1
Regulation of the PI3K, Akt/PKB, FRAP/mTOR, and S6K1 signaling pathways by thyroid stimulating hormone and stimulating-type TSH receptor antibodies in the thyroid gland Jae Mi Suh, Jung Hun Song, Dong Wook Kim, Ho Kim, Hyo Kyun Chung, Jung Hwan Hwang, Jin Man Kim§, Eun Suk Hwang, Jongkyeong Chung¶, Jeung-Hwan Han§§, Bo Youn Cho*, Heung Kyu Ro, Minho Shong Laboratory of Endocrine Cell Biology, Department of Internal Medicine, §Department of Pathology, Chungnam National University, Taejon ,301-721, Korea; ¶ Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea; §§Department of Biochemistry, College of Pharmacy, Sungkyunkwan University, Suwon 440-756, Korea *Department of Internal Medicine, Seoul National University, Seoul, 110-744
running title: Regulation of FRAP/mTOR by TSH and TSAb * supported by National Research Laboratory Program (M1-0104-00-0014) and KOSEF Research Grant (2000-2-20500-007-3) Ministry of Science and Technology, Korea + to whom correspondence should be addressed Minho Shong Laboratory of Endocrine Cell Biology Department of Internal Medicine Chungnam National University School of Medicine 640 Daesadong Chungku Taejon 301-040, Korea Tel) 82-42-220-7161 Fax) 82-42-257-5753 email) [email protected]
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on March 30, 2003 as Manuscript M300805200 by guest on January 16, 2020
http://ww
w.jbc.org/
Dow
nloaded from
2
Abstract
TSH regulates the growth and differentiation of thyrocytes by activating the TSH
receptor. This study investigated the roles of the PI3K, PDK1, FRAP/mTOR and S6K1
signaling mechanism by which TSH and the stimulating-type TSHR antibodies regulate
thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR
immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with
either TSH or 8-Br-cAMP. TSH and cAMP increased the tyrosine phosphorylation of
TSHR and the association between TSHR and the p85α regulatory subunit of PI3K.
TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in
the cells in a PI3K and PKA-dependent manner. TSH induced the PDK1-dependent
phosphorylation of S6K1, but did not induce Akt/PKB phosphorylation. The TSH-
induced S6K1 phosphorylation was inhibited by a dominant negative p85α regulatory
subunit or by the PI3K inhibitors, wortmannin and LY294002. Rapamycin inhibited the
phosphorylation of S6K1 in the cells treated with either TSH or 8-Br-cAMP. The
stimulating-type TSHR antibodies from patients with Graves’ disease also induced
S6K1 activation, whereas the blocking-type TSHR antibodies from patients with
primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1.
In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle
hyperplasia and follicle activity. These findings suggest an interaction between TSHR
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
3
and PI3K, which is stimulated by TSH and cAMP and might involve the downstream
S6K1 but not Akt/PKB. This pathway may play a role in the TSH/TSAb-mediated
thyrocyte proliferation in vitro and in the response to TSH in vivo.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
4
Introduction
The pituitary glycoprotein hormones ACTH, FSH, LH, and TSH control the
function of specific target cells in the adrenal gland, gonads, and thyroid. All of these
hormones are not only important for hormone production but also for maintaining the
glandular weight in their target gland. These hormones bind to ligand-specific cell-
surface G-protein-coupled receptors and activate adenylyl cyclase to produce cAMP.
These glycoprotein hormone receptors also activate the PI3K-dependent signaling
pathways (1). However, it is still unclear how these glycoprotein hormone receptors are
coupled to the PI3K signaling pathways.
The TSH receptor (TSHR) has many important functions that regulate growth,
proliferation, differentiation, and the survival of thyrocytes and increasing hormone
production in the thyroid gland (2, 3). TSHR mediates these activities by activating the
diverse signaling pathways including the PI3K pathway. The signaling components
downstream of TSHR (Gβγ, cAMP, PKA) may overlap with the downstream
components of the PI3K signaling pathway. The Gβγ subunit of heterotrimeric G
proteins specifically activates PI3Kγ in the myeloid-derived cells (4, 5). The role of Gβγ
in PI3Kγ− dependent signaling in thyrocytes is not known. cAMP exerts PKA-
dependent and -independent effects on PI3K signaling in thyroid cells (6-9). The effects
of cAMP on PI3K signaling are cell-type specific (9-11). Both TSH and cAMP induce
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
5
S473 phosphorylation in Akt/PKB, a major phosphorylation site for the regulation of
Akt/PKB by growth factors. TSH and cAMP–induced phosphorylation of S473 is PI3K-
dependent (i.e., wortmannin sensitive) in rat WRT thyroid cells (6). cAMP and TSH also
induce the phosphorylation of ribosomal protein S6 (6) in a PI3K-independent and
PKA-dependent manner in rat WRT thyroid cells (6). These studies raise the possibility
that TSH and cAMP differentially regulate phosphorylation of Akt/PKB and S6, and
that this process may be mediated by PI3K signaling.
PDK1 is a PI3K-dependent serine/threonine kinase (12, 13) whose in vivo
substrates include Akt/PKB and S6K1. The mechanisms that regulate PDK1 are poorly
characterized but may include cellular localization, substrate conformation or
phosphorylation (13, 14). S6K1 is a Ser/Thr kinase that is activated at the G0/G1 of the
cell cycle in mammalian cells (15, 16). It phosphorylates five serine residues in the
ribosomal protein S6 in vitro and is the major S6 kinase in vivo in mammalian cells (17,
18). The S6K1 pathway may regulate the translation of some ribosomal proteins and
ribosome biogenesis (19, 20) thereby regulating cell proliferation. PDK1 and mTOR
(also termed FRAP or RAFT) may regulate the phosphorylation/dephosphorylation of
S6K1 (21, 22). PDK1 activates S6K1 by phosphorylating Thr229, an important residue
in the activation loop of S6K1 (23).
The FRAP/mTOR kinase regulates the initiation and elongation of translation,
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
6
ribosome biosynthesis, and amino acid transport (24), which affect the rate of protein
synthesis. FRAP/mTOR participates in mitogen signaling pathways and acts as a
nutrient sensing checkpoint. (25, 26, 27). Both FRAP/mTOR and PI3K signaling are
required to activate several downstream effector proteins (24, 25). FRAP/mTOR may
stimulate the phosphorylation of downstream targets and repress phosphatase activity
(28). Rapamycin-FKBP12 forms a complex that specifically inhibits FRAP/mTOR in
vivo (29).
This study examines the interactions between the TSHR and the regulatory p85α subunit of PI3K in
the presence and absence of TSH and cAMP. TSH and 8-Br-cAMP stimulate the interaction between
TSHR and PI3K, which leads to a PI3K- and PKA-dependent translocation of PDK1. PDK1
phosphorylation of Akt/PKB and S6K1 appears to be differentially stimulated by TSH and insulin. TSH
stimulates S6K1 through the PI3K-, PDK1- and PKA-dependent but Akt/PKB-independent pathways.
The FRAP/mTOR inhibitor rapamycin inhibits the TSH-stimulated phosphorylation of S6K1 and
inhibits cell cycle progression in the FRTL-5 thyroid cells. Rapamycin also modulates the thyroid
follicle activity, which is induced by elevated endogenous TSH levels in vivo. This study suggests that
S6K1 plays an important role in TSHR-activated-PI3K signaling, which modulates the thyrocyte
proliferation and thyroid follicle activity.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
7
Materials and Methods
Materials
The media and cell culture reagents and materials were purchased from Life
Technologies, Inc. (Gaithersburg, MD), Sigma (St. Louis, MO), Fisher Scientific
(Fairlawn, NJ), Corning, Inc (Corning, NY) and Hyclone Laboratories, Inc (Logan, UT).
Wortmannin and H89 were from Calbiochem (La Jolla, CA). LY294002, and PD98059
were obtained from Sigma (St. Louis, MO) and New England Biolabs (Beverly, MA).
Antibodies for cyclin D1 (sc-8396), p85a (sc-423) and p110gamma (H-119) were from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies for HA (#2363), Akt
(#9272), phospho-Akt-S473 and T308 (#9271 and #9275), phospho-4EBP1-Ser65
(#9451) and 4EBP1 (#9452), Myc (#2276), S6 ribosomal protein (#2212) and phospho-
S6 ribosomal protein-Ser235/236 (#2211), and p44/p42 MAP Kinase (#9102) and
phospho-p44/42 MAP Kinase-Thr202/Tyr204 (#9101) were from Cell Signaling
Technology (Beverly, MA). Mouse anti-human TSHR (MCA1571) was from Serotec
(Kidlington, Oxford) and anti-FLAG antibody (#200472) was from Stratagene (La
Jolla,CA). All other materials, including 8-bromo-cAMP(8-Br-cAMP), N-α-tosyl-L-
phenylalanyl chloromethyl ketone (TPCK) were purchased from Sigma (St. Louis,
MO).
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
8
Preparation of IgG
The control sera were obtained from 5 normal individuals who had no history, clinical,
or chemical evidence (abnormal thyroid hormone and TSH levels) of thyroid disease.
The diagnosis of the 10 patients with Graves’ disease was based on conventional clinical
and laboratory criteria, including elevated serum thyroid hormone levels, undetectable
TSH by a sensitive RIA, testing positive for TBII (TSH binding inhibitory
immunoglobulins), and a diffuse goiter with increased 99mTcO4- uptake at scintiscan.
IgGs were extracted by affinity chromatography using protein A-Sepharose CL-4B
columns; IgG was lyophilized and stored at -20 ℃ until assay. The IgGs were extracted
by affinity chromatography from normal pooled sera (NP), as well as the sera from the
patients with primary myxedema (PM) who tested positive to the blocking TSHR
antibody test. .
Cell Culture and Gene Transfection
A fresh subclone (F1) of FRTL-5 was used in the rat thyroid cells (Interthyr Research
Foundation, Baltimore, MD). After 6 days in medium with no TSH, the addition of 1
mU/mL crude bovine TSH (Sigma, St. Louis, MO) stimulated thymidine incorporation
into DNA by at least 10-fold. The doubling time of the cells with TSH was 36 ± 6 hours;
without TSH, and they did not proliferate. The cells used were diploid and between their
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
9
5th and 20th passage. The cells were grown in 6H medium consisting of Coon's modified
F12 supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture
of six hormones: bovine TSH (1 mU/ml), 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 the cells were
passaged every 7-10 days. In individual experiments, the cells were shifted to 3H
(which is devoid of insulin, TSH and somatostatin), 4H (which is devoid of insulin and
TSH) or 5H medium (which is devoid of TSH) with or without 5% calf serum before
TSH, forskolin (Sigma), or the other agents were added. Chinese hamster ovary (CHO)
cells were maintained at 37 °C and 5% CO2 in Ham's F-12 medium containing 10%
fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Expression
plasmids carrying hTSHR, p85α, PDK1 cDNAs were introduced into the FRTL-5 and
CHO cells using the LipofectAMINE Plus reagents according to the manufacturer
instructions (Life Technologies, Inc.).
PI3K Assay
The cell extracts obtained from the FRTL-5 cells were immunoprecipitated with anti-
TSHR monoclonal antibodies (clone 4C1, Serotec, Oxford) and the control IgG
antibodies. The samples were washed twice with 1% Nonidet P-40 and 1 mM sodium
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
10
orthovanadate in phosphate-buffered saline, twice with washing buffer consisting of 100
mM Tris-HCl (pH 7.5), 500 mM LiCl, and 1 mM sodium orthovanadate, and twice with
ST with 150 mM NaCl and 50 mM Tris-HCl (pH 7.2). The samples were resuspended
in a PI kinase buffer containing 20 mM Hepes (pH 7.2), 100 mM NaCl, 10 µg/mL
leupeptin, and 10 µg/mL pepstatin. A PI/EGTA solution consisting of 1 mg/mL
phosphoinositide and 2.5 mM EGTA was then added, and the samples were incubated at
room temperature for 10 min. A solution containing 20 mM Hepes (pH 7.4), 5 mM
MnCl2, 10 µM ATP, and 20 µCi of [γ-32P]ATP was added, and the samples were
incubated at 30°C for 20 min. The reactions were quenched by the addition of 1 M HCl,
and the phospholipids were extracted using CHCl3. The dried samples were separated
by thin-layer chromatography (TLC). The phosphorylated lipids were visualized by
autoradiography and quantified using a phosphorimager (BAS1500, Fuji).
Immunoprecipitation and Western blot analysis
The following immunoprecipitation procedures were carried out at 4 °C. The cells
grown on the 100-mm dishes were washed with phosphate-buffered saline twice prior to
lysis. The RIPA buffer containing the protease inhibitors (20 µg/ml leupeptin, 10 µg/ml
pepstatin A, 10 µg/ml chymostatin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
11
fluoride) were added for cell lysis. The cell lysate was collected, triturated, and
centrifuged at 1000 × g for 10 min. To preclear the cell lysate, the supernatant was
mixed with 20 µl of protein A/G beads (Santa Cruz), incubated for 30 min with rocking,
and were centrifuged for 15 min at 1000 × g. Precleared samples were incubated with
the primary antibodies for 2 h with rocking, and protein A/G beads were then added,
incubated overnight, and centrifuged at 1000 × g. The immunoprecipitates were
collected and washed three times with RIPA buffer.
For Western blot analysis, the cells were lysed at 4 °C in a mixture of 10 mM
KPO4, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM β-glycerophosphate, 2 mM
dithiothreitol, 1% Nonidet p-40, 1 mM Pefabloc (Roche), and 10 µg each of aprotinin
and leupeptin per milliliter. The total protein lysates were denatured by boiling in a
Laemmli sample buffer, resolved on 7.5% - 15% sodium dodecyl sulfate-
polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The
membranes were blocked in PBS containing 5% (wt/vol) milk and 0.1% Tween and
then incubated for 2 h with the polyclonal antibodies against p70 S6K (supplied by Dr. J.
Blenis, Harvard University, MA)
[3H]-Thymidine Incorporation Assay
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
12
Confluent FRTL-5 thyroid cells in 100 mm dishes were detached by trypsinization,
resuspended in 6H growth medium, seeded at a density of 3 x 104 cells/well in 24-well
plates, and incubated for 2-3 days until 80% confluent. The medium was changed to 5H
medium and incubated for an additional 7days. TSH and/or wortmannin, LY294002,
H89, rapamycin were added to the quiescent cells, which were then incubated for 24 h,
followed by the addition of 2 µCi/mL [3H]thymidine (NEN Life Science Products,
Boston, MA) to pulse the cells for an additional 12 h. The experimental samples were
prepared in triplicate. The cells were washed four times with ice-cold phosphate-
buffered saline, precipitated twice with ice-cold 10% trichloroacetic acid (30 min each
time on ice), briefly washed once with ice-cold ethanol, lysed with 0.2 N NaOH in 0.5%
SDS, and incubated at 37 °C for at least 30 min. The level of radioactivity was
determined by liquid scintillation spectrometry (Beckman Instruments). The results
were measured as the number of counts/min in each well. Each experimental data point
represents triplicate wells from at least four different experiments.
Flow cytometry
The samples were prepared for flow cytometry essentially as described previously (4).
Briefly, the cells were washed with 1x phosphate-buffered saline, pH 7.4, and then fixed
with ice-cold 70% ethanol. The samples were washed with 1x phosphate-buffered saline
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
13
and stained with propidium iodide 60 µg/mL (Sigma) containing 100 µg/mL RNase
(Sigma) for 30 min at 37 °C. Cell cycle analysis was performed using a Becton
Dickinson fluorescence-activated cell analyzer and Cell Quest version 1.2 software
(Becton Dickinson Immunocytometry Systems, Mansfield, MA). At least 10,000 cells
were analyzed per sample. The cell cycle distribution was quantified using the ModFit
LT version 1.01 software (Verity Software House Inc., Topsham, ME).
Confocal microscopy
The FRTL-5 thyroid cells were grown on coverslips and transfected with pEGFP-PDK-
1 and pCDNA3-PDK-1-myc using the LipofectAmine method (Life Technologies, Inc.,
Gaithersburg, MD). Quiescent cells stimulated with TSH were washed three times with
cold phosphate-buffered saline and fixed in 3.7% formaldehyde for 40 min. Fixed cells
were mounted on glass slides with phosphate-buffered saline and observed using laser-
scanning confocal microscopy (Leica TCS SP2).
Protein Kinase Assays
The FRTL-5 thyroid cells were transiently transfected with HA-tagged p70 S6K1 and
immunoprecipitated with an anti-HA monoclonal antibody coupled to protein A/G-
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
14
agarose (Santa Cruz). The samples were washed twice with Buffer A (containing 20
mM Tris-HCl [pH 7.5], 0.1% Nonidet P-40, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2,
50 mM β-glycerophosphate, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 40
µg/mL phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin) and then twice with
Buffer B containing 500 mM NaCl. Finally the immunocomplexes were washed, in
succession, with Buffer C (containing 20 mM Hepes [pH 7.2], 10 mM MgCl2, 0.1
mg/mL bovine serum albumin, and 3 mM β-mercaptoethanol), Buffer D (containing 20
mM Hepes [pH 7.2], 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, and 0.2 mM
EGTA), and Buffer E (containing 50 mM Tris [pH 7.5], 10 mM NaCl, 1 mM
dithiothreitol, and 10% glycerol). The S6 phosphotransferase activities were assayed in
a reaction mixture consisting of 1×Buffer C, 1 µg of S6 peptide, 20 µM ATP, and 5 µCi
of [γ-32P]ATP (specific activity: 3000 Ci/mmol; NEN Life Science Products) at 30 °C
for 20 min. The samples were subjected to liquid scintillation counting (Hewlett-
Packard).
Preparation of thyroid gland and Immunohistochemistry
Male Sprague-Dawley rats (120-130 g) were fed with water containing 0.025% MMI
for 2 wks. Rapamycin (Calbiochem, La Jolla, CA) was delivered once daily by an
intraperitoneal injection at a dose of 1.5 mg/kg dissolved in 2% carboxymethylcellulose
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
15
for one week before histologic examination. Tissue samples of the rat thyroid gland
were fixed in 10% buffered formalin, processed routinely, and embedded in paraffin.
Three-micrometer-thick sections were cut from the paraffin blocks and stained with
hematoxylin and eosin (H&E). The number of follicles in 2 medium power fields
(X200) was counted. Further sections were used for immunohistochemistry (IHC). All
immunostaining steps were carried out at room temperature. After deparaffinization and
antigen retrieval by autoclaving in 10 mM sodium citrate buffer (pH 6.0) at full power
for 10 min, the tissue sections were treated with blocking rabbit serum for 15 min. The
primary antibody, polyclonal rabbit anti-Phospho-S6 ribosomal protein (Cell Signaling
Technology, Inc. USA), was diluted (1:200) with a background-reducing diluent (Dako,
Carpinteria, CA) and incubated for 60 minutes. The sections were then incubated with a
rabbit EnVision-HRP detection system reagent (Dako, Carpinteria, CA) for 30 minutes.
The sections were then sequentially incubated with DAB (3,3-diaminobenzidine)
(DAKO) chromogen for 5 minutes, counterstained with Meyer’s hematoxylin, and
mounted. Careful rinses with several changes of TBS-0.3% Tween buffer were
performed between each step. A negative control that excluded the primary antibody
was used. Cells with cytoplasmic granular staining were considered positive.
Other Assays
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
16
The protein concentration was determined by the Bradford method (Bio-Rad, Hercules,
CA) using recrystallized bovine serum albumin as a standard. The sera were stored -70
oC until immunoglobulin G (IgG) preparation. The IgGs were extracted by affinity
chromatography using protein A-sepharose CL-4B columns (Amersham Pharmacia,
Piscataway, NJ) followed by dialysis. The purity of the IgG preparation was confirmed
by the documentation of undetectable TSH levels with immunoradiometric assay.
Statistical Analysis
All experiments were repeated at least three times with different cells. The values are
the mean ± SE of these experiments. Statistical significance was determined by a two-
way analysis of variance.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
17
Results
Association of PI3K activities with TSHR-– TSHR was immunoprecipitated from
extracts from FRTL-5 thyroid cells exposed to TSH or 8-Br-cAMP using monoclonal
anti-TSHR antibodies and the immunoprecipitate was tested for PI3K activity. The
phosphatidylinositol phosphotransferase activity was detected in the immunoprecipitate,
which was inhibited by the PI3K-inhibitors, wortmannin (100 nM) or LY294002 (0.5
µM) (Fig.1A). The TSHR-associated PI3K activity was barely detectable in the
untreated FRTL-5 thyroid cells (Fig. 1A, lane 1; Fig. 1B, lane 1). Both TSH and 8-Br-
cAMP stimulated the TSHR-associated PI3K activity (Fig. 1B, lane 2, 4, respectively).
H89 did not inhibit the PI3K kinase activity in vitro (data not shown), but the H89
treated cells had a lower level of TSHR-associated PI3K activity in the TSH- or 8-Br-
cAMP-treated cells. (Fig. 1B, lane 3, 5).
The specificity of the interaction between TSHR and PI3K was determined in the
following experiment. The extracts were prepared from the wild-type CHO cells or the
CHO cells expressing human TSHR (CHO-TSHR) (30, 31) and immunoprecipitation
was carried out using anti-TSHR (Fig. 1C, lane 1-4) or the control IgG (Fig. 1C, lane 5).
A low level of PI3K activity was detected in the anti-TSHR immunoprecipitates from
the CHO-TSHR cells (Fig. 1C, lane 2). This activity increased in the cells treated with
TSH (Fig. 1C, lanes 2 vs 3) and was inhibited by 100 nM wortmannin (Fig. 1C, lane 4).
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
18
No PI3K activity was detected in the experiments using the wild-type CHO cells (Fig.
1C, lane 1) or the control IgG (Fig. 1C, lane 5). These findings suggest that TSHR
interacts specifically with PI3K and that TSH and cAMP stimulate this interaction.
TSHR interacts with the p85α regulatory subunit of PI3K - Western blot analysis was
carried out to identify the specific regulatory and catalytic subunits of class I PI3K in
FRTL-5 thyroid cells. The p85α regulatory subunit of class I PI3K (p85α) was
expressed strongly in the cells treated with or without TSH, 8-Br-cAMP and insulin (Fig.
2A). In contrast, the p110γ catalytic subunit of class I PI3K, which is activated by the
Gβγ subunits of the heterodimeric G proteins, was not expressed at a detectable level
in these cells (Fig 2A).
The following experiments examined the association between TSHR and the
regulatory subunit of PI3K. Human TSHR and HA-tagged p85α were co-expressed in
CHO cells. TSHR was immunoprecipitated and the immune complexes were analyzed
with anti-HA antibodies (Fig. 2B). The TSHR immunoprecipitates from the CHO-
TSHR cells included p85α (Fig. 2B lane 4). TSHR Immunoprecipitates from FRTL-5
cells treated with TSH and p85α (Fig. 2C, lane 2, and 3). The cell extracts were also
immunoprecipitated with control IgG; the results show that the interaction between
TSHR and HA-p85α is specific (Fig. 2C, lanes C vs T).
The immune complexes precipitated with the anti-TSHR antibodies were also
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
19
probed with anti-phosphotyrosine antibodies (4G10). In untreated cells, the TSHR had a
low level of tyrosine phosphorylation. In contrast, the TSHR from the cells treated with
TSH or 8-Br-cAMP had a significantly higher phosphotyrosine content (Fig.2D, lanes 1
to 4). Endogenous p85α was detected in the TSHR immunoprecipitates from the TSH-
or 8-Br-cAMP treated FRTL-5 thyroid cells.
These findings suggest that 1) the TSH receptor is specifically associated with the
p85α regulatory subunit of PI3K in the thyroid cells, and the TSHR-transfected CHO
cells. 2) The association between the endogenous p85α and TSHR depends on the
TSH/8-Br-cAMP treatment and on tyrosine phosphorylation status of TSHR.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
20
PDK1 subcellular localization and tyrosine phosphorylation - When PI3K is activated
by growth factors, several events follow. These include the increased synthesis of
PtdIns-3,4,5-P3 and PtdIns-3,4-P2, activation of PDK1, and translocation of PDK1 and
Akt/PKB to the plasma membrane (32, 33). Therefore, the localization of PDK1 was
examined in the TSH-treated cells expressing GFP-tagged PDK1. In the cells treated
with TSH or 8-Br-cAMP, PDK1 rapidly redistributes to the plasma membrane and the
perinuclear region. This redistribution was inhibited by wortmannin, LY294002, and
H89 (Fig. 3A). In the control cells, GFP fluorescence was evenly distributed within the
cytoplasm.
Because tyrosine phosphorylation is associated with the activation of PDK1 (14),
the TSH-treated cells were examined for their the phosphorylation state of PDK1 . The
Myc-tagged PDK1 and TSHR were expressed in CHO cells and tyrosine
phosphorylation was analyzed using anti-phosphotyrosine antibodies (4G10). Myc-
PDK1 was constitutively tyrosine phosphorylated (Fig. 3B) in the TSH or 8-Br-cAMP
treated or untreated cells. Therefore, in the CHO cells, the tyrosine phosphorylation of
PDK1 was not correlated with the cellular localization or TSH treatment.
Akt/PKB and S6K1 activities in TSH-treated cells – Akt/PKB and S6K1 are substrates
of PDK1, which are important downstream effectors of PI3K/PDK1 signaling.
Therefore, Akt/PKB and S6K1 phosphorylation were examined in the TSH-treated
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
21
thyroid cells. Previous studies show that Akt/PKB is phosphorylated on T308 and S473
by PDK1 and PDK2, respectively, in insulin-treated cells. Insulin-treatment had similar
effects on the thyroid cells (Fig. 4B and data not shown). However, TSH treatment did
not induce Akt/PKB T308 and S473 phosphorylation in the thyroid cells (Fig. 4A). This
result was confirmed by comparing the phosphorylation of 4EBP-1/PHAS-1, which is
mediated by Akt/PKB, in the insulin- and TSH-treated thyroid cells. Insulin rapidly
stimulated the phosphorylation of S65 of 4EBP-1/PHAS-1 but TSH did not (Fig. 4D
and 4C). Rapamycin, an inhibitor of FRAP/mTOR, inhibited the phosphorylation of
4EBP-1/PHAS-1 in the insulin-treated thyroid cells (Fig 4D).
S6K1 has two protein subunits, p70 and p85, both of which are phosphorylated
in insulin-treated cells. Figure 5 shows that TSH induces the rapid phosphorylation of
p70 and p85, as detected by the slow-migrating hyperphosphorylated protein species on
a Western blot. The appearance of these species correlated with the increased S6 kinase
activity in the immunoprecipites (Figs. 5A and 5B). The phosphorylation of p70 and
p85 increases with increasing TSH (1 mU/ml) or insulin (10 µg/ml) exposure time (Fig.
5A) (Fig. 5C). After 30 min exposure to TSH, the S6K1 activity reached a maximum,
increasing 3.5-fold (Fig. 5B). The insulin-induced phosphorylation of S6K1 (Fig. 5C)
reached a maximum 15 min after treatment (Fig. 5D). These experiments suggest that
TSH activates S6K1 but not Akt/PKB; in contrast, insulin activates Akt/PKB and S6K1.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
22
Roles of PI3K, PDK1 and PKA in activating S6K1 in TSH-treated cells - The
above results suggest that TSH may stimulate a response in thyroid cells, which
involves TSHR, PI3K, PDK1 and S6K1. The activities upstream of S6K1 were
identified using the inhibitors of PI3K (wortmannin and LY294002) PKA (H89),
FRAP/mTOR (rapamycin) and PDK1 (N-α-tosyl-L-phenylalanyl chloromethyl ketone;
TPCK). LY294002 (500 nM) and wortmannin (300 nM) completely inhibited the TSH-
induced phosphorylation of S6K1 (Figs. 6A and 6B). LY294002 and wortmannin began
to inhibit S6K1 phosphorylation at 500 nM and 100 nM, respectively (Fig. 6A, lane 3;
Fig. 6B, lane 4). Forskolin (10 µM) or 8-Br-cAMP (1 mM) rapidly induced the
phosphorylation of S6K1, and this effect was inhibited by LY294002 (500 nM) and
wortmannin (100 nM) or H89 (Figs. 6D and 6E). The inhibitory effect of H89 was dose-
dependent (Fig. 6F). H89 began to inhibit TSH-induced phosphorylation of S6K1 at 5
µM and completely inhibited S6K1 (and CREB; data not shown) phosphorylation at 50
µM. The PDK-1 inhibitor TPCK also inhibited the TSH-induced phosphorylation of
p70S6K1 (Figs. 7A and 7B) at 20-50 µM and inhibited the phosphorylation of the
ribosomal protein, S6, in a dose-dependent manner (Fig. 7A).
The role of PI3K in TSH-stimulated activation of S6K1 was also examined using
the cells expressing a dominant negative form of HA-tagged-p85α (cell line L5-
∆iSH2p85 expressing pCMV-∆iSH2p85-HA). The cells expressing the wild type p85α
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
23
(cell line L5-WTp85 expressing pCMV-p85-HA) were used as the control (Fig. 6C) (35).
The phosphorylation of S6K1 was measured in the presence and absence of TSH. In the
cells expressing the wild type p85, S6K1 was rapidly phosphorylated in response to
TSH, but S6K1 phosphorylation was strongly reduced in the cells expressing the
dominant negative p85 (Fig. 6C). Similar results were observed in the cells treated
with forskolin or 8-Br-cAMP (data not shown).
Roles of MAPK and FRAP/mTOR in activating S6K1 in TSH-treated cells –
S6K1 is regulated by phosphorylation and has at least eight phosphorylation sites.
PDK1, Akt/PKB, NEK6/7, FRAP/mTOR and MAP kinases act upstream of S6K1. In
vitro, S6K1 is phosphorylated by the MAP kinases (34) and in human thyroid cells,
TSH stimulates the MAP kinase cascade by a cAMP-independent pathway (35). The
thyroid cells were cultured in the presence or absence of TSH and Erk1 and Erk2
phosphorylation were examined. Insulin (10 µg/ml) induced Erk1 and Erk2
phosphorylation at threonine 202 and tyrosine 204 after 15 min (Fig. 8A). In contrast,
TSH did not induce Erk1 and Erk2 phosphorylation until 60 min after treatment (Fig.
8B). In the cells cultured in the absence of TSH, insulin and serum, S6K1 was not
phosphorylated but Erk1/Erk2 were constitutively phosphorylated (Fig. 8). The TSH-
induced S6K1 phosphorylation was not inhibited by a pretreatment with a MEK
inhibitor, PD98059 (Fig. 8C, lanes 2 and 4). These results suggest that the MAP kinase
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
24
pathway does not play a role in the TSH-stimulated phosphorylation of S6K1 in thyroid
cells.
Rapamycin completely inhibited the S6K1 phosphorylation in the cells treated
with TSH (Fig 8C), forskolin or 8-Br-cAMP (Fig. 6D, 6E), suggesting a role for
FRAP/mTOR in the response to TSH in thyroid cells.
Regulation of S6K1 by TSH receptor autoantibodiess – There are two types of
TSH receptor autoantibodies: the stimulating (TSAb) antibodies, which are agonists of
the TSH receptor and associated with Graves’ disease, goiter, and hyperthyroidism; and
the blocking (TSBAb) antibodies, which are antagonists of the TSH receptor and are
associated with primary myxedema, thyroid atrophy, and hypothyroidism. The influence
of the stimulating and blocking TSH receptor autoantibodies on S6K1 phosphorylation
was examined in thyroid cells. Normal pooled IgG (NP) was obtained from normal
adults with no known thyroid disease and were used as the controls. Autoantibodies
from patients with Graves’ disease (GD) significantly enhanced phosphorylation of
S6K1, but the control IgG (NP) did not (Fig. 9A). IgG from 10 patients with Graves’
disease were tested in a radioreceptor assay for TSH-binding inhibitory immunoglobulin
(TBII). Seven patients with TBII-positive Graves’ disease stimulated S6K1
phosphorylation and the ribosomal protein, S6 (Fig. 9A). This activity was completely
inhibited by LY294002 and wortmannin, but not by PD98059 (Fig. 9B). TSBAb IgG
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
25
from patients with primary myxedema (PM) did not induce S6K1 phosphorylation (Fig.
9A, lane PM). TSBAb IgG did not inhibit the insulin-induced phosphorylation of S6K1,
but did inhibit the TSH/TSAb-induced phosphorylation of S6K1 in thyroid cells (Fig.
9C, lane 4, 5, 6).
Effects of S6K1 signaling pathways on thyrocyte proliferation - In thyroid cells,
the cellular proliferation is regulated by TSH, insulin, and other growth factors (36).
The role of signaling kinases in TSH-stimulated proliferation was examined by
measuring the level of DNA synthesis in the presence of wortmannin, LY294002,
rapamycin, or H89. Thyroid cells were cultured in the absence of TSH for 7 days and
then stimulated with TSH in the absence or presence of the kinase inhibitor and 3H-
thymidine. The incorporation of 3H-thymidine increased 17-fold in the cells exposed to
TSH (Fig. 10A). Rapamycin (20 nM), wortmannin (100 nM), LY294002 (500 nM), and
H89 (50 µM) completely inhibited TSH-stimulated DNA synthesis.
TSH regulates the expression of cyclins D and E, which are rate limiting for entry
into the S phase. Cyclin D1 is expressed at a low basal level in the absence of TSH. The
expression of cyclin D1 increases approximately 12 h after exposure to TSH and
decreases to the basal level after 24 h post-exposure (Fig. 10B). Rapamycin inhibits
TSH-induced cyclin D1 expression and does not change the basal level of cyclin D1
(Fig. 10C, lanes 1 and 2). This result is consistent with previous observations showing
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
26
that rapamycin inhibits the G1/S transition and prolongs G1 in several types of cells (37,
38).
The cell cycle distribution was also examined in TSH/insulin-treated thyroid cells in the
presence and absence of rapamycin. The cells maintained in 3H medium with 5% calf
serum (3H5%), which lacks insulin, TSH and somatostatin, were quiescent (G1, 82.9±
5%; S, 1.88± 0.7%) (Fig. 10D). The TSH/insulin treatment promoted cell proliferation
(G1, 49.3 ± 5%; S, 19.5 ± 0.9%) (Fig. 10D). Rapamycin partially inhibited this change
in the cell cycle distribution (G1, 72.6 ± 0.3%) (Fig. 10D).
The phosphorylation of S6K1 was also examined in the experiment shown in
Figure 10. TSH stimulated a lower level of S6K1 phosphorylation than the TSH/insulin
(Fig. 10D lane 4), and rapamycin completely inhibited S6K1 phosphorylation in the
presence of TSH or TSH/insulin (lanes 3 or 5).
Regulation of thyroid follicle activity by rapamycin in vivo. The above results
suggest that rapamycin-sensitive S6K1 signaling plays a major role in the TSH-
stimulated thyrocyte proliferation. The following experiments test the in vivo role of
FRAP/mTOR and S6K1 in the TSH-stimulated thyroid follicles. The in vivo
experimental system involved feeding male Sprague-Dawley rats water containing
0.025% methimazole (MMI); this protocol provides a long-term TSH-stimulation of the
thyroid follicles. In the MMI-treated rats, the thyroid gland showed a prominent dark
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
27
red swelling after 2 weeks of treatment. The thyroid follicle histology revealed the
characteristic findings of long-term TSH stimulation. These include the depletion of
colloids, small hyperplastic collapsed follicles, hypertrophic columnar lining cells, and
crowded hyperchromatic nuclei (Fig. 11A 2nd). The histology was similar in rats
receiving intraperitoneal injections of 2% carboxymethylcellulose for 1 week. However,
the histology was altered in the thyroid glands of the MMI-treated animals administered
2% carboxymethylcellulose containing rapamycin (1.5 mg/kg) once daily. In these
animals, colloid-filled follicles were more abundant (Fig. 11A 4th and B).
The S6K1 kinase activity was estimated in these rats by measuring the quantity of
phosphorylated ribosomal protein S6. In the untreated rats, phosphorylated S6 was not
present in the thyroid follicular cells but was abundant in the parafollicular cells (Fig.
11C Lt). In the MMI-treated rats, a significantly higher percentage of thyroid cells
possessed the phosphorylated S6 protein, (Fig. 11C, Middle) and rapamycin completely
inhibited S6 phosphorylation in the thyroid cells (Fig. 11C, Rt). These observations
suggest that rapamycin-sensitive S6K1 signaling plays a role in the TSH-stimulated
follicles in vivo.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
28
Discussion
The mechanisms of cellular proliferation in the thyroid gland may be relevant to
human thyroid diseases including benign thyroid nodules, differentiated thyroid cancer,
and Graves’ disease (39, 40). Thyrocyte proliferation is regulated by TSH, which is one
of the most important factors regulating the proliferation and function of thyroid cells,
and by other hormones and growth factors that activate the specific signaling pathways
(41). Most studies of thyrocyte proliferation used the rat thyroid cell lines FRTL-5,
WRT, and PC C13 because these cell lines are responsive to TSH (36). TSH activates
TSHR, which activates adenylyl cyclase, elevates cAMP and activates PKA. However,
TSH regulates the thyroid function by activating a complex signaling network.
This study presents evidence showing that PI3K activates S6K1 in response to
TSH in the proliferating thyroid cells (Fig. 12). TSHR interacts with the p85α
regulatory subunit of PI3K and has inducible tyrosine phosphorylation sites (Fig. 2D). It
is possible that a phosphotyrosine residue in the intracellular loop or C-terminal tail of
TSHR interacts with the SH2 domain of p85α. Tyrosine phosphorylation of TSHR is
stimulated by exogenous 8-Br-cAMP and might involve a cAMP-dependent tyrosine
kinase (Fig. 2D). The tyrosine kinase inhibitor genistein inhibits the tyrosine
phosphorylation of TSHR in the cells treated with TSH/8-Br-cAMP and inhibits the
association between PI3K and TSHR (data not shown). These observations suggest that
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
29
TSH/cAMP stimulates the tyrosine phosphorylation of TSHR, which may stimulate the
TSHR-dependent activation of PI3K in thyroid cells. A dominant negative p85α, which
lacks the iSH2 region that binds the p110 catalytic domain of PI3K, strongly inhibits the
TSH-stimulated phosphorylation of S6K1. This suggests that a class I PI3K may be
involved in TSH signaling. To date, there is no evidence showing that the adaptor
molecules Shc, Grb2 and Gab2 facilitate the interaction between TSHR and PI3K,
although these molecules bridge the interactions between other the receptors and PI3K
(42).
This study suggests that PKA may be required for the TSH/cAMP-stimulated
phosphorylation of S6K1 in thyroid cells. LY294002 and wortmannin did not affect the
TSH-stimulated phosphorylation of CREB (data not shown), suggesting that PI3K acts
downstream of PKA. However, H89 inhibits the 8-Br-cAMP/TSH-induced association
between TSHR and PI3K. Therefore, cAMP/PKA may stimulate the formation of an
active TSHR-PI3K complex or modify the subunit composition of PI3K. Recent studies
show that cAMP/PKA phosphorylates p85α and stabilizes the p85α-p110 complex (8,
49). Further investigation is needed to understand the molecular interactions between
TSHR, PI3K and cAMP/PKA in thyroid cells.
cAMP-GEFs play a role in the PKA-independent effects of TSH and FSH in
their target cells (1, 50). The identification of novel cAMP-GEFs (Epacs) raises the
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
30
possibility that cAMP may activate the small GTPases such as Rap1a, Rap2, and
possibly Ras (51, 52). These GTPases activate the downstream signaling kinases
including PI3K (53). TSH activates Rap1 in WRT thyroid cells (7), which induces
Akt/PKB phosphorylation but not S6K1 (7).
This study suggests that PDK1 plays a role in the TSH-stimulated activation of
S6K1 (Fig. 3 and 7). PDK1 is regulated primarily by the substrate conformation,
cellular localization, and tyrosine phosphorylation (13, 14). The PH domain of PDK-1
selectively binds PtdIns-3,4-P2 and PtdIns-3,4,5-P3 (39). PDK1 redistributes from the
cytosol to the plasma membrane and perinuclear region in the TSH-treated cells. This
process is inhibited in the presence of the PI3K inhibitors and might involve the PI3K
lipids since it requires the PH domain of PDK1. PDK1 phosphorylation may also
regulate its activity (14, 43), although TSH did not stimulate the tyrosine
phosphorylation of PDK1 in the thyroid cells overexpressing PDK1 (data not shown) or
in the CHO cells overexpressing TSHR (Fig 3B). S6K1 (p70 and p85) and Akt/PKB are
well known targets of PDK1 in the growth factor signaling pathways (21, 44). It is not
yet understood why TSH does not induce the Thr308 or Ser473 phosphorylated forms of
Akt/PKB (Fig. 4, Fig. 12), but does induce S6K1 phosphorylation. Protein-protein
interactions or structural conformation may influence this specificity. TSH and insulin
also have different effects on these signaling pathways, which may explain why the
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
31
thyroid cells require both TSH and insulin for cellular proliferation.
4E-BP1 (also known as PHAS-1) normally binds eIF4E, initiating cap-
dependent phosphorylation (45). The hyperphosphorylation of 4E-BP1 disrupts this
binding, activating cap-dependent translation. The PI3K/Akt pathway and FRAP/mTOR
kinase regulate 4E-BP1 (46). 4E-BP1 is phosphorylated in vivo on multiple residues.
Phosphorylation by FRAP/mTOR on Thr37 and Thr46 of 4E-BP1 may prime it for the
subsequent phosphorylation sites at Ser65 and Thr70 (47). Insulin induces the
phosphorylation of Ser65 of 4E-BP1 and this phosphorylation is inhibited by rapamycin.
This suggests that insulin induces FRAP/mTOR, which phosphorylates the Ser65 of 4E-
BP1. In contrast, TSH did not induce the phosphorylation of Ser65 of 4E-BP1.
Akt/PKB may also be required for the phosphorylation and activation of 4E-BP1, and
TSH does not activate Akt/PKB, but insulin does. These findings suggest that
FRAP/mTOR is a required downstream effector of Akt/PKB, which phosphorylates 4E-
BP1 (47) .
cAMP exerts different effects on PI3K signaling in the different cell types
(9,38). A recent study on WRT thyroid cells showed that cAMP stimulates S6K1
through a PKA-dependent, PI3K-independent pathway (6). This conclusion was based
on the observation that cAMP-stimulates the phosphorylation of the S6 protein was
repressed by H89 (50 µM) but not by low-dose wortmannin or by a microinjection of
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
32
the N-terminal SH2 domain of the p85 regulatory subunit of PI3K. Cohen et al reported
that H89 (10 µM) is equally effective in inhibiting MSK1, S6K1, ROCK-II and PKA.
Therefore, H89 might inhibit S6K1 directly. However, in this study, H89 inhibited the
association between TSHR and PI3K and the activation of PI3K (Fig. 1), suggesting
that PKA plays a role in the TSHR-stimulated activation of PI3K in thyroid cells.
In contrast to the above results in the WRT thyroid cells, this study shows that
S6K1 activation by cAMP is dependent on PI3K in the FRTL-5 thyroid cells.
Wortmannin and LY294002 are structurally unrelated molecules that, at low
concentrations, are relatively specific, cell-permeable inhibitors of PI3K. Wortmannin
may directly inhibit the FRAP/mTOR autokinase activity at an IC50 approximately 100-
fold higher than its IC50 for inhibiting PI3K (~200 nM in vitro, ~300 nM in vivo).
LY294002 inhibits FRAP/mTOR autokinase activity in vitro with an IC50 of 5 µM (32).
LY294002 and wortmannin inhibit the TSH-stimulated phosphorylation of S6K1 at 500
nM and 100 nM, respectively, which suggests the involvement of PI3K (Fig. 6).
FRAP/mTOR regulates the protein translation by two independent mechanisms
involving the direct or indirect activation of S6K1 and the inactivation of the
translational repressor PHAS-1/4E-BP1. Rapamycin completely inhibits the
phosphorylation of S6K1 induced by TSH, 8-Br-cAMP, forskolin, and insulin in FRTL-
5 thyroid cells (Fig. 6). The mechanisms by which TSH regulates FRAP/mTOR kinase
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
33
is not known. However, FRAP/mTOR senses the cellular ATP level (27) and
mitochondrial dysfunction (54), and 2-deoxyglucose (100mM), which is an inhibitor of
the glycolytic pathway, partially inhibits S6K1 phosphorylation in the TSH-treated cells
(data not shown). Rapamycin may inhibit the S6K1 phosphorylation by activating the
phosphatases in the TSH-treated cells (55). The TSH-induced changes in the cellular
ATP level might affect the TSH -induced phosphorylation of S6K1 in thyroid cells.
These findings suggest that FRAP/mTOR kinase may not act downstream of
PI3K/PDK1 in TSH-treated cells. It is possible that FRAP/mTOR senses the nutrient
signals that change in the TSH-treated cells.
Different thyrocyte cell systems have different requirements for cell
proliferation (i.e., TSH, insulin/IGF-1, serum). TSH and insulin stimulate the
proliferation by mechanisms, which are not completely clear. In dog thyroid cells,
cAMP activates S6K1 but does not promote DNA synthesis (56). TSH also activates
S6K1, and increases the number of cells in the S phase slightly, but does not
significantly increase the number of cells in G2 (Fig. 10). In contrast, TSH/insulin
markedly increases the number of cells in S and G2 and TSH/insulin stimulates the
synergistic prolonged activation of S6K1 (Fig. 10D lane 4). Rapamycin inhibits the
TSH and TSH/insulin-induced cell cycling, suggesting a role for the S6K1 pathways.
However, rapamycin does not inhibit the activation of Akt/PKB in the insulin-treated
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
34
thyroid cells (data not shown), suggesting that the synergistic activation of the S6K1
signaling pathways by TSH/insulin may stimulate the cell cycle progression in FRTL-5
thyroid cells.
Saito et al (57) reported that Akt/PKB was not phosphorylated in the TSH-
treated FRTL-5 thyroid cells. A constitutively active Akt/PKB also promotes the
hormone-independent proliferation in the PC C1 3 thyroid cells (58). These
observations suggest that Akt/PKB might play a role in thyrocyte proliferation.
However, the role of Akt/PKB in TSH and TSH/insulin-treated thyroid cells remains to
be clarified.
This study used Sprague-Dawley rats maintained on MMI-containing drinking
water as an in vivo experimental system to study the long-term stimulation of the
thyroid follicles. The MMI-treated rats had higher TSH levels in response to the
inadequate thyroid hormone production. The thyroid glands from the MMI-treated rats
have a follicular structure that suggests TSH stimulation, and the follicular cells have an
increased phosphorylation of the ribosomal protein S6 (Fig. 11C). These findings
suggest that the endogenous TSH stimulates S6K1 in the thyroid follicles. Follicular
changes induced by sustained TSH stimulation were partially reversed by the short-term
intraperitoneal delivery of rapamycin. This suggests that some of the effects of TSH on
the thyroid follicles are mediated by S6K1.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
35
This study also shows that the TSHR antibodies modulate the S6K1 activity in
thyroid cells. TSAb induces and TSBAb inhibits the phosphorylation of S6K1 and the
S6 protein. The TSAb-stimulated phosphorylation of S6K1 was inhibited by LY294002
and wortmannin but not by PD98059. This suggests that the TSAb-induced activation of
S6K1 requires PI3K. However, it was not possible to determine if the S6K1 and TSAb
activities correlated with each other in this study as a result of insufficient data.
Nevertheless, the TSAb-stimulated activation and TSBAb-stimulated inhibition of
S6K1 might be related to goiter and atrophy in Graves’ disease and primary myxedema,
respectively.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
36
Acknowledgements
The authors wish to acknowledge Dr. John Blenis for providing the polyclonal
S6K1 antibodies, Dr. Leonard D. Kohn for helpful discussion and critical review of the
manuscript, and Yong Mi Kang for technical assistance.
Footnotes
This work was supported by National Research Laboratory Program (M1-
0104-00-0014) and KOSEF Research Grant (2000-2-20500-007-3) Ministry of Science
and Technology, Korea
Abbreviations
The abbreviations used are: CHO, Chinese hamster ovary; cAMP, cyclic adenosine
monophosphaste; CREB, cAMP respose element binding protein; 4EBP, eIF-4E binding
protein; MAPK, mitogen activated kinase; MMI, methimazole; mTOR, mammalian
target of rapamycin; PDK1, 3’-phosphoinositide dependent kinase; PI3K,
phosphatidylinositol 3-kinase; PKA, protein kinase A; PKB, protein kinase B; SDS-
PAGE, sodium dodecylsulfate-polyacrylamide gel; S6K1, ribosomal S6 kinase 1;TSAb;
stimulating-type TSH receptor antibody; TSBAb, blocking-type TSH receptor
antibody;TSH, thyroid stimulating hormone;
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
37
References
1. Richards, J.S. (2001) Mol. Endocrinol. 15, 209-218
2. Kohn, L.D., Shimura, H., Shimura, Y., Hidaka, A., Giuliani, C., Napolitano, G.,
Ohmori, M., Laglia, G., Saji, M. (1995) Vitam Horm 50, 287-384
3. Nagayama, Y., Rapoport, B. (1992) Mol. Endocrinol. 6, 145-156
4. Stephens, L., Smrcka, A., Cooke, F. T., Jackson. T. R., Sternweis, P. C., Hawkins, P. T.
(1994) Cell 77, 83-93
5. Laffargue, M., Calvez, R., Finan, P., Trifilieff, A., Barbier, M., Altruda, F., Hirsch, E.,
Wymann, M. P. (2002) Immunity 16, 441-451
6. Cass, L. A., Summers, S. A., Prendergast, G. V., Backer, J. M., Birnbaum, M. J.,
Meinkoth, J. L. (1999) Mol. Cell. Biol. 19, 5882-5891
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
38
7. Tsygankova, O. M., Saavedra, A., Rebhun, J. F., Quilliam, L. A., Meinkoth, J. L.
(2001) Mol. Cell. Biol. 21, 1921-1929
8. Ciullo, I., Diez-Roux, G., Di Domenico, M., Migliaccio, A., Avvedimento, E. V.
(2001) Oncogene 20, 1186-1192.
9. Cass, L. A., Meinkoth, J. L. (1998) Endocrinology 139, 1991-1998
10. Filippa, N., Sable, C. L., Filloux, C., Hemmings, B. A., Obberghen, E. (1999) Mol.
Cell. Biol. 19, 4989-5000,
11. Kim, S., Jee, K., Kim, D., Koh, H. Chung J (2001) J. Biol. Chem. 276, 12864-
12870.
12. Williams, M. R., Arthur, J. S., Balendran, A, van der Kaay, J., Poli, V., Cohen, P.,
Alessi, D. R. (2000) Curr. Biol. 10, 439-448.
13. Toker, A., Newton, A. C. (2000) Cell. 103, 185-188
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
39
14. Park, J., Hill, M. M., Hess, D., Brazil, D. P., Hofsteenge, J., Hemmings, B. A. (2001)
J Biol Chem 276, 37459-37471
15. Bell, A., Gagnon, A., Dods, P., Papineau, D., Tiberi, M., Sorisky, A. (2002) Am. J.
Physiol. Cell. Physiol. 283, C1056-1064.
16. Susa, M., Olivier, A. R., Fabbro, D., Thomas, G. (1989) Cell 57, 817-824.
17. Bandi, H. R., Ferrari, S., Krieg, J., Meyer, H. E., Thomas, G. (1993) J. Biol. Chem.
268, 4530-4533.
18. Blenis, J., Chung, J., Erikson, E., Alcorta, D. A., Erikson, R. L. (1991) Cell Growth
Differ 2, 279-285.
19. Jefferies, H. B., Fumagalli, S., Dennis, P. B., Reinhard, C., Pearson, R. B., Thomas,
G. (1997) EMBO J 16, 3693-3704
20. Terada, N., Takase, K., Papst, P., Nairn, A. C., Gelfand, E. W. (1995) J. Immunol.
155:3418-3426
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
40
21. Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S. C., Hemmings, B.
A., Thomas, G. (1998) Science 279, 707-710.
22. Brown, E. J., Beal, P. A., Keith, C. T., Chen, J., Shin, T. B., Schreiber, S. L. (1995)
Nature 377, 441-446
23. Han, J. W., Pearson, R. B., Dennis, P. B., Thomas, G. (1995) J. Biol. Chem. 270,
21396-21403
24. Schmelzle, T., Hall, M. N. (2000) Cell 103, 253-262
25. Dufner, A., Thomas, G. (1999) Exp. Cell. Res. 253, 100-109
26. Gingras, A. C., Raught, B., Soneberg, N. (2001) Genes Dev. 15, 807-826.
27. Dennis, P. B., Jaeschke, A., Saitoh, M., Fowler, B., Kozma, S. C., Thomas, G. (2001)
Science 294, 1102-1105
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
41
28. Rohde, J., Heitman, J., Maria, E. C. (2001) J. Biol. Chem. 276, 9583-9586
29. Zhang, H., Stallock, J. P., Ng, J. C., Reinhard, C., Neufeld, T. P. (2000) Genes Dev.
14, 2712-2724.
30. Kim, W. B., Chung, H. K., Lee, H. K., Kohn, L. D., Tahara, K., Cho, B. Y. (1997) J.
Clin. Endocrinol. Metab. 82, 1953-1599.
31. Park, E.S, Kim, H., Suh, J. M., Park, J., You, S. H., Chung, H. K., Lee, K. W., Kwon,
O-Y., Cho, B. Y., Kim, Y. K., Ro, H. K., Chung, J., Shong, M. (2000) Mol.
Endocrinol .14, 662-670.
32. Anderson, K. E., Coadwell, J., Stephens, L. R., Hawkins, P. T. (1998) Curr. Biol. 8,
684-691
33. Lucia, E. R., Lewis, C. C. (1999) J. Biol. Chem. 274, 8347-8350
34. Mukhopadhyay, N.K., Price, D. J., Kyriakis, J. M., Pelech, S., Sanghera, J., Avruch,
J. (1992) J. Biol. Chem. 267, 3325-3335
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
42
35. Saunier, B., Tournier, C., Jacquemin, C., Pierre, M. (1995) J. Biol. Chem. 270, 3693-
3697
36. Medina, D. L., Santisteban, P. (2000) Eur. J. Endocrinol. 143, 161-178.
37. Hidalgo, M., Rowinsky, E. K. (2000) Oncogene 19, 6680-6686
38. Vinals, F., Chambard, J. C., Pouyssegur, J. (1999) J. Biol. Chem. 274, 26776-26782
39. Tonacchera, M., Van Sande, J., Parma, J., Duprez, L., Cetani, F., Costagliola, S.,
Dumont, J. E., Vassart, G. (1996) Clin. Endocrinol. 44, 621-633
40. Parma, J., Duprez, L., Van Sande, J., Cochaux, P., Gervy, C., Mockel, J., Dumont, J.,
Vassart, G. (1993) Nature 365, 649-651
41. Kimura, T., Van Keymeulen, A., Golstein, J., Fusco, A., Dumont, J. E., Roger, P. P.
(2001) Endocr. Rev. 22, 631-656
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
43
42. Okkenhaug, K., Vanhaesebroeck, B. (2001) Sci STKE 65, 1-5
43. Samayor, A., Morrice, N. A., Alessi, D. R. (1999) Biochem. J. 342, 287-292
44. Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F.,
Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., Tepst, P., Coadwell, J.,
Hawkins, P. T. (1998) Science 279, 710-714.
45. Pause, A., Belsham, G. J., Gingras, A. C., Donze, O., Lin, T. A., Lawrence, J. C. Jr.,
Sonenberg, N. (1994) Nature 371, 762-767
46. Brunn, G. J., Hudson, C. C., Sekulic, A., Williams, J. M., Hosoi, H., Houghton, P. J.,
Lawrence, J. C. Jr., Abraham, R. T. (1997) Science 277, 99-101.
47. Gingras, A. C., Gygi, S. P., Raught, B., Polakiewicz, R. D., Abraham, R., Hoekstra
M. F., Aebersold, R., Sonnberg, N. (1999) Genes Dev. 13, 1422-1437.
48. Davies, S. P., Reddy, H., Caivano, M., Cohen, P. (2000) Biochem. J. 351, 95-105
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
44
49. Nedachi, T., Akahori, M., Ariga, M., Sakamoto, H., Suzuki, N., Umesaki, K.,
Hakuno, F., Takahashi, S. I. (2000) Endocrinology 141, 2429-2438
50. Gonzalez-Robayna, I. J., Falender, A. E., Ochsner, S., Firestone, G. L., Richards, J. S.
(2000) Mol. Endocrinol. 14, 1283-1300
51. Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M.,
Housman, D. E., Graybiel, A. M. (1998) Science 282, 2275-2279
52. de Rooij, J., Zwartkruis, F. J., Verheijen, M. H., Cool, R. H., Nijman, S. M.,
Wittinghofer, A., Bos, J. L. (1998) Nature 396, 474-477
53. York, R.D., Molliver, D. C., Grewal, S. S., Stenberg, P. E., McCleskey, E. W., Stork,
P. J. (2000) Mol. Cell. Biol. 20, 8069-8083
54. Desai, B. N., Myers, B. R., Schreiber, S. L. (2002) Proc. Natl. Acad. Sci. U. S. A 99,
4319-4324
55. Peterson, R. T., Desai, B. N., Hardwick, J. S., Schreiber, S. L. (1999) Proc. Natl.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
45
Acad. Sci. U. S. A 96, 4438-4442
56. Coulonval, K., Vandeput, F., Stein, R. C., Kozma, S. C., Lamy, F., Dumont, J. E.
(2000) Biochem. J. 348, 351-358
57. Saito, J., Kohn, A. D., Roth, R. A., Noguchi, Y., Tatsumo, I., Hirai, A., Suzuki, K.,
Kohn, L. D., Saji, M., Ringel, M. D. (2001) Thyroid 11, 339-351
58. De Vita, G., Berlingieri, M. T., Visconti, R., Castellone, M. D., Viglietto, G.,
Baldassarre, G., Zannini, M., Bellacosa, A., Tsichlis, P. N., Fusco, A., Santoro, M.
(2000) Cancer Res. 60, 3916-3920
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
46
Figure Legends
Figure 1. A and B. TSHR-associated PI3K activity in thyroid cells stimulated with
TSH and cAMP. FRTL-5 thyroid cells which were maintained in 4H without serum
condition for 48hr and treated with TSH (1 mU/ml) or 8-Br-cAMP (1mM) for 10 min.
H89 was pretreated for 45min before TSH or 8-Br-cAMP treatment. TSH receptors
were immunoprecipitated with a monoclonal anti-TSH receptor antibody and the
immune complexes were subjected to PI3K lipid kinase assays. Wortmannin and
LY294002 were added in the immune complexes for 10 min. Phosphorylated lipids
were separated by TLC, detected by autoradiography, and quantified with
phosphorimager analyses. Immunoblot analysis showed that similar amounts of human
TSH receptor were used in the assays. W: wortmannin (100 nM), L: LY294002 (500
nM) in vitro, H89 (50 µM) in vivo. C. TSHR associated PI3K activity in CHO and
CHO-TSHR. The activity of TSHR-associated PI3K was measured as above (A, B).
Cell lysates from CHO and CHO-TSHR treated with TSH for 5 min were
immunoprecipitated with anti-TSHR antibody and control IgG for negative control.
Figure 2. A. The expression of p85α regulatory subunit and p110γ catalytic
subunit of class I PI3K isoforms in FRTL5. FRTL-5 cells were grown to 70%
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
47
confluence in complete 6H medium with 5% serum, and then maintained for 6 days in
4H0% medium, which does not contain TSH, insulin and serum. Total cell lysates were
prepared from cells treated for 12h with TSH (1 mU/ml), insulin (10 µg/ml) or 8-Br-
cAMP (1 mM) and analyzed by Western blot using an anti-p85α regulatory subunit of
PI3K antibody and an anti-p110γ catalytic subunit of PI3K antibody. B, C and D.
Interactions between the p85 regulatory subunit of PI3K and TSHR in FRTL5 and
CHO cells. CHO cells were transfected with constructs that express HA-tagged p85α
(HA- p85α) and pcDNA3-hTSHR and after 24 hr incubation, cells were harvested and
immunoprecipitated with an anti-TSHR antibody (B). FRTL5 cells transfected with
HA-tagged p85α (HA- p85α) and were treated with TSH for 5 min (C) and TSH and 8-
Br-cAMP were treated for 5min in FRTL-5 thyroid cells (D). Cells were
immunoprecipitated with an anti-TSHR antibody. The amount of total protein was
measured as described in ‘Materials and Method’. Immunoprecipitates were separated
with SDS-PAGE on a 10% gel and immunoblotted with anti-HA (B, C), anti-
phosphotyrosine antibodies ( D) and anti-p85α (D).
Figure 3. A Subcellular localization of PDK-1 in TSH-treated thyroid cells. FRTL-5
cells were grown on coverslips in 6-well plates and transfected with pEGFP-PDK-1.
Cells were maintained without TSH, insulin and serum after 50% confluence and in
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
48
48hr after transfection, cells were treated with TSH or 8-Br-cAMP for 5 min or treated
with wortmannin (W), LY294002 (L), and H89 for 45 min and further stimulated with
TSH for 5 min as indicated. Fixed cells were examined under a laser-scanning confocal
microscope. The results shown are representative of three independent experiments. B.
Effects of TSH and 8-Br-cAMP on tyrosine phosphorylation of PDK1. CHO-TSHR
cells were transfected with pCDNA3-PDK-1-Myc with lipofection and the cells treated
with TSH and 8-Br-cAMP for 15 min after 24h of transfection. PDK1-Myc was
immunoprecipitated with anti-Myc antibodies and its tyrosine phosphorylation was
analyzed with anti-phosphotyrosine antibody (4G10).
Figure 4. Effects of TSH and insulin on phosphorylation of Akt/PKB and 4E-
BP1/PHAS1 in FRTL-5 thyroid cells. FRTL-5 cells were grown to 70% confluence in
complete 6H medium with 5% serum, and then maintained for 6 days in 4H0% medium,
which does not contain TSH, insulin and serum. Total cell lysates were prepared from
cells treated with TSH (1 mU/ml) (A, C), insulin (10 µg/ml) (B, D) or insulin (10
µg/ml) and rapamycin (20 nM) and analyzed by Western blot using anti-Akt antibodies
(total and phosphospecific) and anti-4E-BP1 antibodies (total and phosphospecific).
Figure 5. Effects of TSH and insulin on the phosphorylation and activation of
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
49
S6K1 in FRTL-5 thyroid cells. FRTL-5 cells were grown to 70% confluence in
complete 6H medium with 5% serum, and then maintained for 6 days in 4H0% medium,
which does not contain TSH, insulin and serum. Total cell lysates were prepared from
cells treated with TSH (1 mU/ml) (A) or insulin (10 µg/ml) (C) and analyzed by
Western blot using a polyclonal anti-S6K1 antibody and an anti-phospho-S6 ribosomal
protein (Ser235/236) antibody. To measure S6K1 activity, cells were transfected with
HA-tagged p70 S6K1 and immunoprecipitated using an anti-HA monoclonal antibody.
Phosphotransferase activity of S6K1 was assayed using recombinant S6 peptide as a
substrate (B, D). The values represent the mean ± standard errors of assays carried out
with three independent cell preparations.
Figure 6. A and B. Effects of PI3K inhibitors on TSH-induced phosphorylation of
S6K1 in FRTL-5 thyroid cells. FRTL-5 cells were prepared as described in the legend
to Figure 3. Total cell lysates were prepared from cells treated with TSH (45 min) and/or
LY294002 and wortmannin and analyzed by Western blot using a polyclonal S6K1
antibody. LY294002 and wortmannin were pretreated for 45 min before TSH addition.
C. Effects of p85α lacking a binding site (iSH2) for the p110 catalytic subunit of
PI3K on the phosphorylation of S6K1 in TSH-treated thyroid cells. FRTL-5 thyroid
cell lines were transfected with constructs that express mutant HA tagged-p85α
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
50
deficient in binding of p110 catalytic subunit of PI3K (L5-∆iSH2p85) or wild-type
p85α (L5-Wp85). Total cell lysates were prepared from cells treated with TSH and
analyzed by Western blot using a polyclonal S6K1 antibody. D and E. Effects of
forskolin and 8-Br-cAMP on the phosphorylation of S6K1 in FRTL-5 thyroid cells.
Total cell lysates were prepared from cells pretreated with rapamycin, wortmannin,
LY294002 or H89 for 45 min and then treated with forskolin (10 µM) or 8-Br-cAMP (1
mM) and analyzed by Western blot using a polyclonal S6K1 antibody. F. Dose-
dependent effects of H89 on TSH-induced phosphorylation of S6K1 in FRTL-5
thyroid cells. Total cell lysates were prepared from cells treated with TSH and/or H89
and analyzed by Western blot using a polyclonal S6K1 antibody. R: rapamycin (20 nM),
W: wortmannin (100 nM), L: LY294002 (500 nM), H89 (50 µM)
Fig. 7. A and B. Effects of N-α-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)
on TSH-induced phosphorylation and activation of S6K1. FRTL-5 cells were
pretreated with TPCK for 45min, and treated with TSH for 30 min before total cell
lysates were prepared and analyzed by Western blot using a polyclonal S6K1 antibody
(A). S6K1 activity was measured as described in the legend to Figure 5 (B).
Figure 8. A and B. Effects of TSH on phosphorylation of Erk1/Erk2 in FRTL-5
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
51
thyroid cells. FRTL-5 cells were grown to 70% confluence in complete 6H medium
with 5% serum, and then maintained for 6 days in 4H0% medium, which does not
contain TSH, insulin and serum. Total cell lysates were prepared from cells treated with
TSH (1 mU/ml) or insulin (10 µg/ml) and analyzed by Western blot using
phosphospecific and total anti-Erk1/Erk2 antibodies. C. Effects of rapamycin and
PD98059 on TSH-induced phosphorylation of S6K1 in FRTL-5 thyroid cells.
FRTL-5 thyroid cells were cultured as described above. Total cell lysates were prepared
from cells treated with TSH (1 mU/ml) for 45 min and pretreated with rapamycin (20
nM) or PD98059 for 45 min before TSH treatment and analyzed by Western blot using a
polyclonal S6K1 antibody.
Fig. 9. Effects of stimulating (TSAb) and blocking (TSBAb) TSH receptor
antibodies on phosphorylation of S6K1 in thyroid cells. (A) FRTL-5 cells were
grown to 70% confluence in complete 6H medium with 5% serum, and then maintained
for 6 days in 4H0% medium, which does not contain TSH, insulin and serum. The IgGs
were extracted by affinity chromatography from normal pooled sera (NP), as well as the
sera from the patients with primary myxedema (PM) and Graves’ disease (GD) who
tested positive to the TSH binding inhibitory immunoglobulins (TBII) test. The total
cell lysates were prepared from the cells treated with the IgGs (10 mg/ml) and analyzed
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
52
by Western blot using polyclonal anti-S6K1 antibodies and an anti-phospho-S6
ribosomal protein (Ser235/236) antibodies. CON; treated with the medium. (B) The
total cell lysates were prepared from the cells treated with TSH (1 mU/ml) or the IgGs
from the patients with Graves’ disease with or without the inhibitors as indicated. The
PI3K inhibitors, wortmannin (W) and LY294002 (L) in addition to the MEK inhibitor,
PD98059 (PD) were preincubated for 45 min and then either TSH or IgG. Which
stimulate the TSH receptor antibody, TSAb, obtained from Graves’ disease patients
were added to the medium for 45 min. (C) The IgG (blocking type TSH receptor
antibody, TSBAb) obtained from the primary myxedema were preincubated for 1h,
which was followed by the addition of insulin (10 µg/ml), TSAb (10 mg/ml) and TSH
(1 mU/ml) for 45 min. The total cell lysates were prepared and analyzed by Western blot
using a polyclonal anti-S6K1 antibodies.
Fig. 10. A. Effects of wortmannin, LY294002, rapamycin, and H89 on TSH-induced
[3H]-thymidine incorporation. FRTL-5 thyroid cells were cultured in medium lacking
TSH for 7days. 80% confluent cells grown in 96-well plates were treated with TSH
and/or wortmannin (0.1 µM), LY294002 (0.5 µM), rapamycin (20 nM), and H89 (50
µM) for 24 h, followed by the addition of 2 µCi/mL [3H]thymidine for an additional 2 h.
Cells were washed and precipitated with ice-cold 10% trichloroacetic acid.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
53
Radioactivity was determined by liquid scintillation spectrometry. Results were
measured as the number of counts/min in each well. Each experimental data point
represents triplicate wells from at least four different experiments. B and C. Effects of
TSH and rapamycin on the level of cyclin D1. FRTL-5 cells were grown to 70%
confluence in complete 6H medium with 5% serum, and then maintained for 6 days in
4H0% medium, which does not contain TSH, insulin and serum. The medium was
replaced with fresh medium including TSH (1 mU/mL). Total cell lysates were prepared
and analyzed by Western blot using an anti-cyclin D1 antibody (B). Total cell lysates
were prepared from cells treated with TSH and/or 20 nM rapamycin for 12 h, and
analyzed by Western blot using anti-cyclin D1 and anti-actin antibodies (C). D. Effects
of rapamycin on TSH/insulin-induced cell-cycle progression and cellular
proliferation. Confluent FRTL-5 thyroid cells in 100 mm dishes were detached by
trypsinization, resuspended in 6H growth medium, seeded at a density of 3 x 104
cells/well in 6-well plates, and incubated for 2-3 days until 80% confluent. The medium
was changed to 3H medium with 5% calf serum and the cells were incubated for an
additional 7 days. TSH and/or insulin plus rapamycin were added to the quiescent cells
and incubated for 48h. Cell cycle analysis was performed using a Becton Dickinson
fluorescence-activated cell analyzer and Cell Quest version 1.2 software. At least 10,000
cells were analyzed per sample, and cell cycle distribution was quantified using the
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
54
ModFit LT version 1.01 software. Representative patterns of cell cycle analysis are
presented. The values represent the mean ± SE of cell counts assays carried out with
three independent cell preparations.
Fig. 11. A and B. Effects of rapamycin on follicle activity in MMI-treated Sprague-
Dawley rats. Male Sprague-Dawley rats (120-130 g) were fed with water containing
0.025% methimazole for 2 wks. Rapamycin dissolved in 2% carboxymethylcellulose
was delivered once daily by intraperitoneal injection at a dose of 1.5 mg/kg for a week
before histologic examination. Cross-sections of the rat thyroid stained with H-E. The
number of colloid-filled follicles were counted in 2 fields at a magnification of x200. C.
Effects of rapamycin on thyroid S6 phosphorylation in MMI treated Sprague-
Dawley rats. Further sections were used for immunohistochemistry (IHC). The primary
antibody, rabbit anti-phospho-S6 ribosomal protein, was diluted (1:200) with a
background-reducing diluent (Dako, Carpinteria, CA) and incubated for 60 minutes,
followed by incubation in the EnVision-HRP reagent. The sections were then
sequentially incubated with DAB (3,3-diaminobenzidine) chromogen for 5 minutes,
counterstained with Meyer’s hematoxylin, and mounted. Careful rinses with several
changes of TBS-0.3% Tween buffer were performed between each step. A negative
control excluded the primary antibody. Cells with cytoplasmic granular staining were
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
55
considered positive.
Fig. 12. Schematic of TSHR-mediated PI3K, PDK-1, and S6K1 activation
pathways. The TSHR-associated PI3K activity is increased by TSH and 8-Br-cAMP.
TSH is able to translocate PDK1 into the plasma membrane via PI3K and PKA
dependent manner and PDK-1 preferentially phosphorylates S6K1, not Akt/PKB.
However, insulin phosphorylates Akt/PKB and 4E-BP1/PHAS1 in a PI3K- and
FRAP/mTOR-dependent manner. Rapamycin inhibits the cooperative actions of 4E-
BP1/PHAS1 and S6K1 and results in the inhibition of TSH-mediated follicle
proliferation and activity. The solid lines represent the pathways preferentially activated
by TSH and the dashed lines represent pathways preferentially activated by insulin in
thyroid cells.
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from
origin
PIP2
1 2 3 4
TSH - + - -
IP: anti-TSHR
A
IB: anti-TSHR
W L
Figure 1
1 2 3 4 5IP: anti-TSHR
- - + - + H89 - + + - -- - - + +
TSH cAMPB
origin
PIP2
C CHO CHO-TSHR0 0 5 5 (W) 5 (min)TSH
PIP2
IP: anti-TSHR IgG IgG1 2 3 4 5
origin
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
Figure 2
p85αp110γ
165105
7655
1 2 3 4 5 6
TSH - + - + - +
cAMP Insulin
A
M(kDa)
IgG
HA-p85α
TSHRHA-p85
- + - + - - + +
FRTL-5
CHO-TSHR1 2 3 4
B
0 5 15 5 5 min
IgG
FRTL-51 2 3 C T
CTSH
p85α
TSHR
TSH cAMP- + - +
FRTL-5
D
HA-p85α
TSHR
pY TSHR
1 2 3 4
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
A
Buffer TSH
Myc-PDK1
IB:anti-pY
IB:anti-MycIP:anti-myc
- - + + +
- + - + -TSH- - - - + cAMP
B
TSH +Wortmannin
TSH +LY294002
TSH+H89
Figure 3
cAMP
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
TSH 0 5 15 30 60 minpT308
pS473
Total
A
B
Figure 4
Insulin 0 15 30 60 120 minpS473
Total
1 2 3 4 5
1 2 3 4 5
Total
pS65
Insulin 0 5 15 15 (min)Rapamycin
1 2 3 4
D
Akt/PKB
Akt/PKB4E-BP1/PHAS1
- - - +
Total
pS65TSH 0 5 15 30 (min)
1 2 3 4
C
4E-BP1/PHAS1
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
TSH 0 5 15 30 60 (min)
1
2
3
4
5
0
2
4
6
10
12
0
TSH (1 mU/ml) 0 5 15 30 60 (min) 0 5 15 30 60 (min)
Insulin (10 µg/ml)
S6K
1 A
ctiv
ity (f
old)
A C
S6K
1 A
ctiv
ity (f
old)medium
TSHmedium Insulin
Figure 5
pS6 pS6
B D
Insulin 0 5 15 30 60 (min)
1 2 3 4 5 1 2 3 4 5
pp70 S6K1
pp85 S6K1
pp70 S6K1
pp85 S6K1
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
Forskolin - + + + + +
R W L H89
R W L H89
cAMP - + + + + +
TSH - + + + + +
H89 (µM)5 10 30 50
D
E
F
pp70 S6K1
pp85 S6K1
pp70 S6K1
pp85 S6K1
pp70 S6K1
pp85 S6K1
pp70 S6K1
pp85 S6K1L5-WTp85 L5-∆iSH2 p85
0 5 15 30 60 0 5 15 30 60 min
TSH - + + + + + 0.5 1 2 5LY294002 (µM)
TSH - + + + + + 0.01 0.1 0.3 0.5Wortmannin (µM)
pp70 S6K1
pp85 S6K1
A
B
1 2 3 4 5 6
1 2 3 4 5 6
pp70 S6K1
pp85 S6K1
C
Figure 6TSH
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
S6K
1 A
ctiv
ity (f
old)
1
2
TSH - + + + +
TPCK (µM)10 20 50
TSH - + - + - - + + TPCK(20 µM)
B
Figure 7
pS6
pp70 S6K1
pp85 S6K1
A
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
- + - + - + Rapamycin
PD98059
1 2 3 4 5 6
0 5 15 30 60 (min)
Insulin
Total
pT202/Y204
pT202/Y204
Total
TSH
TSH- - + + - -- - - - + + 0 5 15 30 60 (min)
A
B
C
Figure 8
pp70 S6K1
pp85 S6K1
ERK1/ERK2
ERK1/ERK2
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
1 2 3 4 5 61 : control 2 : TSH3 : TSBAb4 : Insulin + TSBAb (10 mg/ml)5 : TSAb + TSBAb (10 mg/ml)6 : TSH + TSBAb (10 mg/ml)
TSAb(10 mg/ml)
TSH
L W PDB C
Figure 9
CON NP PM 1 2 3 4 5 6 7 8 9 10
GD
A
pS6
pp70 S6K1
pp70 S6K1
pp85 S6K1 pp70 S6K1
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
TSH - - + +- + - +rapamycin
cyclin D1β-actin
TSH 0 12 24 48 (h)
cyclin D1
1 2 3 4
1 2 3 4
B
C
Figure 10
1
5
10
15
20
TSHL H
Fold
Incr
ease
in[3 H
]-Thy
mid
ine
Upt
ake A
R W- + + + + +
TSH (2)
Control (1) G1: 82.9 %
S: 1.88 %G2: 11.8 %
G1: 72.0 %S: 13.1 %
G2: 9.80 %
G1: 80.3 %S: 5.80 %
G2: 11.8 %
TSH + Rapa (4)
TSH/Insulin (3)
TSH/Insulin + Rapa (5)
D
G1: 49.3 %S: 19.5 %
G2: 19.1 %
G1: 72.6 %S: 13.1 %
G2: 10.5 %
Relative Fluorescence
Cel
l Num
ber
1 2 3 4 5
S6K1
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
ControlMMI
(2 wks)MMI (2 wks)
Vehicle(1 wk)MMI (2 wks)
rapamycin (1wk)
No.
of c
ollo
id-fi
lled
folli
cle/
x200
10
20
30
40
50
60
70
C R RV
MMI
A B
Figure 11
X 400 X 400 X 400
MMI (2 wk) rapamycin (3Days)
MMI (2 wk)vehicle (3Days)Control
C
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
cAMP/PKA
PDK
-1GsAC
p85 p110
S6K1
Thyrocyte proliferationRegulation of follicle activity
TSH/TSAb/TSHR
PI3K
Akt
/PK
B
4E-BP/PHAS1
Figure 12
S6
FRAP/mTOR
INSULIN
ATP
by guest on January 16, 2020 http://www.jbc.org/ Downloaded from
Cho, Heung Kyu Ro and Minho ShongYounHwang, Jin Man Kim, Eun Suk Hwang, Jongkyeong Chung, Jeung-Hwan Han, Bo
Jae Mi Suh, Jung Hun Song, Dong Wook Kim, Ho Kim, Hyo Kyun Chung, Jung Hwanthyroid gland
thyroid stimulating hormone and stimulating-type TSH receptor antibodies in the Regulation of the PI3K, Akt/PKB, FRAP/mTOR, and S6K1 signaling pathways by
published online March 30, 2003J. Biol. Chem.
10.1074/jbc.M300805200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on January 16, 2020http://w
ww
.jbc.org/D
ownloaded from