PI3K Is Involved in the IGF-I Inhibition of TSH-Induced Sodium/Iodide Symporter Gene Expression
Bibian García and
Pilar Santisteban
Instituto de Investigaciones Biomédicas "Alberto
Sols,"
Consejo Superior de Investigaciones Científicas, Universidad
Autónoma de Madrid, Madrid, Spain E-28029
Address all correspondence and requests for reprints to: Dr. Pilar Santisteban, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Arturo Duperier, 4, E-28029 Madrid, Spain. E-mail: psantisteban{at}iib.uam.es
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ABSTRACT
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Here we studied the role of IGF-I on the regulation of the
sodium/iodide symporter (NIS) gene expression in FRTL-5 thyroid cells.
IGF-I did not modify NIS mRNA levels but inhibited TSH- and
forskolin-induced NIS mRNA expression in a dose-dependent manner. We
explored the signaling pathways by which IGF-I mediates the repression
of NIS expression. Inhibition of either the MAPK kinase or PKC
activities had no effect. Interestingly, inhibition of PI3K
blocked IGF-I repression of TSHinduced NIS mRNA and protein
levels. This effect takes place at the transcriptional level, as IGF-I
inhibited TSH-induced transcription of a luciferase reporter construct
containing a 2.8-kb DNA fragment of the rat NIS promoter. The
inhibitory effect of IGF-I on the NIS promoter was blocked by the PI3K
inhibitor LY294002 and was mimicked by overexpression of a vector
harboring the constitutively activated catalytic subunit of PI3K. Using
internal deletions of the NIS promoter, we defined a region from
-1,947 to -1,152 responsible for the observed IGF-I/PI3K inhibitory
effect. When fused to a heterologous promoter, this region inhibits
transcription in response to IGF-I. These results demonstrate a central
role for PI3K in the repression of NIS gene transcription by IGF-I and
suggest the existence, within the above defined promoter region, of
putative PI3K-responsive elements.
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INTRODUCTION
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IODIDE UPTAKE IS the first step in thyroid
hormone biosynthesis (1), the primary function of the
thyroid gland. Iodide transport occurs across the basolateral membrane
of thyroid follicular cells in an active transport process mediated by
the sodium iodide symporter (NIS) (2). Iodide is
subsequently incorporated by thyroid peroxidase (TPO) into the
thyroglobulin (Tg) molecule. All of these steps are stimulated by TSH,
the primary hormone regulating the functions of differentiated thyroid
cells. Although TSH regulation of iodide transport has been widely
reported (3, 4, 5), the cloning of the rat NIS cDNA by Dai
et al. (2) allowed the study of the molecular
mechanisms involved in TSH regulation of NIS gene expression. TSH, via
cAMP, induces the activity of the NIS protein (6, 7) and
increases NIS mRNA levels (8). TSH stimulation of NIS mRNA
expression in FRTL-5 cells is inhibited by RA (9), TNF
,
ceramide, TGFß1, and aging (10). The cloning of the rat
NIS promoter (11, 12) allowed initiation of the study of
NIS transcriptional regulation. A TSH-responsive element was identified
between positions -420 and -385 (13) and a thyroid
transcription factor-1 (TTF-1) binding site was identified between
-245 and -230 (14). In the distal promoter, Ohno
et al. (12) identified a short enhancer, called
NUE (NIS upstream enhancer), another important regulatory element for
TSH-regulated transcriptional activation. This element contains binding
sites for the thyroid-specific transcription factors TTF-1 and
Pax-8, and a cAMP responsive element-like sequence. NUE confers
thyroid-specific transcriptional activity to the NIS gene and responds
to TSH/cAMP. The cAMP response therefore requires the binding of Pax-8
and the integrity of the cAMP-responsive element sequence.
Insulin and IGF-I are additional important factors for thyroid function
that collaborate with TSH in the regulation of thyroid proliferation
and differentiation. The biological effects of insulin and IGF-I are
mediated by activation of their cell surface receptors, which possess
tyrosine kinase activity (15). After ligand binding,
insulin and IGF-I receptor activities converge on the phosphorylation
of insulin receptor substrates (IRS-1 and IRS-2), which then act as
docking proteins for SH2-containing proteins such as PI3K. The
interaction of the PI3K-regulatory subunit p85 with
tyrosine-phosphorylated IRS-1 leads to activation of the p110 catalytic
subunit. This results in activation of several protein kinases,
including Akt and certain PKC isoforms. Grb-2 is another
IRS-1-associated protein; their interaction leads to activation of the
small GTP-binding protein Ras, which then initiates the Raf-1/MAPK
kinase (MEK)/MAPKs ERK1 and ERK2 cascade. Stimulation of any of these
pathways can lead to activation/inhibition of target genes by
transcriptional or posttranscriptional mechanisms (16).
Although the insulin/IGF-I signaling pathways involved in the
regulation of thyroid-specific genes remain unknown, several reports
have shown their importance in regulating thyroid gene transcription.
Insulin and IGF-I stimulate Tg and TPO mRNA expression (17, 18). Both hormones increase Tg transcription, and their effects
are additive to those of TSH/cAMP (17). Our group has
identified response elements for insulin and IGF-I within the Tg
(19) and TPO (20) promoters and reported that
the hormonal regulation of both genes is mediated mainly by the
forkhead thyroid transcription factor-2 (TTF-2), a thyroid-specific
transcription factor that binds to the promoter of both genes
(21, 22). In addition, TTF-2 gene expression is stimulated
by cAMP and insulin/IGF-I (23). Insulin and IGF-I also
stimulate transcription of the TSH receptor gene (24).
Here we studied the role of IGF-I in the regulation of NIS gene
expression and the signaling pathways involved in IGF-I action. The
results show that IGF-I inhibits TSH/forskolin-induced NIS mRNA levels.
This effect is PI3K mediated, as its specific inhibitor, LY294002,
prevents IGF-I inhibition of TSH induction; this effect was also
observed when NIS protein levels were determined. Using transient
transfection assays, we show that IGF-I inhibits TSH-stimulated
transcriptional activity of a 2.8-kb NIS promoter, but no inhibition
was observed using constructs with internal deletions from -1,947 to
-1,152. When fused to a heterologous promoter, this region inhibits
transcription in response to IGF-I, indicating that it contains
IGF-I-responsive elements. Presence of the PI3K inhibitor LY294002
prevents IGF-I inhibition, and cotransfection with a constitutively
active form of the PI3K catalytic subunit reduces TSH-dependent
transcriptional activation. Our results show that via PI3K activation,
IGF-I interferes with TSH induction of NIS gene expression; we define a
region within the promoter responsible for the IGF-I inhibitory
effect.
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RESULTS
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Insulin and IGF-I Do Not Increase NIS mRNA Levels
Insulin and IGF-I are important hormones for thyroid function;
both stimulate expression of the thyroidspecific genes Tg and TPO
(17, 18), as well as the transcription factor TTF-2
(23), and potentiate the stimulatory action of TSH on the
expression of these genes. We performed Northern hybridization assays
to examine the role of insulin and IGF-I in NIS gene expression.
Starved FRTL-5 cells were insulin or IGF-I treated, and NIS mRNA levels
were compared with those observed in untreated cells or cells treated
with TSH or forskolin. TSH and forskolin stimulate NIS expression,
as has been extensively described (8), but neither IGF-I
nor insulin has any effect on NIS mRNA levels compared with untreated
cells (Fig. 1
).

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Figure 1. IGF-I and Insulin Do Not Increase NIS mRNA Levels
Starved FRTL-5 cells were treated with TSH, forskolin (Forsk), IGF-I,
or insulin (Ins) for 24 h and then harvested for RNA extraction. A
representative Northern blot hybridized with the rat NIS probe is
shown. Methylene blue staining of the 18S rRNA shows equal sample
loading.
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IGF-I Inhibits TSH- or Forskolin-Induced NIS mRNA Levels
Although neither insulin nor IGF-I increased NIS mRNA content in
FRTL-5 cells, we analyzed whether these hormones could regulate TSH and
forskolin effects. Starved FRTL-5 cells were treated with TSH (1
nM) or forskolin (10 µM), alone or in the
presence of increasing concentrations of IGF-I. The results show that
IGF-I inhibited the stimulatory effect of TSH or forskolin in a
dose-dependent manner (Fig. 2
, A and B,
respectively). Maximum inhibition of NIS mRNA levels was reached at 100
ng/ml of IGF-I. Like IGF-I, insulin inhibited NIS mRNA expression
induced by TSH or forskolin in a dose-dependent fashion; maximum
inhibition was observed at 10 µM insulin (not shown).
These data suggested that insulin and IGF-I may interfere with cAMP
induction of NIS mRNA expression.

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Figure 2. IGF-I Inhibits TSH- or Forskolin-Stimulated NIS
mRNA Expression
Starved FRTL-5 cells were treated with TSH (panel A) or forskolin
(Forsk; panel B) alone or in the presence of increasing IGF-I
concentrations (1, 10, or 100 ng/ml) for 24 h and then harvested
for RNA analysis. A representative Northern blot hybridized with rat
NIS probe is shown. Methylene blue staining of the 18S rRNA shows equal
sample loading.
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Neither MEK nor PKC Is Involved in IGF-I-Dependent Inhibition of
TSH-Stimulated NIS mRNA Expression
To further analyze the mechanism by which IGF-I inhibits
TSH-induced NIS mRNA expression, we studied the signaling pathways
involved in IGF-I action. After binding of IGF-I to its cell surface
receptors, a signaling cascade is initiated, leading to the activation
of several kinases including MEK and PKC. To assess the involvement of
these kinases in the IGF-I effect, we treated starved FRTL-5 cells with
the specific inhibitors of these kinases, PD98059, which inhibits MEK
activity, and bisindolyl maleimide I (BIS), which inhibits PKC
activity, and determined NIS mRNA levels in response to TSH or TSH plus
IGF-I. Figure 3A
shows the results
obtained with these inhibitors as well as with H89, a specific
inhibitor of PKA. TSH induction of NIS mRNA expression is only
partially inhibited in the presence of H89. This confirms previous
observations by Ohno et al. (12) suggesting
that TSH induction of NIS gene expression occurs in both a
PKA-dependent and -independent manner. TSH retained the ability to
increase NIS mRNA levels in the presence of PD98059 or BIS. None of
these inhibitors (PD98059 or BIS) abolished IGF-I inhibition of TSH
induction, indicating that neither MEK nor PKC is involved in IGF-I
action. The inhibitors have no effect when added alone and do not
substantially affect mRNA levels in cells treated with IGF-I in the
absence of TSH (not shown). The lack of any effect in response to
PD98059 or BIS was not due to a loss of inhibitor function,
demonstrated by immunoblot assays (Fig. 3B
). Starved FRTL-5 cells
treated with IGF-I show increased levels of phospho-ERK1/ERK2,
downstream targets of MEK (25), compared with untreated
cells; the presence of PD98059 abolished this stimulation (Fig. 3B
, upper panel). BIS activity was examined by determining the
activation of the PKC target, protein kinase D (PKD), using a specific
antibody that recognizes phosphorylated PKD (26). The
phorbol ester phorbol-12,13-dibutyrate (PDBu) stimulates PKD
phosphorylation, but no phospho-PKD is detected in the presence of BIS
(Fig. 3B
, lower panel). These results clearly indicate that
both inhibitors were active at the concentrations used.

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Figure 3. The Inhibitors H89, PD98059, and BIS Do Not Block
IGF-I Inhibition of TSH-Induced NIS mRNA Expression
A, Starved FRTL-5 cells were pretreated with the inhibitors H89,
PD98059 (PD), or BIS, 30 min before addition of TSH and IGF-I. After
24 h, cells were harvested for RNA analysis. A representative
Northern blot hybridized with rat NIS probe and methylene blue staining
of the 18S rRNA are shown. B, Starved FRTL-5 cells were pretreated with
inhibitors PD or BIS, 30 min before addition of IGF-I (100 ng/ml) or
PDBu (100 nM) for 15 min. Western blot analyses performed
with 20 µg of total protein and probed with antiphospho-ERK1/ERK2
(upper panel) and antiphospho-PKD (lower
panel) are shown. Immunodetection of ß-actin shows equal
sample loading.
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Inhibition of PI3K Prevents Suppression of TSH-Induced NIS mRNA and
Protein Expression by IGF-I
PI3K is one of the central enzymes implicated in insulin and IGF-I
signaling. To determine whether PI3K is required for IGF-I inhibition
of TSH induction of NIS gene expression, starved FRTL-5 cells were
pretreated with the PI3K inhibitor LY294002 before the addition of TSH,
forskolin, and IGF-I. LY294002 pretreatment increased NIS mRNA levels
in TSH-treated cells and slightly enhanced the messenger level in cells
treated with IGF-I (Fig. 4A
).
Interestingly, IGF-I inhibition of TSH induction is not observed in the
presence of LY294002. Similar results were obtained using wortmannin
(25100 nM), another PI3K inhibitor (not shown). When
forskolin was used instead of TSH, the results resembled those found
for TSH, except that LY294002 did not increase NIS mRNA levels in
forskolin-treated cells. These data suggest that PI3K interferes with
NIS gene expression induced by the cAMP signaling pathway. We next
determined whether the changes in NIS mRNA levels in response to TSH,
IGF-I, and LY294002 correlated with changes in NIS protein levels.
Western blot analyses were performed using a specific anti-NIS antibody
that reacts with two main NIS species, the mature 80- to 90-kDa form
and a partially processed 60- to 65-kDa NIS form (6). Due
to the long half-life of the NIS protein, FRTL-5 cells were maintained
for 7 d in starvation medium, before hormone addition, to
completely deplete cells of NIS protein (7). Cells
maintained for 7 d in starvation medium have no detectable NIS
protein, and TSH treatment of cells led to an increase in NIS protein
levels (Fig. 4C
), as previously reported (6, 7). IGF-I
completely inhibits NIS protein expression in response to TSH, and the
presence of LY294002 prevents this effect. All together, the results
suggest that PI3K interferes with TSH-dependent NIS mRNA and protein
induction and may mediate the IGF-I inhibitory effect observed.

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Figure 4. The PI3K Inhibitor LY294002 Blocks IGF-I Inhibition
of TSH- and Forskolin-Induced NIS mRNA and TSH-Induced Protein
Expression
A and B, Starved FRTL-5 cells were pretreated with LY294002 (LY) 30 min
before addition of TSH, forskolin (Forsk), or IGF-I, and cells were
collected 24 h later. Representative Northern blots hybridized
with rat NIS probe are shown. Methylene blue staining of the 18S rRNA
shows equal sample loading. C, Western blot analysis of NIS expression.
FRTL-5 cells were starved for 7 d and then pretreated with
LY294002 (LY) 30 min before TSH and IGF-I addition. After 24 h,
cells were collected for membrane protein preparation. Membrane protein
(5 µg) was resolved in 12% SDS-PAGE, transferred to nitrocellulose
filters, and probed with anti-NIS antibody. For loading control,
Ponceau S staining of a blot fragment is shown.
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IGF-I Inhibits TSH-Dependent Transcriptional Activation of the NIS
Gene
To study whether IGF-I regulates transcription of the rat NIS
gene, we cloned a 2.8-kb fragment of the rat NIS promoter, as described
in Materials and Methods. This region contains the NUE, a
regulatory element necessary for a full TSH response (12).
Luciferase reporter DNA constructs containing the full-length DNA
fragment or 5'-deletion derivatives were transiently transfected into
FRTL-5 cells and assayed for transcriptional activity in response to
TSH and/or IGF-I (Fig. 5A
). As previously
described (12), the 2,854-bp DNA fragment (pNIS-2.8)
showed significant stimulation of transcription by TSH, whereas the
5'-deletion constructs, pNIS-2, pNIS-1.2, and pNIS-0.5, which lacked
the enhancer, showed no stimulation of transcription or only trace
levels, as was the case for pNIS-0.5. IGF-I had no effect on the
transcriptional activity of any construct when added alone, although
IGF-I inhibited the TSH-induced transcriptional activation of the
construct containing the full promoter, pNIS-2.8; this indicates that
IGF-I signaling interferes with TSH induction of NIS transcription. To
analyze the mechanism of NIS repression by IGF-I, we inserted a region
containing the NUE element upstream of the promoter region in pNIS-1.2
(pNIS-NUE-1.2) and pNIS-0.5 (pNIS-NUE-0.5). Transient transfection
analyses with these two constructs and pNIS-2.8 showed that all three
constructs expressed significant luciferase activity in response to
TSH, but only pNIS-2.8 exhibited a reduction in luciferase activity
when IGF-I was added together with TSH (Fig. 5B
). These results
indicate that IGF-I does not interfere with NUE activation by TSH and
requires the region from -1,947 to -1,152 for the inhibition of the
TSH-dependent stimulation of NIS transcription.

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Figure 5. IGF-I Inhibits TSH-Induced NIS Gene Transcription
A and B, FRTL-5 cells were transfected with 2 µg of RSV-CAT and 2
pmol of the reporter construct containing the complete NIS promoter,
pNIS-2.8, or the 5'-deletion derivatives pNIS-2, pNIS-1.2, or pNIS-0.5
(panel A), or the internal deletion constructs pNIS-NUE-1.2 or
pNIS-NUE-0.5 (panel B). C, FRTL-5 cells were transfected with 2 µg of
RSV-CAT and 2 pmol of pNIS-0.8-TL or the control vector TATA-LUC. After
transfection, cells were maintained 72 h in starvation medium and
then treated with TSH and IGF-I. After 24 h, cells were harvested
and luciferase and CAT activity were determined. Relative luciferase
activity is the value of light units, normalizing the results to CAT
activity derived from RSV-CAT. The NIS promoter activity is expressed
as the x-fold induction over the basal levels (=1 in A and B; =100 in
C) of starved cells (basal). The data represent the mean ±
SE of at least four independent experiments.
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To confirm that this region is IGF-I responsive, the NIS-regulatory
region from -1,947 to -1,152 was inserted upstream from the
ß-globin TATA box of the luciferase expression vector TATA-LUC
(20). This construct (pNIS-0.8-TL) and the control vector
TATA-LUC were transiently transfected into FRTL-5 cells and assayed for
transcriptional activity in response to TSH and/or IGF-I. The results
indicate that pNIS-0.8-TL shows almost 50% inhibition by IGF-I, both
alone or in the presence of TSH, whereas no effect is observed on the
TATA-LUC control vector, which lacked the regulatory region (Fig. 5C
).
These results clearly indicate that the -1,947 to -1,152 region of
the rat NIS gene contains IGF-I-responsive regulatory elements.
PI3K Mediates the Inhibition of TSH-Induced NIS Promoter Activity
by IGF-I
To investigate whether PI3K was required for the observed IGF-I
inhibitory action on NIS promoter activity, we studied the effect of
LY294002 on TSH-induced transcriptional activation of pNIS-2.8, alone
or in the presence of IGF-I. The results (Fig. 6A
) resemble those obtained in Northern
blot assays, i.e. the PI3K inhibitor LY294002 completely
blocked IGF-I repression of the transcriptional activation by TSH.
These data indicate that PI3K activity is necessary for suppression of
TSH-induced NIS gene expression by IGF-I. To ask whether PI3K
activation inhibited TSH induction of NIS gene transcription, we next
transfected FRTL-5 cells with the pNIS-2.8, pNIS-NUE-1.2, or
pNIS-NUE-0.5 constructs, with or without an expression vector for the
constitutively active PI3K catalytic subunit, p110-CAAX. Overexpression
of p110-CAAX caused a striking reduction in the stimulation of pNIS-2.8
transcription by TSH, thus mimicking the effect of IGF-I (Fig. 6B
).
Conversely, the transcriptional activation of pNIS-NUE-1.2 and
pNIS-NUE-0.5 by TSH is not affected by p110-CAAX overexpression. These
results indicate that p110-CAAX does not block NUE activation by TSH,
and that the region between -1,947 and -1,152 responds to PI3K
activation, leading to inhibition of transcriptional activation by
TSH.

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Figure 6. PI3K Involvement in NIS Promoter Repression by
IGF-I
A, FRTL-5 cells were transfected with 2 pmol of the reporter construct
pNIS-2.8 and 2 µg of RSV-CAT. After 72 h in starvation medium,
cells were treated with LY294002 (LY) 30 min before addition of TSH and
IGF-I. B, FRTL-5 cells were transfected with 2 pmol of the reporter
constructs pNIS-2.8, pNIS-NUE-1.2, or pNIS-NUE-0.5, and 2 µg of
RSV-CAT, with 2 µg of either pSG5-p110-CAAX (p110) or the empty
vector. After transfection, cells were maintained 72 h in
starvation medium and then treated with TSH. At 24 h after hormone
treatment, cells were harvested, and luciferase and CAT activity were
determined. Relative luciferase activity was determined as in Fig. 5 .
NIS promoter activity is expressed as the x-fold induction over basal
levels (=1) of starved cells. The data represent the mean ±
SE of at least three independent experiments.
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DISCUSSION
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The data presented here demonstrate that IGF-I inhibits TSH
stimulation of NIS gene expression. These results are in accordance
with previous reports showing that IGF-I inhibited iodide uptake
stimulated by TSH in FRTL-5 cells (27, 28); this effect
was also seen when the cAMP analog 8-Br-cAMP was used instead of TSH
(28). We also demonstrate that IGF-I interferes with cAMP
signaling, because IGF-I prevents the induction of NIS mRNA expression
by forskolin. These results, in addition to the reports mentioned above
(27, 28), thus demonstrate cross-talk between IGF-I
signaling and the cAMP transduction pathway; this cross-talk may take
place downstream of cAMP generation, because IGF-I did not affect the
TSH-induced cAMP levels, as reported by Saji and Kohn
(28).
We studied the signaling pathways involved in the IGF-I inhibitory
effect on the induction of NIS gene expression by TSH. Using specific
inhibitors of the distinct kinases involved in the most important
signaling pathways operating in thyroid cells, we found that the
presence of H89, a PKA inhibitor, decreased but did not abolish TSH
induction of NIS mRNA levels. It is likely that cAMP regulation of NIS
gene expression is mediated by PKA-dependent and -independent pathways,
as suggested by Ohno et al. (12). A new cAMP
cascade has been identified that involves the guanine nucleotide
exchange factor (GEF), called Epac (29) or cAMP-GEF
(30). Epac, which is regulated directly by cAMP, is a GEF
for the small GTPase Rap-1 and is expressed in many tissues, including
thyroid (30). In dog thyroid cells, Rap-1 activation has
been described in response to TSH/cAMP (31) and, in the
WRT rat thyroid cell line, TSH activates Rap1 through a cAMP-mediated,
PKA-independent mechanism (32). It remains to be
determined whether this cAMP-Epac-Rap-1 pathway is involved in
TSH-dependent stimulation of NIS gene expression.
Contrary to observations with H89, the presence of the PI3K inhibitor
LY294002 results in superinduction of NIS mRNA levels by TSH,
suggesting a role for PI3K in TSH-dependent signaling transduction
pathways. These data concur with recent results involving PI3K in the
stimulation of thyroid cell proliferation in response to TSH via cAMP
(33, 34). PI3K signaling is thus required for a mitogenic
response to TSH but, in view of our results, it inhibits the
stimulation of NIS gene expression by TSH. Interestingly, we did not
observe this superinduction when forskolin was used instead of TSH
(Fig. 4
). One explanation for this result may derive from the fact that
forskolin stimulates cAMP production by direct activation of the
adenylyl cyclase, thus eliminating possible PI3K activation by G
protein ß
-subunits, a PI3K activation mechanism (35).
Although it was not the aim of this study to analyze the signaling
pathways involved in TSH-dependent activation of NIS gene expression,
it would be of great interest to investigate the existence of this
possible mechanism of PI3K activation in response to TSH.
IGF-I inhibition of TSH-stimulated NIS mRNA and protein expression was
blocked by the PI3K inhibitor LY294002. We observed similar results
with wortmannin at nanomolar doses (not shown). The absence of effect
in response to PD98059 and BIS should not be attributed to a lack of
function, as we observed an inhibitory effect by both compounds in
Western blots for phospho-ERK1/ERK2 and phospho-PKD, respectively. The
results clearly indicate that PI3K interferes with TSH-induced NIS gene
expression and suggest that PI3K may be involved in IGF-I action.
Previous data from Cass and Meinkoth (36) suggested a role
for PI3K in the regulation of NIS expression. They reported that
transfection of WRT thyroid cells with the Ras mutant RasV12C40, which
signals preferentially through PI3K, resulted in a reduction of NIS
protein levels, whereas Tg protein levels were not affected. It appears
that NIS and Tg gene expression are regulated in a different manner.
The authors proposed that RasV12C40-dependent signals might also
downregulate the expression and/or activity of Pax-8, a
transcription factor involved in the regulation of NIS expression
(12), or affect NIS activity through a posttranscriptional
mechanism. Before the cloning of the NIS cDNA (2), Saji
and Kohn (28) proposed a posttranscriptional mechanism by
which insulin/IGF-I regulated TSH induction of the iodide porter
system. Although these results are not in doubt, an additional
regulatory mechanism at the transcriptional level cannot be ruled out.
Taking advantage of the cloning and characterization of the rat NIS
promoter (11, 12), we cloned a 2.8-kb DNA fragment of the
rat NIS promoter to study the role of IGF-I in regulating NIS gene
transcription. Our results from transient transfection assays, with
reporter constructs containing the full-length DNA fragment or 5'
deletions, confirm the requirement of the NUE for a potent TSH
response, as reported previously (12). We also showed that
transcriptional activation by TSH was inhibited by IGF-I, although the
extent of inhibition was lower than that observed in Northern blot
studies. This difference may be explained by the suggested
posttranscriptional regulation (28) and/or the presence of
regulatory elements upstream of the DNA fragment studied.
The results obtained with the PI3K inhibitor LY294002 indicated that
PI3K activity was required for inhibition of TSH-induced
transcriptional activation by IGF-I. This observation was confirmed by
the results of transient transfection assays with the constitutively
active form of the PI3K catalytic p110
subunit, p110-CAAX, showing
inhibition of transcriptional activation by TSH. Although there are
multiple PI3K isoforms (37), these findings suggest that
signaling pathways leading to activation of the PI3K p110
subunit
could result in the inhibition of NIS gene expression. It nonetheless
remains to be elucidated which PI3K subtype, sensitive to LY294002 and
wortmannin inhibition, mediates IGF-I effects. To confirm the results
observed with PI3K inhibitor, we performed transfection assays with the
dominant negative form of the PI3K-regulatory subunit, p85
iSH2-N.
The results did not provide significant information, as transfection
with this mutant resulted in the loss of the majority of the cells (not
shown), i.e. drastic inhibition of PI3K activity leads to
cell death. This concurs with recent reports on PI3K in cell cycle
progression in thyroid cells (33, 34), in addition to its
role in cell survival signaling (38). Added to our
findings, these results clearly demonstrate the importance of PI3K in
thyroid cell function. Although we have no physiological explanation
for the IGF-I-inhibitory effect observed on NIS gene expression, the
results with LY294002 and the dominant positive mutant of PI3K suggest
that situations leading to activation of the PI3K pathway may affect
NIS expression levels. In this respect, activation of Akt, one of the
downstream targets of PI3K, has been shown in several thyroid tumors
(39). Moreover, the expression level of PTEN, a
3'-phosphatase that converts phosphatidylinositol
(3, 4)P2 to PtIns(4)P, and PtIns
(3, 4, 5)P3 to PtIns (4, 5)P2, thus
inactivating Akt, is decreased in neoplasic thyroid cells and in
thyroid tumors (40). These findings and our results
may explain the down-regulation of NIS expression found in many thyroid
cancers (1). The elucidation of the mechanism by which
PI3K downregulates NIS expression will aid in understanding the absence
of iodide symporter activity in thyroid tumors.
To delimit the region within the NIS promoter responsible for the IGF-I
effect, several deletions of the NIS promoter were studied. The results
indicate that IGF-I does not interfere with the TSH activation of NUE.
The region from -1,947 to -1,152 seems to be necessary for IGF-I/PI3K
inhibition of TSH induction of the NIS promoter; it contains
IGF-I-responsive elements, as it could inhibit transcription of a
heterologous promoter in response to IGF-I. Computer analysis of this
region revealed the existence of several TG/ATTT elements; this
sequence is the core motif of insulin and IGF-I response elements found
in the promoter of several genes that are negatively regulated by
insulin/IGF-I (16). It has recently been reported that a
subfamily of forkhead transcription factors, the FKHR proteins, can
bind this motif and are targets of PI3K (41). This
evidence suggests that these proteins, or related family members, may
confer the inhibitory effect observed on NIS transcription.
Additional observations by our group, indicating that the thyroid
transcription factor TTF-2 mediates transcriptional activation of
thyroid-specific genes by insulin/IGF-I (23), suggest a
general role for forkhead proteins in mediating insulin/IGF-I effects
on transcription. Further studies will elucidate the role of FKHR
proteins on NIS gene expression and will give new insight into the
mechanisms by which forkhead transcription factors regulate the
transcription of cAMP-stimulated genes.
 |
MATERIALS AND METHODS
|
---|
Materials
Tissue culture medium, bovine TSH, and bovine insulin were
purchased from Sigma (St. Louis, MO). IGF-I was obtained
from Peprotech (Rocky Hill, NJ). Forskolin, LY294002, PD98059, H89,
bisindolyl maleimide I HCl, and wortmannin were purchased from
Calbiochem (La Jolla, CA). Donor calf serum and DMEM were
obtained from Life Technologies, Inc. (Gaithersburg, MD),
and Nytran and nitrocellulose filters were obtained from
Schleicher & Schuell, Inc. (Keene, NH). The luciferase
assay kit was purchased from Promega Corp. (Madison, WI).
[
-32P]dCTP and
[
-32P]ATP were obtained from ICN Biochemicals, Inc. Restriction enzymes were obtained from
Roche Molecular Biochemicals (Indianapolis, IN); the
Luminol kit and streptavidin-horseradish peroxidase conjugate,
antiphospho-ERK1/ERK2, and anti-ß-actin antibodies were obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-NIS
antibody was a generous gift from Dr. N. Carrasco (Albert Einstein
College of Medicine, Bronx, NY). Anti-phospho-PKD antibody was kindly
donated by Dr. T. Iglesias (Instituto de Investigaciones
Biomédicas, Madrid, Spain).
Cell Culture
FRTL-5 cells (ATCC CRL 8305, American Type Culture Collection, Manassas, VA) were a generous gift of Dr. L. D.
Kohn (Edison Biotechnical Institute, Athens, OH); these cells
had the properties described (42, 43), were diploid, and
their doubling time with TSH was 36 h. Cells were cultured in
Coons modified Hams F-12 medium supplemented with 5% donor calf
serum and a six-hormone mixture [1 nM TSH, 10 µg/ml
insulin, 10 ng/ml somatostatin, 5 µg/ml transferrin, 10
nM hydrocortisone, and 10 ng/ml
glycyl-L-histidyl-L-lysine acetate; complete
medium (42)]. The effect of hormones and growth factors
was studied by starving near-confluent cells for TSH and insulin in the
presence of 0.2% serum (starvation medium) for 72 h. For NIS
protein analyses, cells were starved for 7 d, after which the
ligands were added to culture medium at the following concentrations: 1
nM TSH, 10 µg/ml insulin, 100 ng/ml IGF-I, 10
µM forskolin, and 100 nM PDBu (unless
otherwise indicated). Inhibitors were added to the cells 30 min before
hormone addition at the following concentrations: 10 µM
H89, 50 µM PD98059, 25100 nM wortmannin, 10
µM LY294002, and 1 µM bisindolyl maleimide
I.
RNA Extraction and Northern Blot Analysis
Total RNA was isolated by the guanidinium-thiocyanatephenol
procedure (44) from FRTL-5 cells after different
treatments. Total RNA (20 µg) was separated in 1% agarose gels
containing 2.2 M formaldehyde. RNA was blotted onto Nytran
filters as suggested by the manufacturer. Methylene blue
staining of the blots revealed the integrity of the RNA and the
presence of equal amounts in each lane. Hybridization and washing were
carried out with the NIS-specific probe (2), labeled by
random oligo priming.
Protein Extraction and Western Blot Analysis
Membranes from FRTL-5 cells were prepared with protease
inhibitors as described previously (45). Briefly, FRTL-5
cells were collected and homogenized in a buffer containing 250
mM sucrose, 10 mM HEPES-KOH (pH 7.5), 1
mM EDTA, 2 µg/ml leupeptin, and 2 µg/ml aprotinin. Cell
homogenates were centrifuged (100,000 x g, 60 min, 4
C) to obtain membrane fractions, and the pellet was resuspended in
buffer as above. Whole-cell extracts were obtained by resuspending the
cell pellet in a buffer containing 50 mM HEPES
(pH 7.0), 2 mM MgCl2, 250
mM NaCl, 0.1 mM EDTA, 0.1
mM EGTA, 0.1% NP40, 1 mM
dithiothreitol, 2 M
Na3VO4, 10
mM NaF, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml pepstatin A, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin. Protein concentration was
determined according to Bradford (46) with the
Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay
kit. For immunodetection of NIS, membrane fractions were diluted 1:2
with loading buffer and heated (37 C, 30 min) before electrophoresis.
Total cell extract proteins (25 µg) or membrane proteins (5 µg)
were separated by 8% or 12% SDS-PAGE for phospho-PKD or
phospho-ERK1/ERK2 and NIS, respectively, and then transferred to
nitrocellulose membrane. Ponceau S staining of the blots for NIS
detection showed equal protein loading. Membranes were blocked (1 h,
room temperature) in TBS-T buffer (20 mM
Tris-HCl, 137 mM NaCl, 0.1% Tween-20, pH 7.5)
containing 5% nonfat milk. Western analyses for NIS detection were
performed with anti-NIS antibody (1:5,000 dilution) (45).
PKD was detected using a noncommercial antibody to phosphorylated PKD
(26). Commercial antibodies were used for ERK1/ERK2 or
ß-actin immunodetection at 1:5,000 or 1:2,000, respectively. After
incubation with antibodies in TBS-T containing 5% nonfat milk,
membranes were washed four times with TBS-T buffer and incubated with a
streptavidin-horseradish peroxidase conjugate (1:2,000), followed by
four washes of 10 min each with TBS-T buffer. Immunoreactive bands were
visualized with the Luminol Western blot detection reagent (Santa Cruz
Biotechnology).
Promoter Constructs
pNIS-2.8 and pNIS-2
DNA fragments from the rat NIS promoter, corresponding to
the regions -2,841 to +13 and -1,941 to +13 (11, 12),
were amplified from FRTL-5 genomic DNA by PCR, using forward primers
with a BamHI (5'-GCTATGGATCCCGAAGTGGCACTCACAACAT GTACC-3')
and SacI sites (5'-TCCTGCGAGCTCTAAGCCTCTGCTAGG-3'),
respectively, and reverse primer with a BamHI site
(5'-CGCAGGATCCATGGAGACAGGTGACTCG-3'). The fragments were cloned
into pGEM-T vector (Promega Corp.), cleaved by
BamHI or SacI and BamHI, and subcloned
into pBSLuc2 vector (22).
pNIS-1.2 and pNIS-0.5
DNA fragments were amplified from pNIS-2 by PCR, using forward
primers with a SacI site (5'-CAACACGA
GCTCCAGCCCTCCCTGGTGGC-3'; 5'-AAGAAGAGCTCCAAGAGAACCTGAGTGC-3') and
the reverse primer described above.
pNIS-NUE-1.2 and pNIS-NUE-0.5
A DNA fragment containing the NUE element was cleaved by
SacI and inserted into the SacI site of pNIS-1.2
(pNIS-NUE-1.2) and pNIS-0.5 (pNIS-NUE-0.5).
pNIS-0.8-TL
The DNA fragment was PCR-amplified from pNIS-2.8, using the
forward primer with a SacI site
(5'-TCCTGCGAGCTCTAAGCCTCTGCTAGG-3') and reverse primer with a
BamHI site (5'-ATGCTGGGATCCTCGCGGTCATGCCGGTGC-3'). The
fragment was cloned into pGEM-T vector, cleaved by SacI, and
subcloned into the TATA-LUC vector (20). The fidelity of
all constructs was confirmed by sequencing on an automatic DNA
sequencer (Perkin-Elmer Corp., Norwalk, CT).
Transfection
FRTL-5 cells were plated at 1.5 x
106 cells per 90 mm-diameter tissue culture dish,
48 h before transfection. Transfections were performed by calcium
phosphate coprecipitation as described previously (47).
Cells were transfected with 2 pmol of test plasmid and 2 µg of Rous
sarcoma virus-chloramphenicol acetyltransferase (RSV-CAT)
(48), used to monitor transfection efficiency. PI3K
pathway involvement was studied using an expression plasmid containing
the constitutively active catalytic PI3K subunit, pSG5-p110-CAAX, or
the dominant negative form, p85
iSH2-N (49). To study
the effect of pSG5-p110-CAAX on pNIS-2.8, pNIS-NUE-1.2, and
pNIS-NUE-0.5, 2 µg of RSV-CAT, 2 pmol of test plasmid, and 2 µg of
pSG5-p110-CAAX or the empty vector pSG5 (to adjust total plasmid
quantity) were used. To study the effects of TSH and IGF-I, transfected
cells were cultured in starvation medium (72 h) and then treated with 1
nM TSH and/or 100 ng/ml IGF-I. The PI3K inhibitor LY294002
was studied by addition at 10 µM, 30 min before hormone
addition. After 24 h, cells were collected for LUC and CAT
activity assay, as described previously (50, 51).
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to Dr. Nancy Carrasco (Albert Einstein College
of Medicine, Bronx, NY) for the NIS cDNA and the NIS antibody, to Dr.
Teresa Iglesias (Instituto de Investigaciones Biomédicas, Madrid,
Spain) for the antiphospho-PKD antibody, and to Dr. Julian Downward
(Imperial Cancer Research Foundation, London, UK) for the p110CAAX and
p85
iSH2-N expression vectors. We thank Catherine Mark for
linguistic assistance.
 |
FOOTNOTES
|
---|
This work was supported by Dirección General de
Investigación Cientifica y Técnica Grants PM97/0065 and
BMC2001-2087 and Comunidad Autónoma de Madrid Grant
08.1/0025/97-99. B.G. is the recipient of a postdoctoral fellowship
from the Comunidad Autónoma de Madrid.
Abbreviations: BIS, Bisindolyl maleimide I; GEF,
guanine-nucleotide-exchange factor; IRS, insulin receptor substrate;
MEK, MAPK kinase; NIS, sodium/iodide symporter; NUE, NIS upstream
enhancer; PDBu, phorbol-12,13-dibutyrate; PKD, protein kinase D;
RSV-CAT, Rous sarcoma virus-chloramphenicol acetyltransferase; Tg,
thyroglobulin; TPO, thyroid peroxidase; TTF-1, TTF-2, thyroid
transcription factors 1 and 2.
Received for publication March 26, 2001.
Accepted for publication October 29, 2001.
 |
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