Involvement of JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) in the Thyrotropin Signaling Pathway
Eun Shin Park,
Ho Kim,
Jae Mi Suh,
Soo Jung Park,
Soon Hee You,
Hyo Kyun Chung,
Kang Wook Lee,
O-Yu Kwon,
Bo Youn Cho,
Young Kun Kim,
Heung Kyu Ro,
Jongkyeong Chung and
Minho Shong
Department of Internal Medicine (E.S.P., H.K., J.M.S., S.J.P.,
H.K.C., K.W.L., Y.K.K., H.K.R., M.S.) Department of Anatomy
(O.-Y.K.) School of Medicine Chungnam National University
Taejon, 301040, Korea
Department of Internal Medicine
(B.Y.C.) School of Medicine Seoul National University
Seoul, 110744, Korea
Department of Biological Sciences
(J.C.) Korea Advanced Institute of Science and Technology
Taejon 305701, Korea
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ABSTRACT
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TSH is an important physiological regulator of
growth and function in thyroid gland. The mechanism of action of TSH
depends on interaction with its receptor coupled to heterotrimeric G
proteins. We show here that TSH induces the phosphorylation of tyrosine
in the intracellular kinases Janus kinase 1 (JAK1) and -2 (JAK2) in rat
thyroid cells and in Chinese hamster ovary (CHO) cells transfected with
human TSH receptor (TSHR). The JAK family substrates STAT3 (signal
transducers and activators of transcription) are rapidly tyrosine
phosphorylated in response to TSH. We also find that JAK1, JAK2, and
STAT3 coprecipitate with the TSHR, indicating that the TSHR may be able
to signal through the intracellular phosphorylation pathway used by the
JAK-STAT cascade. TSH increases STAT3-mediated promoter activity and
also induces endogenous SOCS-1 (suppressor of cytokine signaling-1)
gene expression, a known target gene of STAT3. The expression of a
dominant negative form of STAT3 completely inhibited TSH-mediated
SOCS-1 expression. These findings suggest that the TSHR is able to
signal through JAK/STAT3 pathways.
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INTRODUCTION
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The molecular characterization of the TSH receptor (TSHR) (1, 2, 3)
has considerably increased understanding of the synthesis (4, 5),
structure (6, 7), and function of this molecule (8, 9). TSHR includes a
large hydrophobic extracellular hormone-binding domain, a portion of
which is organized into seven-membrane spanning segments (7, 10). As in
other receptors in this family, interaction of TSH or TSHR antibodies
at the binding site of the extracellular domain activates adenylate
cyclase through heterotrimeric G proteins, including Gs, Gi/o, G12, and
Gq/11 (11, 12, 13). Studies with mutants of TSHR have shown that adenylate
cyclase interacts with the second cytoplasmic loop of TSHR (14) and
that the phosphoinositide-signaling domain is located within the first
loop (15). Over the past 20 yr, the general mechanism has been worked
out for seven-transmembrane domain receptors that are coupled to G
proteins and GTP hydrolysis. Although similar in overall organization,
there is variability in subtype and subcellular localization that
confer consid-erable signaling specificity. Recent studies suggest
that G protein-coupled receptors, e.g. angiotensin II AT1
receptor, may be able to signal through the intracellular
phosphorylation pathway used by cytokine receptors (16, 17). The
signaling by cytokine receptors depends upon their association with
Janus kinases (JAKs), which couple ligand binding to tyrosine
phosphorylation of signaling proteins recruited to the receptor complex
(18, 19, 20). Among these are the signal transducer and activator of
transcription (STAT) factors, a family of transcription factors that
contribute to the diversity of cytokine responses (18, 19, 20). The
JAK/STAT pathway is recognized as one of the major mechanisms by which
cytokine receptors transduce intracellular signals (18, 19, 20). This
system is regulated at multiple levels, including JAK activation
(18, 19, 20, 21), nuclear trafficking of STAT factors (18, 22), and negative
feedback loops (23, 24, 25). Gene deletion studies have implicated selected
STAT factors as predominant mediators for a limited number of
lymphokines (26, 27, 28). This signaling pathway influences normal cell
survival and growth mechanisms (29, 30) and may contribute to oncogenic
transformation (31, 32). Initially identified as the primary mediators
of interferon (IFN)-dependent signaling, JAKs and STATs are now known
to be used by many different extracellular signaling proteins (33).
TSH is a major glycoprotein hormone that plays an important role in
regulating the proliferation and differentiation of thyroid cells (6, 7). It can also modulate immune responses in thyroid cells; for
example, TSH actively suppresses the expression of the MHC class I (34, 35) and the intercellular adhesion molecule-1 (ICAM-1) gene (36) at the
transcriptional level. In the present study, the ability of TSH to
regulate JAK/STAT activity was examined. The results demonstrate that
activation of TSHR by TSH is associated with phosphorylation of JAK1,
JAK2, and STAT3. Furthermore, TSHR is associated with JAK1, JAK2, and
STAT3 through the intracellular loop of TSHR.
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RESULTS
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TSH Stimulates the Tyrosine Phosphorylation of JAK1 and JAK2 in
FRTL-5 Thyroid Cells
To investigate whether TSH stimulates JAK1 and JAK2
phosphorylation, FRTL-5 thyroid cells (37) were exposed to TSH (1
[mult] 10-9 M), and tyrosine
phosphorylation of JAK1 and JAK2 was measured by immunoblot assay (Fig. 1
). Cell lysates were separated by 8%
SDS-PAGE and immunoblotted with polyclonal (rabbit) anti-JAK1 dual
phosphospecific antibody (Affinity BioReagents, Inc.,
Golden, CO), anti-JAK2 dual phosphospecific antibody (Affinity BioReagents, Inc.), anti-JAK1 antibody (Transduction Laboratories, Inc., Lexington, KY), and anti-JAK2 antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (Fig. 1
).
Similar experiments were done in parallel in which FRTL-5 thyroid cells
were exposed to rat IFN-
(100 U/ml). INF-
stimulates tyrosine
phosphorylation in JAK1 and JAK2 as expected (Fig. 1A
, upper, and Fig. 1B
, upper). The induced
phosphorylation of JAK1 and JAK2 was at its highest level within 5 min
after IFN-
exposure.

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Figure 1. Effects of TSH and IFN- on the Tyrosine
Phosphorylation of JAK1 and JAK2 in FRTL-5 Thyroid Cells
FRTL-5 cells were grown to near confluency in complete 6H medium (see
Materials and Method) with 5% serum, and cells were
maintained for 6 days with 5H medium that does not contain TSH. The
medium was replaced with fresh medium including the following additions
as indicated: TSH, 1 x 10-9 M, and
IFN- , 100 U/ml. Total cell lysates were prepared at the indicated
time after treatment and analyzed by SDS/PAGE. Phosphorylated forms of
JAK1 and JAK2 were detected using phosphospecific antibodies (see
Materials and Methods).
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Surprisingly, exposure of FRTL-5 cells to TSH significantly induced
phosphorylation of JAK1 (Fig. 1A
, lower) and JAK2 (Fig. 1B
, lower). The phosphorylation of JAK1 and JAK2 was noted as
soon as 5 min after TSH treatment. To determine whether the TSH-induced
phosphorylation of JAK1 and JAK2 is a secondary effect mediated by
cAMP, FRTL-5 cells were treated with forskolin (100
µM); after forskolin treatment, no significant
phosphorylation of JAK1 or JAK2 was detected (data not shown). These
findings suggest that the phosphorylation of JAK1 and JAK2 in response
to TSH are probably not mediated by cAMP. FRT thyroid cells that do not
express the functioning TSHR did not show any significant
phosphorylation of JAK1 or JAK2 in response to TSH (data not
shown).
Similar experiments were carried out using hTSHR-CHO cells, which
express functional human TSHR (hTSHR). After treatment with TSH, the
phosphorylation of JAK1 and JAK2 was identified with anti-JAK1 and
anti-JAK2 dual phosphospecific antibody (Fig. 2
). hTSHR-CHO cells showed a low level of
preexisting phosphorylation of JAK1 and JAK2, and this phosphorylation
was enhanced with TSH treatment (Fig. 2
). These findings suggest that
TSH-induced phosphorylation of JAK1 and JAK2 was specifically mediated
by the TSHR.

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Figure 2. Effects of TSH on the Tyrosine Phosphorylation of
JAK1 and JAK2 in hTSHR-CHO Cells
After hTSHR-CHO cells were grown to near confluency in Coons modified
Hams F12 medium with 5% serum, medium was replaced with fresh medium
including the following additions as indicated: TSH, 1 x
10-9 M. Cell lysates were prepared as
described in the text, and phosphorylated forms of JAK1 and JAK2 were
detected using phosphospecific antibodies (see Materials and
Methods).
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TSH Induces Association of JAK1, JAK2, and STAT3 with TSHR in
hTSHR-CHO Cells
To test whether JAK1 or JAK2 is associated with TSHR, a rabbit
monoclonal antibody (Novocastra, New Castle, UK) against hTSHR was used
for immunoblot analysis after JAK1, JAK2, and STAT3 immunoprecipitation
(Fig. 3
). hTSHR-CHO cells, which are
stably transfected with hTSHR, were treated with TSH, the cell lysate
was immunoprecipitated with anti-JAK1 (Transduction Laboratories, Inc.) or anti-JAK2 (Santa Cruz Biotechnology, Inc.
Santa Cruz, CA) and the precipitated proteins probed with anti-TSHR
antibody. Western blot analysis with this antibody revealed a major
band corresponding to molecular mass of 80 kDa. The results show
that TSHR coprecipitates with JAK1 and JAK2 in hTSHR-CHO cells treated
with TSH but not in untreated cells (Fig. 3A
, lane 1 vs.
lanes 2 and 3; Fig. 3B
, lane 1 vs. lane 2 and 3). The
results also suggest that the association between hTSHR-JAK1 and
hTSHR-JAK2 is mediated by TSH (Fig. 3A
, lanes 2 and 3; Fig. 3B
, lanes 2
and 3).

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Figure 3. Interaction between JAK1, JAK2, STAT3, and TSHR
in Vivo
After hTSHR-CHO cells were grown to near confluency in Coons modified
Hams F12 medium with 5% serum, medium was replaced with fresh medium
including the following additions as indicated: TSH, 1 x
10-9 M. The cell lysates were prepared as
described in the text and immunoprecipitated with anti-JAK1 (A),
anti-JAK2 (B), and anti-STAT3 (C). Immunoprecipitated complexes were
separated by SDS-PAGE and immunoblotted with anti-TSHR antibody.
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Similar immunoprecipitation experiments were carried out to determine
whether STAT3 binds with TSHR in hTSHR-CHO cells. For this experiment,
STAT3 was overexpressed with the expression vector, RcCMV-STAT3 (38) in
hTSHR-CHO cells (39). The lysate from these transfected hTSHR-CHO shows
that STAT3 binds to TSHR (Fig. 3C
, lane 1), and addition of TSH
slightly increases the hTSHR-STAT3 association (Fig. 3C
, lanes 2 and
3). Coprecipitation of TSHR and STAT1 using anti-STAT1 was not observed
in response to TSH (data not shown).
TSH Stimulates the Tyrosine (Y705) Phosphorylation of STAT3, but
not STAT1, in FRTL-5 Thyroid Cells
FRTL-5 cells are cytokine responsive. IFN-
stimulates JAK1 and
JAK2, leading to phosphorylation of STAT1 and STAT3 proteins. To
determine whether exposure to TSH stimulates the phosphorylation of
STAT1 and STAT3 in FRTL-5 cells, the amount of the phosphorylated forms
of STAT1(Y701) and STAT3(Y705) were quantitated using phosphospecific
antibodies (New England Biolabs, inc., Beverly, MA) (Fig. 4
) in cells treated with or without TSH.
IFN-
stimulates the phosphorylation of Y701 in STAT1 (Fig. 4
, upper, lane 2) and of Y705 in STAT3 (Fig. 4
, lower, lane 2) as reported in other cells (18, 19, 20, 21). TSH
induces an increase in tyrosine(Y705)-phosphorylation of STAT3 within
30 min of TSH treatment (Fig. 4
, lower, lane 3) but STAT1
was not phosphorylated after TSH treatment (Fig. 4
, upper,
lane 3). STAT5 and STAT6 were not phosphorylated in response to TSH in
FRTL-5 cells (data not shown).

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Figure 4. TSH-Mediated Tyrosine Phosphorylation of STAT3
After FRTL-5 cells were grown to near confluency in complete 6H medium
with 5% serum, cells were maintained for 6 days with 5H medium that
does not contain TSH. Medium was replaced with fresh medium including
the following additions as indicated: IFN- , 100 U/ml, and TSH,
1 x 10-9 M. Total cell lysates were
prepared and analyzed by SDS-PAGE. Phosphorylated forms of STAT1 and
STAT3 were detected by phosphospecific antibodies.
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The ability of TSH to induce nuclear translocation of STAT3 was tested
in the following experiment. FRTL-5 cells were exposed to TSH, a
nuclear extract was prepared, and an electrophoretic mobility shift
assay (EMSA) was performed with radiolabeled oligonucleotide with the
IFN-
-activated site (GAS) from the 5'-flanking region of the ICAM-1
gene (36). The nuclear extracts from FRTL-5 cells cultured in 5H5%
(lacking TSH) had a minimal basal level of the STAT3/DNA complex (Fig. 5A
, lane 1). The addition of forskolin
(100 µM) and 8-bromo-cAMP (5 µM) does not
induce the formation of the STAT3/DNA complex (Fig. 5A
, lanes 2 and 4).
In contrast, a 30-min treatment with TSH and interleukin-6 (IL-6) with
its receptor (50 nM) induced significant STAT3/DNA complex
formation relative to the control (Fig. 5A
, lanes 4 and 6). Treatment
with IFN-
induced a STAT1/DNA complex; this complex was detected by
anti-STAT1 antibody supershift (data not shown) and forms a faster
migrating complex below the STAT3 homodimer complex (Fig. 5A
, lane 7).
The presence of STAT3 in the TSH-induced complex is demonstrated by an
antibody supershift experiment (Fig. 5B
, lane 5); anti-STAT3 antibody
(Santa Cruz Biotechnology, Inc.) inhibits the formation of
the STAT3/DNA complex.

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Figure 5. Effects of TSH on STAT3/DNA Complex Formation
The ability of TSH to modulate the formation of STAT3/DNA complexes
between nuclear extracts and a 32P-radiolabeled probe
containing the 5'-flanking region of rat ICAM-1 was measured by EMSA.
The nuclear extracts were prepared (see Materials and
Methods) and incubated with radiolabeled oligonucleotides with
the GAS sequence of the rat ICAM-1 promoter (-154 to -120 bp). EMSA
was carried out as described in Materials and Methods.
A, After growth to near confluency, FRTL-5 thyroid cells with a low
passage number (<20) were maintained for 6 days in 5H medium plus 5%
calf serum (no TSH) and treated with various agents: forskolin 100
µM, TSH, 1 x 10-9 M,
8-bromo cAMP, 5 µM, IL-6 with receptor, 50
nM, and IFN- , 100 U/ml for 30 min. B, The nuclear
extract from FRTL-5 cells cultured in 6H5% medium were preincubated
with the following antisera from Santa Cruz Biotechnology, Inc. (detailed in Materials and Methods) before
being evaluated for its ability to form complexes: anti-STAT1,
anti-STAT2, and anti-STAT3.
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TSH Increases the GAS-Derived Transcriptional Activity in FRTL-5
Thyroid Cells
STAT factors participate in cytokine-stimulated regulation of
transcription by binding to the GAS in promoters of specific genes such
as class II transactivator (CIITA), ICAM-1, and IFN regulatory factor 1
(IRF-1). To address whether TSH activates the GAS-dependent promoter
activity, FRTL-5 cells were stably transfected with luciferase reporter
constructs (36) that include different forms of the 5'-flanking region
of the rat ICAM-1 promoter (see Fig. 6A
):
pCAM-175 has a single copy of palindromic GAS sequence; pCAM-175 GAS
mut is a derivative of pCAM-175 in which the GAS sequence is mutated to
a nonpalindromic form that does not bind STAT1 or STAT3;
8xGAS-luciferase construct contains eight GAS sequences linked to a
minimal PRL promoter; and pCAM-97 includes the first 97 bp of the
ICAM-1 promoter (Fig. 6A
).

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Figure 6. Effects of TSH and AG490 on GAS-Derived Promoter
Activity in FRTL-5 Thyroid Cells
FRTL-5 cells were transfected with pCAM-175, pCAM-175 GAS mut, pCAM-97,
and pGAS(x8)-luciferase constructs and selected by G418 for stable
expression of the reporter construct. Cells were treated with TSH and
used to prepare whole-cell lysates to measure the luciferase activity.
A, This figure shows the fragments of the ICAM-1 promoter region cloned
upstream of a promoterless firefly luciferase cDNA in the pGL2-basic
vector (see Materials and Methods). B, Stable FRTL-5
cells stably expressing reporters constructs pCAM-175 and pCAM-97 were
treated with or without TSH and/or AG490, and whole-cell lysates
were prepared to measure luciferase activity. The transfection
efficiency was corrected by cotransfection of pTK-GH expression vector
and determination of GH expression. Promoter activity is represented as
fold increase in value relative to the value in untreated cells.
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FRTL-5 cells expressing these reporter constructs were treated with TSH
and IFN-
for 4 h and the luciferase activity was measured. The
response of the constructs of pCAM-175 and 8xGAS-luciferase to TSH is
biphasic: TSH increases the promoter activity of pCAM-175, and
8xGAS-luciferase up to 12 h and then decreases their activity
after 24 h of TSH treatment (40). In the experiment shown in Fig. 6B
, the activity of promoter constructs was compared after a 4-h TSH
treatment. TSH increases the promoter activity of pCAM-175 (Fig. 6B
)
and 8xGAS-luciferase (Fig. 6D
) 7- to 9-fold, but TSH had no effect on
the promoter activity of pCAM-175 GAS mut. In addition, TSH-mediated
GAS activity was significantly inhibited by tyrphostin AG490 (100
µM), which is a JAK2 inhibitor (Fig. 6C
).
TSH Induces STAT3-Dependent Suppressor of Cytokine Signaling-1
(SOCS-1) Expression, but a Dominant Negative Form of STAT3 Inhibits
TSH-Mediated SOCS-1 Gene Expression in FRTL-5 Thyroid Cells
The above results suggest that TSH stimulates the phosphorylation
of JAK1, JAK2, and STAT3. In the next experiment, we explored whether
TSH stimulates expression of the endogenous SOCS-1 gene, which is
STAT3-dependent in nonthyroidal cells (41, 42, 43). The addition of TSH
significantly induced SOCS-1 gene expression in FRTL-5 thyroid cells in
a time-dependent manner (Fig. 7A
), and
the addition of IL-6 with its receptor, which is known to activate
STAT3, also induces SOCS-1 RNA in thyroid cells (Fig. 7A
, lane 1
vs. lane 4). To determine whether TSH-mediated induction of
SOCS-1 is through the activation of STAT3, we transfected the FRTL-5
cells with a dominant negative form of STAT3 (38) (RcCMV STAT3Y, which
is mutated at tyrosine residue 705), and the transfected cells were
treated with TSH and analyzed for induction of SOCS-1 RNA. The FRTL-5
thyroid cells transfected with wild-type STAT3 expression vector showed
TSH-induced SOCS-1 RNA expression (Fig. 7B
, lane 4), but the cells
transfected with the dominant negative form of STAT3
(RcCMV-STAT3Y) did not; therefore, RcCMV-STAT3Y completely blocked the
TSH-mediated SOCS-1 induction (Fig. 7B
, lane 5).

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Figure 7. Effect of TSH and a Dominant Negative Form of STAT3
on SOCS-1 expression in FRTL-5 Thyroid Cells
A, FRTL-5 cells were grown to near confluency in complete 6H medium
with 5% serum, and cells were maintained for 6 days with 5H medium
that does not contain hydrocortisone, insulin, or TSH. The medium was
replaced with fresh medium containing TSH, 1 x 10-9
M at the indicated time. B, The FRTL-5 cells were
transfected with RcCMV, RcCMV-STAT3, and RcCMV-STAT3Y by
electroporation. Stable transfectants were selected in the presence of
G418 (400 ng/ml), and pooled. RNA was isolated 2 h after the
TSH treatment and analyzed by Northern blot (20 µg/lane) using probes
for SOCS-1 and rat ß-actin. Representative Northern analyses are
shown.
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DISCUSSION
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This work demonstrates that the TSHR signaling pathway involves
JAK1, JAK2, and STAT3 activation and is mediated by direct association
with TSHR in FRTL-5 thyroid cells and hTSHR-transfected CHO cells. In
previous studies, we suggested that TSH has multiple regulatory
mechanisms in the JAK/STAT pathway, which is mainly activated by
cytokines, especially IFN-
(44). It was shown that TSH induces
multiple molecules, such as SOCS and cytokine-inducible SH2 domain
containing protein (CIS), which is involved in negative feedback
regulation of cytokine signaling (44). The expression of SOCS molecules
is dependent on the activation of STAT. This study supports the notion
that TSH induces SOCS molecules via JAK/STAT3 activation.
JAK1 and/or JAK2 is activated via members of the cytokine receptor
superfamily including IFN-
, GH, PRL, erythropoietin, granulocyte
colony-stimulating factor (G-CSF), interleukins, leptin, angiotensin
II, and ligands whose receptor include gp130 (IL-6, oncostain M, and
leukemia-inhibitory factor) (33). Some of these receptors show a marked
preference and/or requirement for JAK2, including GH, PRL,
erythropoietin, and angiotensin II. JAK2 activation by G
protein-coupled receptors has been studied in the case of the
angiotensin II receptor, AT1 (16). AT1 has multiple effects on cardiac
hypertrophy and other cardiac actions through its activation of a
multiple signal cascade (45). The AT1 receptor has a preferential
effect on JAK2 activation and leads to STAT3 phosphorylation (16, 17).
The intracellular loop of the AT1 receptor does not show any
significant homology to the intracellular domain of TSHR. The motif
YIPP, which is present in the C-terminal tail of the AT1 receptor, is
known as the JAK2 binding site. The primary sequence of TSHR does not
include JAK2 binding consensus sequences, box1 and box2 (46, 47), which
were identified from the IFN receptor and the YIPP motif, which is
present in the AT1 receptor (17). The intracellular domain of the
-subunit of the IFN-
receptor has a JAK1 binding domain (46), but
the intracellular C-terminal tail of human and rat TSHR has no
homologous domain with a consensus JAK1 binding site.
These findings suggest that TSHR might have a unique unidentified
binding site for JAK2 or the existence of adaptor molecules that
mediate the binding between JAK2 and TSHR. The recently identified
SH2-Bß protein (48), which has a Src homology (SH2) and pleckstrin
homology (PH) domain, is known as a potent JAK2 activator through
direct SH2-Bß-JAK2 binding during GH signaling. However, the adaptor
or activators for JAK2 during TSHR-mediated signaling have not yet been
identified, and the relationship between TSH and SH2-Bß is
unknown.
Deletion of JAK1 and JAK2 by gene disruption causes perinatal and
embryonic lethality (49, 50), indicating that JAKs are essential for
animal development. Although JAK1-deficient cells
(jak1-/-) are responsive to many
cytokines, they fail to manifest biological responses of class II
cytokine receptors (49). JAK2-deficient mice lack definitive
erythropoiesis in fetal liver, presumably because of a deficiency in
response to cytokines that are required during erythropoiesis (51).
Taken together, these findings indicate that JAKs plays an essential
role in many biological functions. Although JAKs are an essential
signaling kinase, their role in the thyroid gland has not been
precisely elucidated. TSH-mediated JAK activation may be important
during the development and maintenance of differentiated function and
immunological tolerance in the thyroid gland.
One effect of JAK1 and JAK2 activation by TSH is tyrosine
phosphorylation of STAT3 in thyrocytes. STAT5 and STAT6 were not
phosphorylated by TSH in FRTL-5 cells (data not shown). The role of
STAT3 activation by TSH in the thyroid gland is largely unknown.
However, it has been demonstrated that STAT3 plays an important role in
cellular growth and transformation (52, 53); and it can also inhibit
programmed cell death in specific cells (54, 55). TSH is a well known
mitogen in the presence of insulin in FRTL-5 cells (37), and it also
suppresses apoptotic cell death in response to various stimuli (56).
These findings suggest that STAT3 activated by TSH may play a role in
growth promotion and suppression of apoptosis in the thyrocyte.
By analogy to growth factor receptors, TSHR may affect the kinase
cascade by binding through adapter proteins and/or recruitment of
cytosolic tyrosine kinases. The TSHR contains tyrosine residues in its
C-terminal domain, but their roles as phosphorylation sites during
signal transduction are unknown. The human, dog, and rat TSHR have a
putative STAT3 binding motif (YXXQ) in the C-terminal tail region
(Y706-Q709) (1, 2, 3). This putative STAT3 binding motif is also present
in a limited number of other G protein-coupled receptors including the
-adrenergic receptor and endothelin-1 (45). Endothelin-1 is capable
of activating STATs in CHO cells stably transfected with the
ETA receptor. The YXXQ regions of
ETA receptor could serve as docking sites to
facilitate phosphorylation of STAT3 by recruited tyrosine kinases. The
YXXQ motif (Y706-Q709) in TSHR is under investigation in our laboratory
as to whether it provides a docking site for STAT3.
In conclusion, TSHR signaling involves the activation of JAK1, JAK2,
and STAT3 in response to TSH. Furthermore, there is a direct
association of JAK1, JAK2, and STAT3 with TSHR. This novel pathway of
TSHR signaling may be important for the maintenance of normal growth
and function in thyroid cells.
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MATERIALS AND METHODS
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Materials
Highly purified bovine TSH (NIDDK-bTSH I-1; 30 U/mg) was
obtained either from the hormone distribution program of the National
Institute of Diabetes and Digestive and Kidney Diseases (NIH, Bethesda,
MD) or was a previously described preparation, 26 ± 3 U/mg,
homogeneous in the ultracentrifuge, about 27,500 in mol wt, with the
amino acid and carbohydrate composition of TSH. Rat recombinant IFN-
was from Life Technologies, Inc., Inc. (Gaithersburg, MD).
[
-32P] dCTP (3000 Ci/mmol) and
[
-32P] ATP were from NEN Life Science Products (Boston, MA). The source of all other materials
was Sigma (St. Louis, MO) unless otherwise noted.
Cell Culture
FRT rat thyroid cells were grown in Coons modified F12 medium
supplemented with 5% calf serum and 1 mM nonessential
amino acids (Life Technologies, Inc., Gaithersburg, MD).
FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD)
were a fresh subclone (F1) that had all properties previously detailed
(37). Their doubling time with TSH was 36 ± 6 h; without
TSH, they did not proliferate. After 6 days in medium with no TSH,
addition of 1 x 10-10 M TSH
stimulated thymidine incorporation into DNA by at least 10-fold. Cells
were diploid and between their 5th and 20th passage. Cells were grown
in 6H medium consisting of Coons modified F12 supplemented with 5%
calf serum, 1 mM nonessential amino acids, and a mixture of
six hormones: bovine TSH (1 x 10-10
M), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin
(5 µg/ml), glycyl-L-histidyl-L-lysine acetate
(10 ng/ml), and somatostatin (10 ng/ml). Fresh medium was added to all
cells every 2 or 3 days, and cells were passaged every 710 days. In
individual experiments, cells were shifted to 5H medium with no TSH and
5% calf serum, during or after which time TSH, IFN-
, forskolin, or
other agents were added, as noted.
Construction of Promoter/Luciferase Chimeric Plasmids
Chimeric expression constructs using fragments of pCAM-1822,
containing 1,822 bp of the 5'-flanking region of the rat ICAM-1 gene,
were made by high-fidelity PCR (36). Promoter segments were amplified
with Pfu polymerase using appropriate forward and reverse primers
including a BglII site (5'-end) and HindIII site
(3'-end). Mutations of promoter sequences were generated by PCR using
primers incorporating the mutated sequence. Amplified fragments were
ligated into the pGL2-basic vector containing a luciferase reporter
gene and the correct DNA sequence confirmed by DNA sequencing analysis.
The 5'-deletion mutants included pCAM-175, pCAM-175 GAS mut, and
pCAM-97, containing the indicated fragment of the ICAM-1 promoter
starting from the numbered nucleotide at the 5'-end and extending to +1
bp, the start of protein translation. Eight copies of the consensus
sequence TTCTCGGAA (18, 20) were placed upstream of the minimal
promoter to generate 8xGAS-luciferase. All plasmid preparations were
purified twice by CsCl gradient centrifugation.
Transfection
Stably transfected FRTL-5 cells were selected after
electroporation of pGL2-basic, pCAM-175, pCAM-175 GAS mut, pCAM-97, and
8xGAS-luciferase plasmid constructs. Near-confluent FRTL-5 cells in 6H
medium were cotransfected with 20 µg of plasmid DNA and 10 µg of
pRcNeo. PRcNeo contains a portion of human early cytomegalovirus
promoter from pRc/CMV vector. After 2 days, 400 µg/ml of G418
(Life Technologies, Inc.) were added to the medium, and
after 3 weeks the G418-resistant colonies were pooled and used for
experiments. To test the effect of cytokines and hormones, cells were
grown to 7080% confluency in 6H medium, and then maintained without
TSH (5H medium) for 5 days, at which time they were exposed to various
concentrations of the noted agents (IFN-
, TSH) for 4 h before
luciferase activity was measured.
Nuclear Extracts
FRTL-5 cells were grown in the presence of complete 6H medium
until approximately 80% confluency, and then maintained in 5H medium
(lacking TSH) for the time periods as noted. After cells were exposed
to TSH, nuclear extracts were prepared from the FRTL-5 cells as
described (50), with the exception that they were washed with
Dulbeccos modified PBS without Mg2+ and
Ca2+. After centrifugation at 500 x
g, the cells were resuspended in 5 pellet volumes of 0.3
M sucrose and 2% Tween-40 in Buffer A [10
mM HEPES-potassium hydroxide (KOH); pH 7.9,
containing 10 mM KCl, 1.5
mM MgCl2, 0.1
mM EGTA, 0.5 mM
dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl
fluoride (PMSF), 2 µg/ml leupeptin, and 2 µg/ml pepstatin A].
After freezing, thawing, and gently homogenizing, nuclei were isolated
by centrifugation at 25,000 x g in Buffer A with a 1.5
M sucrose cushion. Isolated nuclei were lysed in
Buffer B [10 mM HEPES-KOH; pH 7.9, 420
mM NaCl, 1.5 mM
MgCl2, 0.1 mM EGTA, 10%
glycerol, 0.5 mM DTT, 0.5
mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml
pepstatin A], and centrifuged at 100,000 x g for
1 h. The supernatant was dialyzed against a buffer containing 10
mM Tris (pH 7.9), 1 mM
MgCl2, 1 mM DTT, 1
mM EDTA, and 5% glycerol, and aliquoted. Samples
were stored at -70 C.
EMSA
Oligonucleotides derived from the rat ICAM-1 gene,
5'-CGAGGTTTCCGGGAAAGTGGCCCC-3' (a putative GAS is
underlined), were used as the DNA oligonucleotide for EMSA.
Gel-purified oligonucleotides were labeled with
[
-32P] ATP using T4 polynucleotide kinase,
and purified on an 8% native polyacrylamide gel. EMSA was performed as
previously described (49, 50). Binding reactions in high salt with
detergent were carried out in a solution of 1.5 fmol
[32P] DNA, 2 µg nuclear extract, and 0.5 µg
poly(dI-dC) in 10 mM Tris-Cl (pH 7.9), 5
mM MgCl2, 50
mM KCl, 1 mM DTT, 1
mM EDTA, 0.1% Triton X-100, and 12.5% glycerol.
Where indicated, unlabeled double-stranded oligonucleotides were also
added as competitor to the binding reaction and incubated for 20 min
before the addition of labeled DNA. After incubation, reaction mixtures
were analyzed by electrophoresis on 4 or 4.5% native polyacrylamide
gels at 160 V in 0.5x Tris-borate-EDTA buffer. Gels were dried
and autoradiographed. For the supershift assay, extracts were incubated
in the same buffer containing specific antibodies or a control antibody
at 20 C for 1 h before being processed as indicated above.
Immunoprecipitation and Western Blot Analysis
For immunoprecipitation, cells were washed with ice-cold STE
[NaCl 150 mM, 50 mM Tris (pH 7.4), 1
mM EDTA] and were lysed in lysis buffer [20
mM Tris (pH 7.4), 1 mM EDTA, 5 mM
EGTA, 10 mM MgCl2, 50 mM
ß-glycerophosphate, 2 mM DTT, 1 mM
Na3VO4, 1 mM
PMSF, 4 µg/ml aprotinin]. Cell lysates were incubated with
antibodies against JAK1 (Transduction Laboratories, Inc.,
Lexington, KY), JAK2 (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), STAT1 (New England Biolabs, Inc.,
Beverly, MA), and STAT3 (New England Biolabs, Inc.)
at 4 C for 2 h, and immune-complexes were precipitated by
protein A or G. They were washed twice in lysis buffer with 500
mM NaCl and once with ST (50 mM Tris, pH 7.4,
150 mM NaCl). Precipitated proteins were released from
protein A beads by boiling in the presence of 1x SDS sample buffer and
separated by SDS-PAGE. Immunoblot analyses were performed using
anti-TSHR (Novocastra, New Castle, UK).
For the Western blot, adherent FRTL-5 cells were stimulated in the
presence or absence of TSH and IFN-
for the indicated period of time
at 37 C. The treated cells were scraped, lysed by addition of SDS
sample buffer [62.5 mM Tris-HCl (pH 6.8), 6% (wt/vol)
SDS, 30% glycerol, 125 mM DTT, 0.03% (wt/vol) bromophenol
blue] and separated by 10% SDS-PAGE along with biotinylated mol wt
standards. The proteins were transferred to a nitrocellulose membrane
by electrotransfer for 2 h. After soaking the membrane in blocking
buffer (1x Tris-buffered saline, 0.1% Tween-20 with blocking reagent
5% milk) the membrane was incubated with the primary antibody
overnight at 4 C. Primary antibodies were rabbit polyclonal IgG,
affinity purified (Affinity BioReagents, Inc., Golden, CO)
specifically for phosphorylated forms of JAK1 or JAK2. Blots were
developed using horseradish peroxidase (HRP)-linked antirabbit
secondary antibody and chemiluminescent detection system (Phototope-HRP
Western Blot Detection Kit, New England Biolabs, Inc.).
Other Assays
Protein concentration was determined by Bradfords method
(Bio-Rad Laboratories, Inc. Hercules, CA) and used
recrystallized BSA as the standard.
Statistical Significance
All experiments were repeated at least three times with
different batches of cells. Values are the mean ± SE
of these experiments. Significance between experimental values was
determined by two-way ANOVA.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Ick Dong Yoo, Korea Research Institute of
Bioscience and Biotechnology, for helpful discussions and
critical reading of this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Minho Shong, M.D. Associate Professor, Department of Internal Medicine, School of Medicine, Chungnam National University, 640 Daesadong Chungku Taejon 301040, Korea.
Supported by Biotech 2000 (98-N102-04-A-01), Molecular Medicine
Research Group Program, Ministry of Science and Technology, and Korea
Research Foundation (KRF-99041-F00159 F1104), Korea.
Received for publication August 6, 1999.
Revision received January 7, 2000.
Accepted for publication February 1, 2000.
 |
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