Departments of 1 Biochemistry, Microbiology, and Immunology, 2 Medicine, and 3 Cellular and Molecular Medicine, Ottawa Health Research Institute, University of Ottawa, Ottawa Ontario, K1Y 4E9, Canada
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ABSTRACT |
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Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 µM TSH. In the presence of 20 µM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.
p70 S6 kinase; protein kinase B; phosphoinositide 3-kinase; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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THE THYROID-STIMULATING hormone (TSH) receptor (TSHR) is a G protein-coupled receptor (GPCR) of the seven-transmembrane domain family. The glycoprotein hormone TSH binds to TSHR on thyrocytes in the thyroid and activates the classic G protein effectors adenylyl cyclase and phospholipase C. TSH induces proliferation, iodine uptake, thyroid hormone release, and cell survival in the thyrocyte (15).
Extrathyroidal TSHR protein expression has been reported in orbital fibroblasts (3, 9, 41). Orbital fibroblasts can differentiate into adipocytes (39, 40), and TSHR protein expression may increase with orbital adipogenesis (9, 41). TSHR has also been detected in guinea pig, mouse, and rat adipocytes through binding assays and mRNA analysis (12, 19, 35). More recently, TSHR mRNA was found in human abdominal adipose tissue, where it may regulate lipolysis (21, 22). We previously showed (3) that TSHR mRNA is expressed in human subcutaneous abdominal adipose tissue and that TSHR protein is expressed in primary cultures of human abdominal preadipocytes. Addition of TSH to these cells activates p70 S6 kinase (p70 S6K), a Ser/Thr protein kinase identified as a novel downstream target of TSHR in thyroid cells (5).
The murine 3T3-L1 preadipocyte cell line is a well-established model of adipogenesis that differentiates into mature, lipid-laden adipocytes when appropriately cued (36). In this study, we have used 3T3-L1 preadipocytes to investigate proximal signaling events leading to p70 S6K activation by TSH and to determine whether TSH modulates preadipocyte survival.
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METHODS |
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Materials.
3T3-L1 preadipocytes were obtained from American Type Culture
Collection (Manassas, VA) and maintained at low passage. FRTL-5 thyrocyte (Dr. R. Germinario, McGill University, Montreal, QC, Canada),
J774 mouse macrophage (Dr. Y. Marcel, University of Ottawa, Ottawa, ON,
Canada), and CHO-vector control and human (h)TSHR-transfected CHO
(CHO-hTSHR) (Dr. J. E. Dumont, Erasme University Hospital, Free
University of Brussels, Brussels, Belgium) cell lines were kindly given
to us for these studies. DMEM, Ham's F12 medium, fetal bovine
serum (FBS), calf serum (CS), newborn calf serum, PMSF, HEPES,
penicillin-streptomycin (PS), and nystatin were from GIBCO BRL
(Burlington, ON, Canada). Experiments were normally performed with
commercial bovine TSH from Sigma (St. Louis, MO), except when bovine
TSH (TSH-NIH; AFP8755B) from the National Institute of Diabetes and
Digestive and Kidney Diseases National Hormone and Peptide Program
(Torrance, CA) was used, as indicated. Mouse monoclonal TSH-R2
(211-414) was from Novacastra Laboratories
(Newcastle, UK). Rabbit polyclonal phospho-protein kinase B (PKB/Akt)
(Ser473), rabbit polyclonal phospho-p70 S6K, and rabbit
polyclonal phospho-PKB/Akt (Thr308) antibodies were from
New England Biolabs (Mississauga, ON, Canada). Mouse monoclonal
phosphotyrosine antibody PY20 was from BIO/CAN Scientific (Mississauga,
ON, Canada). Rabbit polyclonal janus kinase (JAK)1 antibody was a gift
from Dr. M. Kozlowski (University of Ottawa, Ottawa, ON, Canada), and
rabbit polyclonal p70 S6K antibody was from Santa Cruz Biotechnology
(Santa Cruz, CA). Rabbit polyclonal caspase 3 antibody was from
PharMingen International (Mississauga, ON, Canada). EGTA, sodium
pyrophosphate (NaPPi), sodium orthovanadate
(Na3VO4), leupeptin, benzamidine,
-glycerophosphate, Triton X-100, protein A Sepharose, protein G
agarose, IBMX, cAMP, potassium hydroxide (KOH), phosphatidylinositol,
transferrin, glycyl-L-histidyl-L-lysine,
forskolin, LY-294002, and 3,3',5-triiodo-DL-thyronine (T3)
were from Sigma. Somatostatin and hydrocortisone-PO4 were from Calbiochem (San Diego, CA). Microcystin, sodium fluoride (NaF),
perchloric acid, and Nonidet P-40 were from VWR (Ville Mont-Royal, QC,
Canada). Wortmannin was from Kamiya Biomed (Thousand Oaks, CA).
Aprotinin and ATP were from Boehringer-Mannheim (Laval, QC).
[
-32P]ATP, [14C]cAMP,
[3H]adenine, and nitrocellulose were from Amersham
Pharmacia Biotech (Baie d'Urfé, QC, Canada). The p70 S6K in
vitro kinase assay kit was from Upstate Biotechnology (Lake Placid,
NY). The in situ cell death detection kit was from Roche Molecular
Biochemicals (Laval, QC, Canada). The enhanced chemiluminescence (ECL)
detection kit was from NEN Life Science Products (Boston, MA). X-Omat
film was from Kodak, and Molecular Analyst was from Bio-Rad
(Mississauga, ON, Canada).
Cell culture and stimulations. 3T3-L1 preadipocytes were grown to confluence in DMEM supplemented with 10% CS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. The FRTL-5 cell line was grown in Ham's F12 medium supplemented with 5% newborn calf serum, 1 mU/ml TSH, 834 nM insulin, 5 µg/ml transferrin, 10 ng/ml somatostatin, 10 ng/ml glycyl-L-histidyl-L-lysine, and 10 ng/ml hydrocortisone-PO4. The J774 mouse macrophage cell line was grown in DMEM supplemented with 10% FBS and PS. The CHO-vector control and CHO-hTSHR cell lines were grown in Ham's F12 medium supplemented with 10% FBS, PS, nystatin (50 U/ml), and 400 µg/ml G418. For stimulation experiments, confluent 3T3-L1 preadipocytes were placed in DMEM-0.5% CS-PS, and confluent CHO-vector control and CHO-hTSHR cell lines were placed in Ham's F12-0.5% FBS-PS for 16 h and stimulated with TSH for the times and concentrations indicated. For phosphoinositide 3-kinase (PI3K) inhibition experiments, preadipocytes were preincubated with 100 nM wortmannin, 10 µM LY-294002, or vehicle (0.1% DMSO) for 15 min before stimulation.
Western blot analysis. For TSHR and anti-phosphotyrosine immunoblot analysis, cells were lysed in Laemmli buffer (24) supplemented with 0.2 mM Na3VO4. For all other immunoblots, cells were lysed in Laemmli buffer supplemented with 5 mM EGTA, 5 mM NaPPi, 50 mM NaF, and 1 µM microcystin. Lysate protein was quantified, and equal amounts of solubilized protein were resolved on SDS-PAGE, followed by electrophoretic transfer to nitrocellulose. Blots were probed overnight at 4°C with mouse monoclonal TSH-R2 (1:60), rabbit polyclonal phospho-PKB/Akt (Ser473; 0.1 µg/ml), rabbit polyclonal p70 S6K (1:500), rabbit polyclonal phospho-p70 S6K (1:250), rabbit polyclonal phospho-PKB/Akt (Thr308; 1:500), mouse monoclonal phosphotyrosine (1 µg/ml), rabbit polyclonal JAK1 (1:1,000), or rabbit polyclonal caspase 3 (1:500) primary antibody. For phospho-PKB/Akt analysis, immunoblots were normally probed with anti-phospho-PKB/Akt (Ser473) antibody except when anti-phospho-PKB/Akt (Thr308) antibody was used, as indicated. After incubation with the appropriate peroxidase-conjugated secondary antibody, membranes were processed for ECL detection. When indicated, blots were stripped according to the manufacturer's directions and subsequently reprobed.
Immunoprecipitation and in vitro kinase assay of p70 S6K and
PI3K.
To determine p70 S6K activity, preadipocytes were stimulated with TSH
and the reaction was terminated by the addition of lysis buffer (PBS,
1% Nonidet P-40, 0.2 mM Na3VO4, 0.1 mg/ml
PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 4 µg/ml
benzamidine, 5 mM NaPPi, 50 mM NaF, 1 µM microcystin, and 1 mM
-glycerophosphate). To determine PI3K activity, preadipocytes were
stimulated with TSH and lysed in the p70 S6K lysis buffer described
above with the following modifications: 1% Triton X-100 was
substituted for 1% Nonidet P-40, and NaPPi and microcystin were
omitted. Lysates were centrifuged at 15,000 g for 10 min at
4°C, and supernatants were precleared for 1 h at 4°C with
protein A Sepharose in the case of p70 S6K or protein G agarose in the
case of phosphotyrosine. Samples were incubated for 90 min at 4°C
with 2 µg of rabbit anti-p70 S6K antibody or 5 µg of mouse
anti-phosphotyrosine antibody preadsorbed to protein A Sepharose and
protein G agarose, respectively. For measurement of immunoprecipitated
p70 S6K activity, the Upstate Biotechnology in vitro kinase assay
commercial kit was used (3). After this assay,
immunoprecipitated p70 S6K was resuspended in immunoblot lysis buffer
and subjected to SDS-PAGE and Western blot analysis to detect gel
mobility shifts. For measurement of coimmunoprecipitated PI3K in the
anti-phosphotyrosine immunoprecipitates, a PI3K kinase assay was used
(27). The reaction was initiated with the addition of
Mg-ATP cocktail (10 mM MgCl2, 10 µM cold ATP, 20 µCi
[
-32P]ATP) to immunoprecipitated
phosphotyrosine-containing proteins suspended in PI3K assay buffer (20 mM Tris, 0.1 M NaCl, 0.5 mM EGTA, 0.2 mg/ml phosphatidylinositol).
After a 3-min incubation period, chloroform-methanol-HCl (50:100:1
vol/vol/vol) was added to terminate the reaction. The lipid product was
extracted, spotted onto a silica gel plate, and resolved by thin-layer
chromatography. The silica gel plate was exposed to X-Omat film for
autoradiographic detection. The relative intensity of the band was
measured with Molecular Analyst imaging software, and data are
expressed as integrated optical density (IOD) units.
cAMP assay. 3T3-L1 preadipocytes and their differentiated counterparts were labeled overnight in DMEM containing 5% FBS and 2 µCi/ml [3H]adenine (20). The next day, cells were incubated at 37°C in DMEM containing 20 mM HEPES and 1 mM IBMX and treated with vehicle (H2O), TSH (5 µM), or forskolin (10 µM) for 30 min. The reaction was terminated on addition of cold stop solution containing 2.5% (vol/vol) perchloric acid, 100 µM cAMP, and ~10,000 cpm of [14C]cAMP. After a 30-min incubation period at 4°C, acid-cell lysates were transferred to tubes containing 4.2 M KOH for neutralization. Sequential chromatography on Dowex and alumina columns was performed to determine intracellular cAMP levels. Data are expressed as ([3H]cAMP/total uptake per well) × 1,000.
Cell enumeration.
3T3-L1 preadipocytes were grown to confluence in the absence or
presence of T3 (2 nM) as indicated and either left in serum or serum deprived in the absence or presence of TSH (1, 5, 10, and 20 µM) for 6 h. Cell enumeration was performed as described previously (13). The following equation was used to
calculate % cell death
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TUNEL assay. Confluent 3T3-L1 preadipocytes were either left in serum or serum deprived in the absence or presence of TSH (20 µM) for 3 h. The time period for serum withdrawal was shortened from 6 h to 3 h to optimize the number of adherent cells staining positively for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), thus avoiding cell detachment associated with the end stages of apoptosis. Preadipocytes were subjected to TUNEL according to the in situ cell death detection kit (13). Briefly, preadipocytes were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% sodium acetate-0.1% Triton X-100, and treated with TdT for 60 min. Cells were incubated with alkaline phosphatase conjugated with anti-fluorescein antibody and then stained with the alkaline phosphatase substrate mix nitro blue tetrazolium chloride-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP). TUNEL-positive cells were counted in 10 random fields by two independent observers, and the average from duplicate samples was calculated.
Statistical analysis. Statistical analysis by ANOVA was performed with GraphPad InStat version 3.00 for Windows 98 (GraphPad Software, San Diego, CA). Values with P < 0.05 were considered significant.
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RESULTS |
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We first examined whether TSHR protein is expressed in 3T3-L1
preadipocytes. TSHR mRNA expression has been detected in 3T3-L1 preadipocytes on adipogenic stimulation (18). Solubilized
protein from confluent 3T3-L1 preadipocyte cultures was subjected to
immunoblot analysis (Fig. 1). A 100-kDa
band representing the mature processed form of the receptor was
observed in 3T3-L1 preadipocytes and the positive control FRTL-5 rat
thyroid cell line, but not in the negative control J774 mouse
macrophage cell line (2).
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Previously, we demonstrated (3) that TSH activates p70 S6K
in human preadipocytes. Activity was measured by in vitro kinase assay
after immunoprecipitation as well as by the extent of
Ser389 phosphorylation, indicative of kinase activation
(11). TSH also stimulated p70 S6K in 3T3-L1 preadipocytes
as demonstrated by phospho-p70 S6K immunoblot analysis (Fig.
2). Consistent with these data, there was
a 20-fold increase in TSH-stimulated p70 S6K activity with the in vitro
kinase assay (data not shown).
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PKB/Akt is positioned upstream of p70 S6K in many cell types, and
phosphorylation of Ser473 indicates its activation
(42). Immunoblot analysis revealed a dose-dependent rise
in phospho-PKB/Akt levels on TSH stimulation (Fig.
3). A recent report suggests that
contaminants in commercially available TSH preparations could possibly
induce signal transduction pathways independent of TSHR
(7). To confirm that TSH activation of PKB/Akt did not
result from potential contaminants in the commercial TSH preparation,
we examined the effect of TSH on CHO cells transfected with hTSHR
(CHO-hTSHR). Consistent with our results in 3T3-L1 preadipocytes, we
observed strong activation of PKB/Akt in CHO-hTSHR cells. Although a
slight increase in pPKB/Akt levels was observed in empty-vector control
CHO cells, this activation was not significant (P > 0.05). These results demonstrate that the TSH-stimulated rise in
pPKB/Akt levels is dependent on the presence of TSHR and argue against
the possibility of a contaminant in the TSH preparation leading to
activation of PKB/Akt. Stimulation of preadipocytes with the highly
purified TSH-NIH preparation also activated PKB/Akt (Fig. 3), further
demonstrating the specificity of the response to TSH.
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PKB/Akt is a major downstream target of PI3K, and the
PI3K-PKB/Akt pathway is involved in a number of cellular responses
including cell survival (42). To determine whether PI3K is
involved in TSH signal transduction in 3T3-L1 preadipocytes, we tested
the effects of two structurally unrelated PI3K inhibitors (Fig.
4; Ref. 29). Wortmannin or
LY-294002 completely blocked the TSH activation of p70 S6K and PKB/Akt.
These inhibitor studies point to a role for PI3K in mediating the TSH
activation of p70 S6K and PKB/Akt.
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Activation of PI3K occurs on its association with
phosphotyrosine-containing proteins after agonist stimulation of
receptor tyrosine kinases (42). This is
increasingly recognized to occur downstream of GPCRs. After TSH
stimulation of 3T3-L1 preadipocytes, tyrosine-phosphorylated proteins
were immunoprecipitated and then immunoblotted with
anti-phosphotyrosine antibody. A prominent increase in signal intensity
in the 125-kDa region was observed (Fig.
5). Insulin-like growth factor-I (IGF-I)
is known to stimulate tyrosine phosphorylation in 3T3-L1 preadipocytes.
We previously showed (13, 14) that PI3K-PKB/Akt pathway is
activated by IGF-I in these cells. However, IGF-I did not stimulate
tyrosine phosphorylation in the 125-kDa region. A recent report
implicating TSH in the activation of the JAK/signal transducer and
activator of transcription (STAT) pathway found that TSH induced
tyrosine phosphorylation of the 125-kDa protein JAK1 in rat thyrocytes and CHO-hTSHR cells (31). We therefore immunoprecipitated
JAK1 from control and TSH-stimulated 3T3-L1 preadipocytes but did not detect any TSH-stimulated tyrosine phosphorylation of JAK1 (data not
shown). Further studies are necessary for identification of this
125-kDa tyrosine phosphorylated protein.
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Given that TSH increases the level of tyrosine-phosphorylated
proteins, we tested whether it also promotes their association with
PI3K. After TSH stimulation, tyrosine-phosphorylated proteins were immunoprecipitated and the presence of PI3K was evaluated by in
vitro lipid kinase assay. The fourfold increase in PI3K activity
observed (Fig. 6), along with the
inhibitor studies above, indicates that activation of PI3K by TSH is
essential for stimulation of p70 S6K and PKB/Akt in 3T3-L1
preadipocytes.
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The activation of p70 S6K by TSH in thyrocytes has been positioned
downstream of adenylyl cyclase (5, 6, 8). TSH had no
effect on cAMP levels in 3T3-L1 preadipocytes (Fig.
7), suggesting that adenylyl cyclase is
not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. TSH
did increase cAMP levels fourfold in differentiated 3T3-L1 adipocytes
(Fig. 7). Forskolin, a direct activator of adenylyl cyclase, raised
cAMP levels by ~50-fold in preadipocytes (data not shown) but had no
effect on phospho-PKB/Akt levels (Fig.
8).
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In experiments assessing phospho-PKB/Akt levels described thus far, we have used anti-phospho-PKB/Akt (Ser473) antibody as an indicator of PKB/Akt activation. Previous studies demonstrated that phosphorylation of both Ser473 and Thr308 is required for full activation of the enzyme (42). We show that TSH induces a clear increase in the levels of both phospho-PKB/Akt (Thr308) and phospho-PKB/Akt (Ser473), as expected (Fig. 8).
Our laboratory (13) has implicated PI3K and PKB/Akt in
IGF-I-mediated survival of growth factor-deprived 3T3-L1 preadipocytes. We examined the effect of TSH on 3T3-L1 preadipocyte apoptosis induced by serum deprivation (Fig. 9).
After 6 h of serum starvation, the presence of TSH (1-20
µM) during serum deprivation of 3T3-L1 preadipocytes reduced cell
death by 29-76% (P < 0.05 at all doses with the
exception of 10 µM). TUNEL was used to confirm that TSH-reduced cell
death was due to decreased apoptosis. After 3 h of serum deprivation, 13 ± 1 (±SE) TUNEL-positive cells/field were
observed. The presence of TSH during the serum starvation period
reduced the number to only 6 ± 1 TUNEL-positive cells/field,
comparable to that seen in the presence of serum, an 88% reduction.
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A number of cytokines or hormones may enhance or oppose the effects of TSH on cell survival. For example, T3 has been shown to increase apoptosis in lymphocytes (28). We examined the effect of T3 on preadipocyte apoptosis in the absence and presence of TSH (Fig. 9). T3 did not significantly alter TSH-induced survival of serum-starved 3T3-L1 preadipocytes (P > 0.05).
Most apoptotic cell death involves caspase 3 activation. To
determine whether TSH reduces caspase 3 activation, 3T3-L1
preadipocytes were serum starved for 24 h in the absence or
presence of TSH and then subjected to immunoblot analysis to
detect the 17-kDa cleaved activated caspase 3 (Fig.
10). Levels of activated caspase 3 increased fivefold when preadipocytes were serum starved for 24 h.
The addition of TSH reduced the level of cleaved activated caspase 3 by
93%.
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DISCUSSION |
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We have characterized TSH-dependent signaling and function in the 3T3-L1 preadipocyte model. TSH activates p70 S6K and PKB/Akt in a PI3K-dependent manner. In addition, TSH induces tyrosine phosphorylation of an unidentified protein(s) in the 125-kDa region and increases the association of PI3K activity in phosphotyrosine immunoprecipitates. When serum-starved 3T3-L1 preadipocytes are exposed to TSH, the level of activated caspase 3 and apoptosis are reduced.
The well-established TSH signal transduction pathway in thyrocytes is characterized by G protein activation of adenylyl cyclase, leading to elevated cAMP levels and activation of protein kinase A (PKA) (15). Recent studies have described novel cAMP targets, in addition to PKA, including guanine nucleotide exchange factors for the small GTPases Rap1a, Rap2, and potentially Ras (34). p70 S6K has been implicated in cAMP-mediated TSH signaling in Wistar rat thyroid cells and primary dog thyrocytes (5, 8). Furthermore, our laboratory (3) has identified p70 S6K as a target of TSH signaling in human orbital and abdominal preadipocytes.
p70 S6K is implicated in the control of transcription and translation, and its phosphorylation leads to a complex sequence of conformational changes that activate the enzyme (11). Phosphorylation of Ser/Thr residues in the COOH-terminal domain facilitates phosphorylation of Thr389 and Thr229 residues that are critical for enzyme activation. The kinase(s) responsible for the initial COOH-terminal phosphorylations are currently unknown, but other upstream regulators have been identified for two important Thr residues. Mammalian target of rapamycin (mTOR) governs the phosphorylation of Thr389 on p70 S6K, but it remains unclear whether mTOR directly phosphorylates p70 S6K or inhibits the Ser/Thr protein phosphatase 2A (4, 32). Phosphorylation of p70 S6K at Thr229 has been attributed to 3-phosphoinositide-dependent protein kinase-1 (PDK-1). PI3K production of 3-phosphorylated phosphoinositides induces translocation of PDK-1 to the plasma membrane. PKB/Akt is simultaneously recruited to the membrane for phosphorylation and activation. Complete activation of PKB/Akt requires phosphorylation at Thr308 and Ser473. PDK-1 is known to phosphorylate Thr308, but the nature of the kinase responsible for Ser473 phosphorylation is currently unresolved (42).
TSH activation of p70 S6K and PKB/Akt in Wistar rat thyroid cells is mediated by cAMP, as demonstrated by the mimicking effect of cAMP-elevating agents on p70 S6K and PKB/Akt activation (6). Two additional studies with thyrocyte models have also implicated cAMP in TSH-stimulated p70 S6K activation (5, 8). In contrast, we observed activation of p70 S6K and PKB/Akt, with no change in cAMP levels in TSH-stimulated 3T3-L1 preadipocytes. Therefore, at the preadipocyte stage, which is the focus of these studies, TSH was able to activate p70 S6K and PKB/Akt in the absence of any changes in cAMP. Furthermore, the addition of forskolin, which resulted in a prominent increase in cAMP, had no effect on phospho-PKB/Akt levels. Our data demonstrate that elevated cAMP levels are not required for TSH activation of PKB/Akt in 3T3-L1 preadipocytes. However, we do note that, on differentiation into mature adipocytes, these cells did exhibit TSH-dependent changes in cAMP. This indicates that TSHR coupling to cAMP, perhaps owing to changes in adenylyl cyclase or one of the regulators of G protein signaling (RGS protein), may be differentiation stage-specific (38) and merits further investigation in the future.
Our data demonstrate that the TSH signaling pathway in 3T3-L1 preadipocytes includes PI3K-PKB/Akt-p70 S6K. Preincubation with PI3K inhibitors abrogated TSH activation of p70 S6K and PKB/Akt. Direct measurement of PI3K activity in anti-phosphotyrosine immunoprecipitates revealed that TSH promotes the association of PI3K activity with tyrosine-phosphorylated proteins.
Tyrosine kinase receptors, such as those for insulin and IGF-1, have
been shown to induce activation of PI3K on agonist stimulation. Recent
studies position PI3K-PKB/Akt-p70 S6K downstream from GPCRs. G proteins
can activate class IA and class IB PI3K through
indirect and direct mechanisms, respectively (16). The
details of these pathways have yet to be fully elucidated, but some of
the targets have been identified. Nonreceptor tyrosine kinases comprise
one set of targets that play a role in the GPCR activation of class IA PI3K. For example, the gastrin/CCKB receptor
initiates src family nonreceptor tyrosine kinase phosphorylation of
IRS-1, followed by docking and activation of PI3K (10).
Alternatively, GPCR activation of class IB PI3K may be
direct, as is the case for the G subunit activation
of PI3K
(25). It should be noted that, unlike our data
demonstrating PI3K activation in TSH-stimulated preadipocytes, studies
on TSH signaling in thyrocyte models have not detected PI3K activation,
despite the inhibitory effects of wortmannin and LY-294002 (5,
7).
TSH stimulation of thyroid epithelial cell lines results in activation of nonreceptor tyrosine kinases (1, 30). Interestingly, it was reported that TSH increased the tyrosine phosphorylation of an unidentified 125-kDa tyrosine-phosphorylated protein(s) (30). A separate report has implicated the ~125-kDa protein JAK1 in rat thyrocytes and CHO-hTSHR cells (31). Results from our immunoblot analysis indicate that JAK1 is not a target of TSH signaling in 3T3-L1 preadipocytes. Further studies are warranted to determine the identity of this protein(s). It may provide the link between TSHR and PI3K in our cell system, although at this point, we cannot rule out other routes of PI3K activation.
The antiapoptotic properties of TSH have been previously demonstrated in FRTL-5 thyroid cells (26, 37). TSH-mediated survival of serum-deprived FRTL-5 cells is mediated by cAMP-dependent events. The possibility of PI3K involvement in TSH-induced cell survival was not addressed in these studies. However, many groups have implicated PI3K-PKB/Akt in antiapoptotic mechanisms in various cell types (42). Our laboratory (13) recently showed that PI3K is necessary for IGF-I-mediated antiapoptotic mechanisms in 3T3-L1 preadipocytes subjected to serum withdrawal. We found that TSH also protects 3T3-L1 preadipocytes from apoptosis induced by serum withdrawal and reduces the levels of activated caspase 3. It appears that TSH may converge with IGF-I survival signaling pathways. A prior study using a model of rat preadipocytes reported that TSH stimulated proliferation, inhibited differentiation (lipoprotein lipase mRNA), and had no effect on apoptosis (DNA laddering) (17). The partly differentiated nature of their cells, and the culture conditions and assays they used, might account for the differences compared with our data.
Although the concentrations of TSH (1-20 µM) we used in our cell culture experiments are much greater than those observed in vivo, the presence of other growth factors/cytokines in vivo may augment TSH signaling. For example, in vitro studies with thyrocyte cell lines require IGF-I to be used in conjunction with TSH (23).
It has been proposed that adipose tissue is subject to remodeling that may involve cellular turnover (33). TSH, via potential effects on reducing apoptosis, may influence adipose tissue growth normally, as well as in hypothyroid states in which TSH is elevated. Future studies are necessary to further delineate the TSH signaling pathway and the mechanism by which TSH regulates survival in these cells.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Canada. A. Sorisky is a Career Investigator of the Heart and Stroke Foundation of Ontario. A. Gagnon was the recipient of a Canadian Diabetes Association Postdoctoral Research Fellowship during the time of this work. A. Bell is the recipient of a Heart and Stroke Foundation of Canada/Canadian Institutes of Health Research Doctoral Research Award.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Sorisky, Ottawa Health Research Institute, 725 Parkdale, Ottawa ON, K1Y 4E9, Canada (E-mail: asorisky{at}ohri.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00058.2002
Received 11 February 2002; accepted in final form 22 May 2002.
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