Thus, the nuclear factor(s) interacting with the upstream region may be involved in a part of TSH-responsiveness, or the upsream site may be necessary for full activity of the TSH-increased transcription factor characterized herein.

To the best of our knowledge, this is the first report describing a TRE and a nuclear factor interacting with the TRE in thyroid-specific proteins, using deletion analyses of the 5'-flanking region and gel mobility shift assays. We demonstrate a protein whose ability to form a complex with the TRE is increased by TSH. We establish that this is functionally relevant by showing that mutations of the NIS TRE, which eliminate its ability to form the complex, also abolish the ability of TSH to stimulate promoter activity. We also show, most importantly, that the TSH-increased FRTL-5-specific nuclear protein interacting with the NIS TRE is distinct from TTF-1, TTF-2, and Pax-8, which are thyroid-specific transcription factors previously identified (12, 13, 14, 15, 16, 17, 18, 19); oligonucleotides C, DS, or K do not compete for the protein interacting with the NIS TRE (Fig. 3BGo) and vice versa (data not shown). We tentatively term the protein, NIS TSH-responsive factor-1 or NTF-1. The cis element determined as contact points by methylation interference contains a G-rich region, GNNCGGANG, -410 to -402 bp, with homology to the Ets family consensus sequence, GAGGAA (29). However, an oligonucleotide containing Ets family consensus sequence does not prevent formation of the protein-DNA complex involving the NIS TRE and TSH-treated FRTL-5 cell extracts at a 100-fold excess over probe (data not shown). Further, the oligonucleotide with the sequence of the consensus cAMP response element does not compete for the protein interacting with the NIS TRE (Fig. 3CGo). These suggest that proteins interacting with these sequences do not interact with the NIS TRE and are distinct from NTF-1. The NIS TRE appears, therefore, to be a novel cis element whose identification may have broader significance.

There is controversy as to which nuclear proteins and DNA elements are involved in TSH regulation of thyroid-specific genes. TSH regulation of thyroid-specific genes, TSHR, TG, and TPO, has been studied as follows. TSHR is autoregulated by TSH and its cAMP signal, which increase and then decrease TSHR gene expression as a function of time (30). Kohn and co-workers (16, 17) reported that TSH regulation of TTF-1 is involved in TSH-positive and -negative autoregulation of the TSHR gene. Within the first 2 h after TSH is given to FRTL-5 cells maintained without TSH, TSH-induced, protein kinase A-mediated TTF-1 phosphorylation results in increased binding of TTF-1 to the TSHR DNA in association with increased TSHR gene expression. After 2 h, there is a TSH-induced decrease in TTF-1 mRNA levels and in TTF-1/TSHR DNA complex formation, which is coincident with TSH-induced down-regulation of the TSHR gene (16, 17). They also reported that TSH decreases the mRNA levels and DNA-binding activity of a single-strand DNA-binding protein (SSBP), thereby decreasing TSHR gene expression (31, 32). Lalli and Sassone-Corsi (33) showed that TSH-directed induction of the cAMP response element modulator (CREM) isoform participates in the down-regulation of the TSHR. Saiardi et al. (34) showed that TSH-induced down-regulation of the TSHR is mediated by thyroid hormone receptor (TR{alpha}1). TSH up-regulates TG and TPO gene expressions at the transcriptional levels (35, 36). TSH/cAMP inducibility of TG and TPO genes has been shown to be dependent on TTF-1, whose DNA-binding activity is stimulated by phosphorylation by protin kinase A (37, 38). Kambe et al. (20) reported that the redox regulation of Pax-8 and TTF-1 by TSH is involved in their increased DNA-binding activities and in an increase in TG promoter activity. It is likely that none of the above studies identified a TRE of each thyroid-specific gene because of the different strategy. In the present study we showed that a novel thyroid transcription factor is involved in the TSH-induced up-regulation of NIS gene.

The NIS TRE exists about 170 bp upstream of TTF-1 site, -245 to -230 bp, which confers the cell-type-selective expression of the NIS gene (11). Most importantly, the NIS TRE has properties that relate it to the TTF-1-mediated cell type-selective expression of the NIS gene. Thus, we show that TTF-1 stimulates NIS promoter activity in the presence of the intact TRE, more than that in the absence of the TRE. This links cell type-selective-TSH-increased regulation of the NIS gene. Definitive interactions of NTF-1 with TTF-1, TTF-2, Pax-8, or other transcription factors must await cloning of NTF-1 interacting with the NIS TRE, which we have characterized in this report. Similarly, the role of NTF-1 in non-thyroid-specific genes is incompletely defined and requires further investigation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Bovine TSH is a highly purified preparation obtained from Sigma (St. Louis, MO). [{gamma}-32P]ATP (3000 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). A TTF-1 expression vector, RcCMV-THA (13) was kindly donated by Dr. R. Di Lauro (Naples, Italy). The sources of other materials were detailed previously (11, 32).

Promoter-LUC Chimeric Plasmids
Genomic sequence of NIS gene from -1968 to -36 bp was cloned into SacI–BglII sites of plasmid pGL2-Basic (Promega, Madison, WI); this luciferase (LUC) construct is designated p(-1968) (11). p(-1128) was obtained by internal deletion of p(-1968). Genomic sequences from -590 to -36, -420 to -36, -370 to -36, -320 to -36, and -210 to -36 bp were amplified by PCR using pBluescript plasmid containing the 15-kb fragment including the rat NIS 5'-flanking sequence (11) together with forward and reverse primers containing a 5'-adaptor sequence (KpnI or BglII) to facilitate directional cloning. PCR products were cloned into KpnI–BglII sites of plasmid pGL2-Basic; these LUC constructs are designated p(-590), p(-420), p(-370), p(-320), and p(-210), respectively. Cloned inserts were sequenced in their entirety to ensure the PCR-generated misincorporations. To generate a chimera containing mutations in the sequence, p(-420MT), objective promoter segments with mutation (Fig. 5AGo) were generated by PCR using a forward primer that had the mutated sequence with KpnI site on the 5'-end and the reverse primer described above. All plasmids were prepared using QIAGEN Plasmid Maxi Kit (Qiagen, Chatsworth, CA).

Cells
FRTL-5 [CRL 8305, American Type Culture Collection (ATCC), Rockville, MD (39)] and FRT rat thyroid cells (40) were grown in Coon’s modified Ham’s F-12 supplemented with 5% calf serum (GIBCO BRL, Grand Island, NY). The FRTL-5 cell medium includes a six-hormone mixture (6H medium), containing bovine TSH (10 mU/ml), insulin (1.3 x 10-6 M), cortisol (10-6 M), transferrin (6.3 x 10-11 M), glycyl-L-histidyl-L-lysine acetate (2.5 x 10-6 M), and somatostatin (6.1 x 10-9 M). Buffalo rat liver cells (BRL 3A, CRL 1442, ATCC) were in Coon’s modified Ham’s F-12 supplemented with 5% FCS.

Stable Expression Analysis
Stable transfection used FRTL-5 cells. Before transfection, FRTL-5 cells were grown to 80% confluency in 6H medium and then shifted to 5H medium (6H medium minus TSH) for 4 days. One day before transfection they were returned to 6H medium. Transfection used an electroporation technique (Gene Pulser, Bio-Rad, Richmond, CA), previously described (11, 32). FRTL-5 cells were harvested, washed, and suspended, 1.5 x 107 cells/ml, in 0.8 ml PBS. Fifty micrograms of the NIS promoter-LUC chimeric plasmids and 5 µg pMC1neopolyA (Stratagene, Menasha, WI) were added. The cells were pulsed (300 V; 960 microfarads), plated, and cultured in 6H medium. Two days after transfection, 400 µg/ml G418 (GIBCO BRL) were added to the medium. Four weeks after transfection, 20–30 G418-resistant clones were pooled and used for luciferase assays described herein. Luciferase assay was performed as described previously (41).

Transient Expression Analysis
Ten micrograms of pRc/CMV or RcCMV-THA (13) were cotransfected with 3 µg pCH110-ß-gal into 0.8 x 107 FRTL-5 cells stably expressing p(-420) or p(-420MT), as described above. After 65–72 h, the cells were harvested for both luciferase assay and ß-galactosidase (ß-gal) assay. ß-gal assay was performed as described previously (42). Luciferase activity was normalized by ß-gal activity.

Nuclear Extracts
Nuclear extracts were prepared basically as previously described (11, 32). The 6H extract from FRTL-5 cells was prepared using cells grown in 6H medium until near confluence. The 5H extract was from FRTL-5 cells maintained in 5H medium (6H medium minus TSH) for 7 days after near confluency in 6H medium was achieved. FRT and BRL cells were grown to near confluence in their appropriate medium. Cells were washed with PBS, pH 7.4, scraped, and, after centrifugation at 500 x g, suspended in five pellet vol of 0.3 M sucrose and 2% Tween-40 in buffer A [10 mM HEPES-KOH, pH 7.9, containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonylfluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin-A]. After the cells were frozen in liquid nitrogen, thawed, and gently homogenized, the suspension was layered onto 1.5 M sucrose in buffer A and centrifuged at 25,000 x g in a swinging bucket rotor. Nuclei were washed with buffer A and lysed in 2.5 vol of buffer B (10 mM HEPES-KOH, pH 7.9, containing 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 2 µg/ml leupeptin, and 2 µg/ml pepstatin-A). Lysed nuclei were centrifuged at 15,000 x g for 1 h, and used in EMSAs or methylation interference assays.

EMSA
EMSAs were performed basically as previously described (11, 32). Synthesized, double-stranded oligonucleotides were labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase, and then purified on an 8% native polyacrylamide gel. Two micrograms of nuclear extract were incubated in a 30-µl reaction volume for 20 min at room temperature in the following buffer with or without unlabeled competitor oligonucleotides: 10 mM Tris-HCl (pH 7.6), 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 5% glycerol, 0.1% Triton X-100, and 0.5 µg polydeoxyinosinic-deoxycytidylic acid [poly (dI-dC)]. Labeled probe (50,000 cpm; ~0.5 ng DNA) was added, and incubation was continued for an additional 20 min at room temperature. DNA-protein complexes were separated on 5% native polyacrylamide gels.

Methylation Interference Assays
Methylation interference assays were performed as described (13, 31). To obtain double-stranded probe spanning -420 to -385 bp with coding or noncoding strand labeled, the single-stranded oligonucleotides end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase were annealed with their cold complementary strand and then purified on an 8% native polyacrylamide gel. The probes were modified with dimethyl sulfate (43) for 20 min on ice. For the preparative mobility shift, 5 x 105 cpm of modified oligonucleotides were incubated with 12 µg poly(dI-dC) and 20 µg FRTL-5 nuclear extract. The undried gel was exposed to a Bas 2000 Image Analyzer (Fuji Film Co., Tokyo, Japan) for 30 min, and the region corresponding to the expected protein-DNA complex and unbound probe in the gel were excised, eluted, and then precipitated. Base elimination and strand scission reactions at adenines and guanines (G > A) were performed (43) before samples were lyophilized, resuspended in water, relyophilized (three times), and analyzed on an 8% sequencing gel.

Other Analyses
Protein concentration was determined by Bradford’s method (Bio-Rad), and recrystallized BSA was used as a standard. All experiments were repeated at least three times with different batches of cells. Where noted, values are the mean ± SE of these experiments; significance (P < 0.05) between experimental values was determined by Student’s paired t test.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Masato Ikeda (Yamanashi Medical University, Yamanashi, Japan) for helpful advice and discussions.


    FOOTNOTES
 
Address requests for reprints to: Toshimasa Onaya, Professor and Chairman, Third Department of Internal Medicine, Yamanashi Medical University, 1110 Shimokato, Tamaho, Yamanashi 409–38, Japan.

Received for publication October 28, 1997. Accepted for publication January 14, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Dumont JE, Lefort A, Libert F, Parmentier M, Raspe E, Reuse F, Maenhaut C, Roger P, Corvilain B, Laurent E, Mockel J, Lamy F, Van Sande J, Vassart G 1990 Transducing systems in the control of human thyroid cell function, proliferation, and differentiation. In: Ekholm R, Kohn LD, Wollman S (eds) Control of the Thyroid: Regulation of its Normal Growth and Function. Plenum Press, New York, pp 357–372
  2. Vassart G, Dumont JE 1992 The thyrotropin receptor and regulation of thyrocyte function and growth. Endocr Rev 13:596–611[Medline]
  3. Kohn LD, Shimura H, Shimura Y, Hidaka A, Giuliani C, Napolitano G, Ohmori M, Laglia G, Saji M 1995 The thyrotropin receptor. Vitam Horm 50:287–384[Medline]
  4. Carrasco N 1993 Iodide transport in the thyroid gland. Biochim Biophys Acta 1154:65–82[Medline]
  5. Halmi NS, Granner DK, Doughman DJ, Peters BH, Muller G 1959 Biphasic effect of TSH on thyroidal iodide collection in rats. Endocrinology 67:70–81
  6. Knopp J, Stolc V, Tong W 1970 Evidence for the induction of iodide transport in bovine thyroid cells treated with thyroid-stimulating hormone or dibutyryl cyclic adenosine 3', 5'- monophosphate. J Biol Chem 245:4403–4408[Abstract/Free Full Text]
  7. Weiss SJ, Philp NJ, Grollman EF 1984 Iodide transport in a continuous line of cultured cells from rat thyroid. Endocrinology 114:1090–1098[Abstract]
  8. O’Neill B, Magnolato D, Semenza G 1987 The electrogenic, Na+-dependent I- transport system in plasma membrane vesicles from thyroid glands. Biochim Biophys Acta 896:263–274[Medline]
  9. Dai G, Levy O, Carrasco N 1996 Cloning and characterization of the thyroid iodide transporter. Nature 379:458–460[CrossRef][Medline]
  10. Kogai T, Endo T, Saito T, Miyazaki A, Kawaguchi A, Onaya T 1997 Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells. Endocrinology 138:2227–2232[Abstract/Free Full Text]
  11. Endo T, Kaneshige M, Nakazato M, Ohmori M, Harii N, Onaya T 1997 Thyroid transcription factor-1 activates the promoter activity of rat thyroid Na+/I- symporter gene. Mol Endocrinol 11:1747–1755[Abstract/Free Full Text]
  12. Civitareale D, Lonigro R, Sinclair AJ, Di Lauro R 1989 A thyroid-specific nuclear protein essential for tissue-specific expression of the thyroglobulin promoter. EMBO J 8:2537–2542[Abstract]
  13. Guazzi S, Price M, De Felice M, Damante G, Mattei M-G, Di Lauro R 1990 Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J 9:3631–3639[Abstract]
  14. Mizuno K, Gonzalez FJ, Kimura S 1991 Thyroid-specific enhancer-binding protein (T/EBP): cDNA cloning, functional characterization, and structural identity with thyroid transcription factor TTF-1. Mol Cell Biol 11:4927–4933[Medline]
  15. Francis-Lang H, Price M, Polycarpou-Schwarz M, Di Lauro R 1992 Cell-type-specific expression of the rat thyroperoxidase promoter indicates common mechanisms for thyroid- specific gene expression. Mol Cell Biol 12:576–588[Abstract]
  16. Shimura H, Okajima F, Ikuyama S, Shimura Y, Kimura S, Saji M, Kohn LD 1994 Thyroid- specific expression and cyclic adenosine 3',5'-monophosphate autoregulation of the thyrotropin receptor gene involves thyroid transcription factor-1. Mol Endocrinol 8:1049–1069[Abstract]
  17. Ohmori M, Shimura H, Shimura Y, Ikuyama S, Kohn LD 1995 Characterization of an up- stream thyroid transcription factor-1-binding site in the thyrotropin receptor promoter. Endocrinology 136:269–282[Abstract]
  18. Santisteban P, Acebron A, Polycarpou-Schwarz M, Di Lauro R 1992 Insulin and insulin-like growth factor 1 regulate a thyroid-specific nuclear protein that binds to the thyroglobulin promoter. Mol Endocrinol 6:1310–1317[Abstract]
  19. Zannini M, Francis-Lang H, Plachov D, Di Lauro R 1992 Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters. Mol Cell Biol 12:4230–4241[Abstract]
  20. Kambe F, Nomura Y, Okamoto T, Seo H 1996 Redox regulation of thyroid-transcription factors, Pax-8 and TTF-1, is involved in their increased DNA-binding activities by thyrotropin in rat thyroid FRTL-5 cells. Mol Endocrinol 10:801–812[Abstract]
  21. Kosower NS, Kosower EM 1987 Formation of disulfides with diamide. Methods Enzymol 143:264–270[Medline]
  22. DeGroot LJ 1989 Endocrinology. Grune & Stratton, Orlando, FL
  23. Werner SC, Ingbar S 1991 A fundamental and clinical text. In: Braverman LE, Utiger RD (eds) The Thyroid. JB Lippincott, Philadelphia, pp 1–1365
  24. Wolff J 1964 Transport of iodide and other anions in the thyroid gland. Physiol Rev 44:45–90[Free Full Text]
  25. Wilson B, Raghupathy E, Tonoue T, Tong W 1968 TSH-like actions of dibutyryl cAMP on isolated bovine thyroid cells. Endocrinology 83:877–884[Medline]
  26. Bagchi N, Fawcett DM 1973 Role of sodium ion in active transport of iodide by cultured thyroid cells. Biochim Biophys Acta 318:235–251[Medline]
  27. Endo T, Kogai T, Nakazato N, Saito T, Kaneshige M, Onaya T 1996 Autoantibody against Na+/I- symporter in the sera of patients with autoimmune thyroid disease. Biochem Biophys Res Commun 224:92–95[CrossRef][Medline]
  28. Endo T, Kaneshige M, Nakazato M, Kogai T, Saito T, Onaya T 1996 Autoantibody against thyroid iodide transporter in the sera from patients with Hashimoto’s thyroiditis possesses iodide transport inhibitory activity. Biochem Biophys Res Commun 228:199–202[CrossRef][Medline]
  29. Scott GK, Daniel JC, Xiong X, Maki RA, Kabat D, Benz CC 1994 Binding of an ETS- related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells. J Biol Chem 269:19848–19858[Abstract/Free Full Text]
  30. Saji M, Akamizu T, Sanchez M, Obici S, Avvedimento E, Gottesman ME, Kohn LD 1992 Regulation of thyrotropin receptor gene expression in rat FRTL-5 thyroid cells. Endocrinology 130:520–533[Abstract]
  31. Shimura H, Shimura Y, Ohmori M, Ikuyama S, Kohn LD 1995 Single strand DNA-binding proteins and thyroid transcription factor-1 conjointly regulate thyrotropin receptor gene expression. Mol Endocrinol 9:527–539[Abstract]
  32. Ohmori M, Ohta M, Shimura H, Shimura Y, Suzuki K, Kohn LD 1996 Cloning of the single strand DNA-binding protein important for maximal expression and thyrotropin (TSH)-induced negative regulation of the TSH receptor. Mol Endocrinol 10:1407–1424[Abstract]
  33. Lalli E, Sassone-Corsi P 1995 Thyroid-stimulating hormone (TSH)-directed induction of the CREM gene in the thyroid gland participates in the long-term desensitization of the TSH receptor. Proc Natl Acad Sci USA 92:9633–9637[Abstract]
  34. Saiardi A, Falasca P, Civitareale D 1994 The thyroid hormone inhibits the thyrotropin receptor promoter activity: evidence for a short loop regulation. Biochem Biophys Res Commun 205:230–237[CrossRef][Medline]
  35. Van Heuverswyn B, Leriche A, Van Sande J, Dumont J, Vassart G 1985 Transcriptional control of thyroglobulin gene expression by cyclic AMP. FEBS Lett 188:192–196[CrossRef][Medline]
  36. Gerard CM, Lefort A, Libert F, Christophe D, Dumont JE, Vassart G 1988 Transcriptional regulation of the thyroperoxidase gene by thyrotropin and forskolin. Mol Cell Endocrinol 60:239–242[CrossRef][Medline]
  37. Avvedimento EV, Musti AM, Ueffing M, Obici S, Gallo A, Sanchez M, DeBrasi D, Gottesman ME 1991 Reversible inhibition of a thyroid-specific trans-acting factor by Ras. Genes Dev 5:22–28[Abstract]
  38. Gallo A, Benusiglio E, Bonapace IM, Feliciello A, Cassano S, Garbi C, Musti AM, Gottesman ME, Avvedimento EV 1992 v-Ras and protein kinase C dedifferentiate thyroid cells by down-regulating nuclear cAMP-dependent protein kinase A. Genes Dev 6:1621–1630[Abstract]
  39. Ambesi-Impiombato FS 1986 Fast-growing thyroid cell strain. US Patent 4,608,341
  40. Ambesi-Impiombato FS, Coon HG 1979 Thyroid cells in culture. Int Rev Cytol [Suppl] 10:163–171[Medline]
  41. de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:725–737[Medline]
  42. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning-A Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  43. Maxam AM, Gilbert W 1980 Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560[Medline]