Thyrotropin Induces SOCS-1 (Suppressor of Cytokine Signaling-1) and SOCS-3 in FRTL-5 Thyroid Cells
Eun Shin Park,
Ho Kim,
Jae Mi Suh,
Soo Jung Park,
O-Yu Kwon,
Young Kun Kim,
Heung Kyu Ro,
Bo Youn Cho,
Jongkyeong Chung and
Minho Shong
Department of Internal Medicine (E.S.P., H.K., J.M.S., S.J.P.,
Y.K.K., H.K.R., M.S.) Department of Anatomy (O.-Y.K.)
Chungnam National University School of Medicine Taejon, 301040,
Korea
Department of Internal Medicine (B.Y.C.) Seoul
National University School of Medicine 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 has multiple physiological roles: it is
required for growth, differentiation, and function of the thyroid
gland, and it regulates transcription of interferon-
(IFN-
)-responsive genes in thyrocytes, including genes for the major
histocompatibility complex and intercellular adhesion molecule-1. This
report demonstrates that TSH induces the expression of suppressor of
cytokine signaling (SOCS)-1 and -3 proteins and alters the
phosphorylation state of signal transducer and activator of
transcription (STAT) proteins STAT1 and STAT3. The expression of SOCS-1
and SOCS-3 and the phosphorylation state of STAT1 and STAT3 were
examined after treatment with TSH or IFN-
in either TSH-sensitive
FRTL-5 thyroid cells or TSH-insensitive FRT and buffalo rat liver (BRL)
cells, which lack functional TSH receptors. SOCS-1 and SOCS-3 are
constitutively expressed in FRTL-5 cells, but not in FRT and BRL cells.
IFN-
up-regulated SOCS-1 and SOCS-3 RNA and protein in FRTL-5 cells,
as reported previously for nonthyroid cells. Interestingly, TSH
also significantly induced SOCS-1 and SOCS-3 in FRTL-5 cells, but not
in FRT and BRL cells. When SOCS-1 or SOCS-3 was overexpressed in FRTL-5
cells, STAT1 phosphorylation at Y701 and STAT1/DNA complex formation in
response to IFN-
were reduced. Furthermore, overexpression of either
SOCS-1 or SOCS-3 significantly inhibited the IFN-
-mediated
transactivation of the rat ICAM-1 (intercellular adhesion molecule-1)
promoter. TSH and IFN-
had different effects on STAT1 and STAT3
phosphorylation. The phosphorylation of Y701 in STAT1, which is
responsible for homodimer formation, nuclear translocation, and DNA
binding, was specifically stimulated by IFN-
, but not by TSH or
forskolin. However, the phosphorylation of S727 in STAT1 was induced by
IFN-
, TSH, and forskolin. TSH induced phosphorylation of both Y705
and S727 in STAT3, while IFN-
phosphorylated only the Y705. In
addition, we found that SOCS-3 was associated with JAK1 and JAK2 and
that these associations were stimulated by TSH. These findings
demonstrate that TSH induces SOCS in thyroid cells and provides the
evidence of signal cross-talk between TSH and cytokines in thyroid
cells.
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INTRODUCTION
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Cytokines are elicited during the progression of autoimmune
thyroid diseases, and they exert multiple biological effects such as
abnormal regulation of immune response genes and altered growth and
function of thyroid cells (1, 2). The interactions of type I and type
II interferons and interleukin-6 type cytokines with their
specific receptors activate receptor-associated Janus kinases (JAKs)
(3, 4). The activation of the JAKs is crucial for propagation of the
signal cascade via proteins such as STAT (signal transducer and
activator of transcription) factors (3, 5, 6, 7, 8, 9). The activated JAKs
phosphorylate receptors and STATs, leading to altered gene expression,
cellular differentiation, and cell growth properties (5, 6, 7, 8, 9).
The proinflammatory cytokine interferon-
(IFN-
) has pleiotropic
effects on cells and is involved in a variety of pathophysiological
conditions associated with autoimmune thyroid disease (10). The
thyrocyte response to IFN-
is mediated by two distinct classes of
proteins, Janus kinases (JAK1 and JAK2) and STAT1 (11). The activation
of STAT1 participates in the regulation of many genes by binding to an
IFN-
-activated site (GAS) in the promoter region (12, 13, 14, 15, 16).
IFN-
-activated genes include interferon regulatory factor-1 (IRF-1)
(12), IRF-2 (13), intercellular adhesion molecule-1 (ICAM-1) (14), and
MHC class II transactivator (CIITA) (15, 16). Recently, four proteins
were identified that are members of the suppressors of cytokine
signaling family, SOCS-1, SOCS-2, SOCS-3, and cytokine-inducible SH2
domain-containing protein (CIS)(17, 18, 19, 20, 21, 22). These proteins appear to
provide negative regulation of cytokine signal transduction pathways
(17, 18, 19, 20, 21, 22).
TSH has long been regarded as an important physiological regulator of
the growth and function of the thyroid gland (2). The function of TSH
depends on the receptor-mediated activation of adenylate cyclase
leading to cAMP synthesis and protein kinase A (PKA)-mediated
phosphorylation of transcription factors that regulate thyroid gene
expression (23, 24). Despite the pivotal roles of cAMP and PKA in TSH
signaling, it is likely that additional mechanisms are involved in the
regulation of thyroid-specific gene expression and cellular function in
the thyroid. It was recently observed that TSH also negatively
regulates the expression of MHC class I (25, 26, 27) and ICAM-1 (28, 29) by
inhibiting STAT1 (30). These findings suggest that TSH down-regulates
the IFN-
-activated JAK-STAT signaling pathway.
This report demonstrates that TSH induces SOCS (suppressor of cytokine
signaling) proteins, which are negative regulators of cytokine
signaling, and alters the phosphorylation state of STAT factors. These
observations indicate that TSH signaling interacts with the JAK-STAT
pathway; thus, there is signaling cross-talk between TSH and cytokines,
and this functional interaction contributes to maintaining thyrocyte
function during normal and pathological conditions.
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RESULTS
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TSH-Mediated Expression of SOCSs and CIS RNA in FRTL-5, FRT, and
BRL Cells
The expression of SOCS-1, SOCS-2, SOCS-3, and CIS was examined by
Northern blot using total RNA from FRTL-5 cells maintained in 5% calf
serum and 3H medium lacking hydrocortisone, insulin, and TSH. IFN-
increases SOCS-1, SOCS-3, and CIS RNA (Fig. 1A
, lanes 2 and 7). After addition of
IFN-
, CIS RNA expression was greater at 2 h than at 4 h
(Fig. 1A
, lane 2), but SOCS-1 and SOCS-3 RNA expression was greater at
4 h than at 2 h (Fig. 1A
, lane 7). SOCS-2 RNA was not
detected in FRTL-5 cells even after stimulation with IFN-
(data not
shown). TSH, which is important for the growth and function of FRTL-5
cells (24, 25, 26), significantly augmented the SOCS-1 and SOCS-3 RNA
levels (Fig. 1A
, lane 3 and lane 8) as compared with the control (Fig. 1A
, lane 1 and lane 6). Forskolin (100 µM) mimicked the
action of TSH on SOCS-1 and SOCS-3 RNA. In contrast to SOCS-1 and
SOCS-3, CIS RNA was not induced by TSH or forskolin at 2 h or
4 h (Fig. 1A
, lanes 4 and 9). The induction of SOCS-1 and SOCS-3
expression by TSH and forskolin was transient; maximal induction was
within 2 h of treatment, and within 4 h of treatment
near-basal expression was restored. The antithyroid drug, methimazole
(MMI), had no effect on SOCS-1, -3, or CIS RNA expression.

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Figure 1. Effects of IFN- , TSH, Forskolin (FSK), and
Methimazole (MMI) on SOCS-1, SOCS-3, and CIS RNA Levels in Rat FRTL-5,
FRT, and BRL Cells
A, After FRTL-5 cells were grown to near confluency in complete 6H
medium with 5% serum, cells were maintained for 6 days with 3H medium
that does not contain hydrocortisone, insulin, or TSH. The medium was
replaced with fresh medium including the following additions as
indicated: 100 U/ml IFN- , 1 x 10-9 M
TSH, 100 µM FSK (forskolin), and 0.5 mM MMI.
B, FRT and BRL cells were grown to near confluency in their
respective media. The cells were then treated with or without 100 U/ml
IFN- or 1 x 10-9 M TSH. RNA was
isolated 2 h after the final treatment and subjected to Northern
analysis (20 µg/lane) using probes for ICAM-1, MHC class I, and rat
ß-actin. Representative Northern analyses are shown.
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Expression of SOCS-1, -2, -3, and CIS was also examined in FRT thyroid
cells and BRL cells, which lack thyroid transcription factor-1 (TTF-1)
(31, 32) and thyroid-specific gene expression (TSH receptor,
thyroglobulin, and thyroperoxidase). In these cells, IFN-
induced
SOCS-1 RNA (Fig. 1B
, lanes 2 and 5), but TSH did not (Fig. 1B
, lanes 3
and 6). SOCS-2, SOCS-3, and CIS RNA were not detectable in FRT or BRL
cells with or without IFN-
stimulation (data not shown).
Western blot analysis (Fig. 2
)
demonstrated the TSH-mediated induction of SOCS-1 and SOCS-3 proteins
in FRTL-5 cells (Fig. 1
). The SOCS-1 and -3 proteins were readily
detectable in FRTL-5 cells maintained in 3H 5% medium (Fig. 2
, lane
1), and the level increased and was maintained at a high level after
the addition of TSH (Fig. 2
, lanes 24). As expected, IFN-
augmented the expression of SOCS-1 and SOCS-3 protein; the level of
expression at 6 h after treatment of IFN-
was higher than with
treatment of TSH (Fig. 2
, lane 2 vs. lane 4). SOCS-2 protein
was not detected by Western blot, which is consistent with its Northern
blot analyses (data not shown).

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Figure 2. Effects of TSH and IFN- on the Level of SOCS-1
and SOCS-3 Proteins
FRTL-5 cells were treated with TSH and IFN- as indicated. Untreated
cells are shown in lane 1. Total cell lysates were resolved by SDS/PAGE
and analyzed using anti-SOCS-1 and anti-SOCS-3 antibodies.
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Overexpression of SOCS-1 and SOCS-3 Protein in FRTL-5 Cells
Inhibits Tyrosine Phosphorylation, Nuclear Translocation, and
Transactivation of STAT1 in Response to IFN-
STATs participate in cytokine-stimulated signaling pathways in the
thyroid. The following experiments were designed to test whether SOCS-1
and SOCS-3 may be involved in signaling in FRTL-5 cells stimulated by
TSH and IFN-
. In particular, the effect of SOCS RNA and protein
induction on tyrosine 701 phosphorylation of STAT1 was directly
examined using a transient transfection experiment. FRTL-5 cells were
transfected with expression vectors for SOCS-1 (pEF-SOCS-1) or SOCS-3
(pEF-SOCS-3) (17) and treated with IFN-
(100 U/ml) for 30 min.
Western blot and electrophoretic mobility shift assays (EMSA) were then
carried out with whole-cell lysates and nuclear extracts, respectively.
The level of phosphorylation of Y701 in STAT1 was evaluated using an
antibody that specifically recognizes the Y701 phosphotyrosine in
STAT1. IFN-
stimulated the phosphorylation of Y701 on STAT1 in
FRTL-5 cells transfected with the empty expression vector, pEF (Fig. 3A
, lane 3). However, FRTL-5 cells
overexpressing SOCS-1 or SOCS-3 had a significantly lower level of Y701
phosphorylation after IFN-
stimulation (Fig. 3A
, lanes 4 and 5).
EMSA was carried out with nuclear extracts from these cells and a
radiolabeled probe including the GAS from the 5'-flanking region of the
rat ICAM-1 gene. Overexpression of SOCS-1 or SOCS-3 significantly
decreased the amount of STAT1/DNA complex detected in extracts from
IFN-
-treated FRTL-5 cells (Fig. 3B
, lane 3 vs. lanes 4
and 5).
Reporter constructs were prepared with promoters containing or lacking
a functional GAS element and the luciferase reporter gene. The function
of these constructs was tested in FRTL-5 cells after cotransfection in
the presence or absence of SOCS-1 and SOCS-3 expression vectors. The
promoter variants included 5'-deleted fragments of the rat ICAM-1
promoter, pCAM-431, pCAM-175, pCAM-175 GAS mut, and pCAM-97 (Fig. 4
). pCAM-431 includes Sp1, nuclear
factor-
B (NF-
B), GAS, and TATA regulatory elements, but pCAM-175
includes only the GAS element (11-bp core element, 5'-tttccgggaaa-3')
and the TATA element. pCAM-175 GAS mut has a mutated nonpalindromic GAS
element. Figure 5A
shows that the
promoter activities of pCAM-431 and pCAM-175, but not pCAM-175 GAS mut,
were strongly stimulated by IFN-
, suggesting that the GAS is
required for IFN-
inducibility of this promoter. Addition of TSH
dramatically inhibited the promoter activities of pCAM-431 and pCAM-175
in the presence or absence of IFN-
(Fig. 5A
). Thus, TSH inhibits
both the induced expression level and the basal expression level of
these constructs. These results strongly suggest that the promoter
function of the GAS element is positively regulated by IFN-
and
negatively regulated by TSH.

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Figure 4. Schematic Representation of the 5'-Region of the
Rat ICAM-1 Promoter-Luciferase Reporter Constructs
The fragments of the ICAM-1 promoter region cloned upstream of a
promoterless firefly luciferase cDNA in the pGL2-basic vector is shown
(see Materials and Methods).
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The promoter activity and IFN-
-responsiveness of pCAM-431 and
pCAM-175 were also tested in the absence or presence of SOCS-1 or
SOCS-3 overexpression. The activities of pCAM-431 and pCAM-175 were
increased about 8- and 7-fold in the presence of IFN-
, respectively.
However, cotransfection of pEF-SOCS-1 or pEF-SOCS-3 with the reporter
plasmids strongly decreased not only the basal activities of pCAM-431
and pCAM-175 (Fig. 5B
), but also their induced activities by IFN-
(Fig. 5B
). The inhibitory abilities of SOCS-1 and SOCS-3 to the
IFN-
-mediated stimulation of the promoter activities of pCAM-431 and
pCAM-175 were similar (Fig. 5B
).
TSH Induces Phosphorylation of Tyrosine 705 and Serine 727 Residues
of STAT3, but Only Serine 727 of STAT1
The results presented above show that TSH stimulates the
expression of SOCS-1 and SOCS-3 in FRTL-5 cells (Fig. 1
) and that SOCS
overexpression inhibits IFN-
-mediated tyrosine phosphorylation, DNA
binding, and transactivation activities of STAT1 (Figs. 3
and 5
). As
previously described by others (17, 18, 19), the expression of SOCS is
dependent on the activation of STAT1 and STAT3 by various cytokines. To
understand whether the TSH-mediated induction of SOCS-1 and SOCS-3 was
also regulated by STAT(s), we examined phosphorylation of tyrosine 701
(Y701) and serine 727 (S727) residues of STAT1 and phosphorylation of
tyrosine 705 (Y705) and S727 of STAT3 in FRTL-5 cells. Figure 6A
shows that IFN-
, but not TSH or
forskolin, increased phosphorylation of tyrosine 701 in STAT1. In
contrast, IFN-
, TSH, and forskolin all stimulated phosphorylation of
serine 727 in STAT1 (Fig. 6A
). Different results were observed for
STAT3 (Fig. 6B
); TSH induced the phosphorylation of both Y705 and S727
in STAT3 (Fig. 6B
, lane 3), and IFN-
induced phosphorylation of Y705
without affecting S727 phosphorylation (Fig. 6B
, lane 2). To address
whether stimulation of FRTL-5 cells with TSH interferes with the
phosphorylation of STAT1, proteins from lysates were immunoblotted with
a specific antibody against tyrosine-phosphorylated (Y701) STAT1 (Fig. 6C
). Stimulation of FRTL-5 cells with IFN-
(100 U/ml) resulted
in a dramatic increase of phosphorylation in tyrosine 701 of STAT1
(Fig. 6C
, lane 2), as expected. However, pretreatment with TSH
significantly inhibited the IFN-
-induced phosphorylation of Y701
(Fig. 6C
, lane 4).

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Figure 6. TSH Modulates the Phosphorylation of STAT1 and
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
did not contain TSH. The medium was replaced with fresh medium
including the following additions as indicated: 100 U/ml IFN- ,
1 x 10-9 M TSH, and 100 µM
FSK. Total cell lysates were prepared 30 min after treatment and
analyzed by SDS/PAGE. Phosphorylated forms of STAT1 were detected by
phosphospecific antibodies. In panel C, the FRTL-5 cells were
pretreated with TSH for 4 h, and medium was replaced with fresh
medium including the following additions as indicated: 100 U/ml
IFN- , 1 x 10-9 M TSH, for 30 min.
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For many cytokines, the signaling pathway involves the following steps:
binding to their receptors, activation of receptor-associated
cytoplasmic kinases (JAKs), and JAK-dependent phosphorylation of the
receptor cytoplasmic domain and its associated STATs. SOCS proteins
recognize and inhibit activated signaling molecules, including JAKs and
cytokine receptors, through their SH2 and N-terminal domains. Thus, the
effect of TSH on the association between SOCS-3 and JAK1/JAK2 was
investigated in FRTL-5 cells. This experiment demonstrated that SOCS-3
was associated with JAK1 and JAK2 and that these associations increased
in cells treated with TSH (Fig. 7
).

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Figure 7. Association of JAK1 with SOCS-1 and SOCS-3
in Vivo
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
did not contain TSH. Medium was replaced with fresh medium including
1 x 10-9 M TSH as indicated. Cell
extracts were immunoprecipitated (IP) with antibodies specific for JAK1
and immunoblotted with anti-SOCS-1 (panel A) and anti-SOCS-3 (panel B).
The filter was washed and reprobed with anti-JAK-1 and anti-IgG.
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DISCUSSION
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The present report shows that TSH treatment of FRTL-5 cells
induces SOCS proteins and alters the phosphorylation state of STATs and
suggests that TSH-induced SOCS-1 and -3 are negative regulators of
IFN-
signaling in these cells. Somatic cell genetics coupled with
expression cloning has identified proteins that mediate interferon
signal transduction (3, 4, 5, 6, 7, 8). Several groups have described a new family
of cytokine-inducible proteins that inhibit the Jak-STAT signaling
cascade. These proteins have been termed suppressors of cytokine
signaling (SOCS) (17, 20, 21), STAT-induced STAT inhibitors (SSI) (19),
CIS (18), and Jak binding protein (JAB) (18). The basal expressions of
SOCS-1, SOCS-2, SOCS-3, and CIS RNA were observed in limited organs;
SOCS-1 in thymus, spleen, and lung, SOCS-2 in testis, liver, and lung,
SOCS-3 in the lung, spleen, and thymus, whereas CIS expression was more
widespread, including testis, heart, lung, and liver. In this study,
IFN-
increased the expression of SOCS-1, SOCS-3 and CIS, but not
SOCS-2, in FRTL-5 thyroid cells. All of these expressions are related
to the negative regulation of the IFN-
signaling cascade, but the
relative potencies and time-dependent mechanisms for inhibiting
JAK-STAT signaling cascade have not yet been determined. TSH increases
the expression of SOCS-1 and SOCS-3, but not CIS, through the
activation of TSH signaling in thyroid cells. These findings suggest
that SOCS-1 and SOCS-3 are more important than CIS in TSH-mediated
negative regulation of IFN-
signaling.
Recent studies show that TSH has multiple effects on the regulation of
immune response genes in thyroid cells, including MHC class I (25, 26, 27),
MHC class II (33, 34), and ICAM-1 genes (28). TSH suppresses MHC class
I and ICAM-1 gene expression by modulating the action of specific
transcription factors for these genes (25, 26, 27). These studies also
showed that TSH can interfere with the induction of these genes by
IFN-
(28). TSH typically triggers the cAMP signaling pathway, which
can interrupt JAK-STAT, and NF-
B activation (35, 36). This study
shows that induction of SOCS-1 and SOCS-3 is another mechanism
contributing to signal interference and cross-talk between TSH and
IFN-
in thyroid cells.
The SOCS proteins are relatively small molecules comprised mainly of an
SH2 domain and a C-terminal homology domain termed SOCS box (20, 37).
SOCS proteins recognize activated signaling molecules including JAK
kinases and cytokine receptors through their SH2 and N-terminal domains
and inhibit their activity (18, 19, 37). Zhang et al. (39)
reported that the SOCS box mediates interactions with elongins B and C,
which in turn may couple SOCS proteins and their substrates, JAK
kinases or cytokine receptors, to the proteosomal degradation pathway
(37, 38). SOCSs expression is induced in response to a range of
cytokines (17, 18, 19, 20, 21, 22) or hormones (22), and these proteins are therefore
thought to form part of a negative feedback loop. In addition to their
roles as negative regulators of cytokine signal transduction, SOCSs may
have multiple biological functions mediated through their SOCS box, SH2
domain, and GTPase domains.
Although the regulation of SOCS-1 and -3 in specific tissues is not yet
fully identified, their expression is dependent on STAT1 or STAT3
activation (19). With respect to the current study, it appears that
TSH-mediated induction of SOCS-1 and -3 may be related to TSH-dependent
phosphorylation of S727 in STAT1 and Y705 and S727 in STAT3. The
phosphorylation of S727 of STAT1 and STAT3 is required for maximal
transcriptional activation (5, 7) and cell survival (39, 40). It has
been demonstrated that the MEK inhibitor PD98059-sensitive kinase can
phosphorylate S727 in STAT1 and STAT3 (7), but it is not clear which
kinase(s) mediates the TSH-induced phosphorylation of S727 in FRTL-5
thyroid cells. TSH strongly induced the phosphorylation of Y705 in
STAT3 but not the phosphorylation of Y701 in STAT1 (Fig. 6A
, lane 3,
vs. Fig. 6B
, lane 3). The molecular mechanism of the
phosphorylation of Y705 tyrosine in STAT3 by TSH is not currently
known, but it may involve the activation of the TSH receptor and JAKs,
especially JAK2 (data not shown). IFN-
is more potent than TSH in
the induction of SOCS-1 and SOCS-3 in FRTL-5 cells (Fig. 1A
). When we
treated the cells with IFN-
and TSH in FRTL-5 cells, they did not
show synergistic effects on SOCS RNA induction (data not shown) because
TSH inhibits IFN-
-mediated STAT1 tyrosine phosphorylation (Fig. 3C
).
If TSH acts as a general negative regulator of cytokine action in
thyroid tissue, then this study may be relevant to the pathogenesis of
autoimmune thyroid diseases such as Graves disease and Hashimotos
thyroiditis. Many cytokines are involved in the amplification or
maintenance of the autoimmune process in the thyroid gland. Without
doubt, TSH is one of the most important physiological factors required
to maintain cellular survival and differentiated functions in thyroid
gland. STAT1 activation (both constitutive and cytokine-stimulated) is
involved in growth inhibition or apoptosis in other cell types, and
IFN-
induced STAT1 activation is also involved in growth inhibition
in FRTL-5 thyroid cells (our unpublished data). IFN-
is
involved in many immunological perturbations during autoimmune thyroid
diseases. Many cytokines, including type 1 interferons and
interleukin-6 type cytokines, act through the same JAK-mediated
signaling pathway as IFN-
. It is possible that TSH-mediated negative
regulation via SOCS-1 and SOCS-3 may apply to other cytokine signals
beside IFN-
. Thus, TSH could be a major rescue factor for
maintaining thyrocyte survival and function during autoimmune thyroid
disease.
One of the major factors in the pathogenesis of Graves
hyperthyroidism is thyroid- stimulating autoantibodies. The effect of
these autoantibodies on thyroid signal transduction is not known, but
it is possible that they could induce SOCSs in thyroid cells. Continued
studies of TSH regulation of cytokine signaling may elucidate the
pathogenesis of such autoimmune thyroid diseases.
In summary, this study demonstrates the stimulatory effects of TSH on
SOCS-1 and SOCS-3 expression in FRTL-5 cells and its effect on the
TSH-induced phosphorylation/activation state of STAT1 and STAT3.
Further studies of these cytokines and their effects on thyroid tissue
will improve understanding of the mechanisms and physiological
importance of cross-talk between the hormone and cytokine signaling
pathways.
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MATERIALS AND METHODS
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Materials
Highly purified bovine TSH was obtained either from the hormone
distribution program of the National Institute of Diabetes and
Digestive and Kidney Diseases, NIH (NIDDK-bTSH I-1; 30 U/mg) 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 (Gaithersburg, MD).
[
-32P] dCTP (3000 Ci/mmol) and
[
-32P]ATP were from DuPont-Merck
Pharmaceutical Co. (Wilmington, DE). The source of all other
materials was Sigma (St. Louis, MO) unless otherwise
noted.
Cell Culture
Buffalo rat liver cells (BRL 3A, ATCC No. CRL 1442)
were grown in Coons modified Hams F-12 (Sigma, St.
Louis, MO) supplemented with 5% FCS (Biofluids,
Rockville, MD). 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.). FRTL-5
rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a
fresh subclone (F1) that had all properties previously detailed (42).
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
x10-10 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
x10-10 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 passed every 710 days. In individual
experiments, cells were shifted to 3H (which is devoid of
hydrocortisone, insulin, and TSH) or 5H medium (which is devoid of TSH)
with 5% calf serum during or after TSH, IFN-
, and forskolin, or
other agents were added.
RNA Isolation and Northern Analysis
Total cellular RNA was isolated by standard procedures and
Northern analysis was performed as described (25, 26, 27). Final washes
were carried out at 65 C in 1x SSPE (150 mM NaCl,
10 mM NaH2PO4,
1 mM EDTA, pH 7.4). The SOCS-1, -2, -3, and CIS probes were
the purified insert fragments of the expression vectors pEF-SOCS-1, -2,
-3, and CIS, respectively [provided by Dr. Starr, The Walter Eliza
Hall Institute for Medical Research, Victoria, Australia (17)].
All probes were radiolabeled by a random priming protocol
(Amersham Pharmacia Biotech, Arlington Heights, IL).
Construction of Promoter/Luciferase Chimeric Plasmids
Chimeric expression constructs using fragments of pCAM-1822,
containing 1822 bp of the 5'-flanking region of the rat ICAM-1 gene,
were made using high-fidelity PCR (28). 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 was confirmed
by DNA sequencing analysis. The 5'-deletion mutants included pCAM-431,
pCAM-175, pCAM-175 GAS mut, and pCAM-95 (see Fig. 4
), 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. All plasmid preparations were purified twice by CsCl
gradient centrifugation.
Transfection
Transient transfections were performed in FRTL-5 or FRT cells at
80% confluency. For transfection, 20 µg of pCAM-431 or equivalent
molar amounts of the deletion mutants or pGL2-basic (negative control)
were cotransfected with 5 µg pSVGH, and then cultured for 48 h.
Medium was taken for RIA of hGH (Nichols Institute Diagnostics, San Juan Capistrano, CA), and cells were harvested
for luciferase assay. Luciferase assays were performed as described
previously (25, 26, 27).
Stably transfected FRTL-5 cells were constructed with pGL2-basic,
pCAM-431, pCAM-175, or pCAM-95. 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) was 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 24 h before
luciferase activity was measured.
Nuclear Extracts
FRTL-5 cells were grown in the presence of complete 6H medium to
approximately 80% confluency and then maintained in 5H medium (lacking
TSH) for the time periods as noted. After cells were exposed to
IFN-
, nuclear extracts were prepared from the FRTL-5 cells as
described (25, 26, 27) 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'-CGAGGTTTCCGGGAAAGT GGCCCC-3' (a putative GAS is
underlined), were used as the DNA binding substrate 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 (25, 26, 27). Binding reactions in low salt without
detergent were carried out in a volume of 20 µl for 30 min at room
temperature. The reaction mixtures contained 1.5 fmol of
[32P] DNA, 4 µg cell or nuclear extract, and
0.5 µg poly (dI-dC) in 10 mM Tris-Cl (pH 7.9),
1 mM MgCl2, 1
mM DTT, 1 mM EDTA, and 5%
glycerol. Binding reactions in high salt with detergent were carried
out in a solution of 1.5 fmol of [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 TBE 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
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) 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-SOCS-1 or anti-SOCS-3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For Western blot, adherent
FRTL-5 cells were stimulated in the presence or absence of TSH or
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 TBS, 0.1%
Tween-20 with blocking reagent 5% milk) the membrane was incubated
with the primary antibodies overnight at 4 C. Primary antibodies were
rabbit polyclonal IgG, affinity purified (New England Biolabs, Inc.,
Beverly, MA; and Upstate Biotechnology, Inc., Lake
Placid, NY) specific for STAT1 or STAT3 or phosphorylated forms of
STAT1 (Y701, S727) or STAT3 (Y705, S727). Blots were developed using
horseradish peroxidase-linked antirabbit secondary antibody and
chemiluminescent detection system (Phototope-HRP Western Blot Detection
Kit, New England Biolabs).
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
|
---|
Acknowledgements
We thank Drs. Starr and Hilton for the gift of expression
vectors pEF-mSOCS-1, pEF-mSOCS-2, pEF-mSOCS-3, and pEF-mCIS, and
helpful suggestions. We are also grateful to Dr. Kohn for helpful
discussion 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.
This work was supported by the Biotech 2000 project, Grant
98-N102-04-A-01, and Molecular Medicine Research Group Program,
Ministry of Science and Technology, Korea.
Received for publication June 23, 1999.
Revision received December 6, 1999.
Accepted for publication December 13, 1999.
 |
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