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 40938, Japan
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ABSTRACT
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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 36 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.
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INTRODUCTION
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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 50100% 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
50100% 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 1224 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.
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RESULTS
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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 612 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 67 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 1B
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.
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TSH significantly stimulates the luciferase activity of a 1968-bp rat
NIS promoter chimera, p(-1968) (Fig. 1B
). 5'-Deletion mutants between
-1968 and -420 bp retain TSH responsiveness, approximately 2- to
3-fold higher than in cells without TSH (Fig. 1B
). The TSH response is
lost with p(-370), p(-320), and p(-210), approximately 0.5- to
0.7-fold (Fig. 1B
). These results suggest a TRE exists between -420
and -370 bp of the rat NIS promoter (Fig. 1A
).
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. 2A
). 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. 2A
). 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. 2A
). The TSH effect is cAMP mediated,
since it is duplicated by forskolin (Fig. 2B
), 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. 2C
). 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.
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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. 3A
). 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. 3
, B and C). The TSH-induced protein-DNA complex A was FRTL-5
cell-specific (Fig. 3A
, solid arrow). Thus, nuclear extracts
from FRTL-5 cells, but not from FRT or BRL cells, formed a protein-DNA
complex A (Fig. 3A
). 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. 3A
, 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.
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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. 3B
, 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. 3B
, lanes 4 and 5 vs. 1), oligo DS
(Fig. 3B
, lanes 6 and 7 vs. 1), and oligo K (Fig. 3B
, 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. 3C
, 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. 4A
). 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. 4A
). As
summarized in Fig. 4B
, NTF-1-binding was inhibited by methylation of
nucleotides at -410, -406, -405, -404, and -402 bp on the coding
strand (Fig. 4B
, top line, open circles) and by methylation
of nucleotide at -407 bp on the noncoding strand (Fig. 4B
, 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.
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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. 5A
). The mutation (MT) changes four
of six residues of the NTF-1-binding site that were identified as
contact points by methylation interference (Fig. 5A
, open
circles, vs. Fig. 4
). 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. 5B
, lanes 1 and 2,
vs. Figs. 2A
and 3A
). The complex is no longer evident when
the radiolabeled mutant oligonucleotide, MT, is a probe (Fig. 5B
, 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. 5C
, lanes 2 and 3 vs. 1), but not
at all by a 100-fold excess of the mutant oligonucleotide MT (Fig. 5C
, 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. 5D
),
whereas p(-420) exhibited both TSH (Fig. 1B
) and forskolin
responsiveness (Fig. 5D
). 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.
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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. 6
). 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. 6
, 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. 6
, 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. 6
, 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.
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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. 7
). 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. 7
). 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. 7
). 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. 5 . 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).
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DISCUSSION
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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 Hashimotos 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. 3B
) 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. 3C
). 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
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
|
---|
Materials
Bovine TSH is a highly purified preparation obtained from Sigma
(St. Louis, MO). [
-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 SacIBglII 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 KpnIBglII 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. 5A
) 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
Coons modified Hams 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 Coons modified Hams 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, 2030 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 6572 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
[
-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 [
-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 Bradfords 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 Students 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 40938, Japan.
Received for publication October 28, 1997.
Accepted for publication January 14, 1998.
 |
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