Growth Hormone-dependent Differentiation of 3T3-F442A
Preadipocytes Requires Janus Kinase/Signal Transducer and Activator of
Transcription but Not Mitogen-activated Protein Kinase or p70 S6 Kinase
Signaling*
Stephen J.
Yarwood
,
Elizabeth M.
Sale§,
Graham J.
Sale§,
Miles
D.
Houslay
,
Elaine
Kilgour¶, and
Neil G.
Anderson
**
From the
Division of Biochemistry and Molecular
Biology, Institute of Biological and Life Sciences, University of
Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom,
§ Department of Biochemistry, University of Southampton,
Bassett Crescent East, Southampton SO16 7PX, United Kingdom,
¶ Zeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield,
Cheshire SK10 4TG, United Kingdom, and
Department of Surgery,
University of Manchester, Manchester M13 9PT, United Kingdom
 |
ABSTRACT |
The signals mediating growth hormone
(GH)-dependent differentiation of 3T3-F442A preadipocytes
under serum-free conditions have been studied. GH priming of cells was
required before the induction of terminal differentiation by a
combination of epidermal growth factor, tri-iodothyronine, and insulin.
Cellular depletion of Janus kinase-2 (JAK-2) using antisense
oligodeoxynucleotides (ODNs) prevented GH-stimulated JAK-2 and signal
transducer and activator of transcription (STAT)-5 tyrosine
phosphorylation and severely attenuated the ability of GH to promote
differentiation. Although p42MAPK/p44MAPK
mitogen-activated protein kinases were activated during GH priming, treatment of cells with PD 098059, which prevented activation of these
kinases, did not block GH priming. However, antisense ODN-mediated
depletion of mitogen-activated protein kinases from the cells showed
that their expression was necessary for terminal differentiation.
Similarly, although p70s6k was activated during GH priming,
pretreatment of cells with rapamycin, which prevented the activation of
p70s6k, had no effect on GH priming. However, rapamycin did
partially block epidermal growth factor, tri-iodothyronine, and
insulin-stimulated terminal differentiation. By contrast, cellular
depletion of STAT-5 with antisense ODNs completely abolished the
ability of GH to promote differentiation. These results indicate that
JAK-2, acting specifically via STAT-5, is necessary for
GH-dependent differentiation of 3T3-F442A
preadipocytes. Activation of p42MAPK/p44MAPK
and p70s6k is not essential for the promotion of
differentiation by GH, although these signals are required for
GH-independent terminal differentiation.
 |
INTRODUCTION |
The differentiation of adipocyte precursor cells (preadipocytes)
into mature adipocytes involves a coordinated program of gene induction
and repression, which is under the influence of hormonal, neural, and
dietary signals (1, 2). A major regulator of this process is growth
hormone (GH),1 which has been
shown in several studies to promote the differentiation of
preadipocytes both in vitro and in vivo
(3-9). The dual effector theory has been proposed to explain the
actions of GH upon differentiation of preadipocyte cells (3). GH is
thought to induce a primed state in the preadipocytes (Gp)
in which cells acquire increased responsiveness to insulin and
insulin-like growth factor 1 (IGF-1), which then promote terminal
differentiation. However, the intracellular signaling mechanisms
underpinning these actions have not been established. We have been
studying the differentiation of 3T3-F442A preadipocytes, which is
dependent on both GH and insulin, with other factors exerting a
modulatory influence (6). Recent work from several laboratories has
begun to define the signaling pathways induced by GH in various cell
types. GH induces activation of the non-receptor tyrosine kinase JAK-2,
an event that is believed to initiate multiple downstream signaling
pathways including activation of STAT transcription factors and the MAP
kinase and p70s6k cascades (reviewed in Ref. 10). However,
the roles played by these signals in biological actions of GH, such as
the promotion of preadipocyte differentiation, have not been fully
elucidated. Studies in this area have been hampered by the lack of a
system allowing the action of GH to be studied in the absence of other extracellular factors. The development of serum-free media capable of
supporting adipocyte differentiation has permitted the analysis of
specific combinations of agents on the process (6, 7). In this study,
we describe a serum-free protocol for the differentiation of 3T3-F442A
cells in which the priming effects of GH can be separated from the
actions of other factors that subsequently induce terminal differentiation. This system has been used to investigate the importance of JAK-2 and the STAT, MAP kinase, and p70S6k
cascades in regulating GH-induced priming of 3T3-F442A preadipocytes.
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EXPERIMENTAL PROCEDURES |
Materials--
Culture media, newborn calf serum, fetal calf
serum, Lipofectin, and epidermal growth factor (EGF) were obtained from
Life Technologies, Inc. Tri-iodothyronine (T3) and insulin
were obtained from Sigma. PD 098059 and rapamycin were obtained from
Calbiochem. Recombinant bovine GH was a gift from Monsanto (St. Louis, MO).
Antibodies--
Rabbit polyclonal antibodies to
p44MAPK, p70s6k/p85s6k, and JAK-2
have been described previously (11). Rabbit antiserum to recombinant bovine GH (anti-rabbitGH) (12) and the monoclonal antibody to p42MAPK were generous gifts from Dr. D. J. Flint
(Hannah Research Institute, Ayr, Scotland) and Prof. Ailsa Cambell
(Institute of Biological and Life Sciences, University of Glasgow,
Glasgow, Scotland), respectively. Donkey anti-serum to rabbit IgG was
from the Scottish Antibody Production Unit (Carluke, Scotland).
Anti-mouse IgG-horseradish peroxidase conjugate was obtained from
Amersham plc. Anti-rabbit IgG-horseradish peroxidase conjugate was
obtained from Sigma. Antisera to STAT-1, STAT-3, and STAT-5a were from
Transduction Laboratories (Lexington, KY).
Standard Procedure for Cell Culture and
Differentiation--
3T3-F442A cells (Dr. Howard Green, Harvard
Medical School) were grown in Dulbecco's Modified Eagle's Medium
(DMEM) containing 10% newborn calf serum. Confluent cultures were
washed three times in phosphate-buffered saline and incubated in a
defined differentiation medium (DDM) (13). Under these conditions, at
least 70% of the cells exhibited adipocyte morphology and were
positive for Oil Red O staining after 6-10 days.
Two-Phase Protocol for Cellular Differentiation--
For these
studies, cells were grown to confluence in the presence of 10% newborn
calf serum that had been depleted of GH. This was prepared by
incubating the serum with anti-rbGH (1:1000 dilution) for 24 h at
room temperature. Antiserum to rabbit IgG was then added to a final
dilution of 1:10. After 4 h, the precipitate was pelleted by
centrifugation at 3000 × g for 30 min, and the supernatant (GH-depleted calf-serum) was carefully removed. Cells were
passaged at least twice in medium containing GH-depleted calf serum
before use for differentiation studies. Confluent cells were washed
three times in phosphate-buffered saline and then incubated for 2 days
in standard serum-free medium (see above) with or without 2 nM GH. Cultures were then washed, as before, and the medium
was replaced with maturation medium (standard serum-free medium
containing 1.8 µM insulin, 50 ng/ml EGF, and 0.1 ng/ml T3). Differentiation was measured after an additional 6-8 days.
Immunoblotting--
Cells were washed once in ice-cold
phosphate-buffered saline and then lysed at 4 °C in Buffer A (25 mM HEPES, pH 7.5, 50 mM NaCl, 2.5 mM EDTA, 50 mM NaF, 30 mM sodium
pyrophosphate, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml pepstatin
A, 2 µg/ml leupeptin, 10% (v/v) glycerol, and 1% (v/v) Nonidet
P-40), clarified by centrifugation (14,000 rpm, 10 min), and denatured
by adding 0.25 volume of 5× concentrated sample buffer. Equal
quantities of lysate protein (10-25 µg) were electrophoresed on
polyacrylamide gels as follows: MAP kinases, 10% (final acrylamide
concentration), 30:0.32 (acrylamide:bis-acrylamide ratio);
p70s6k, 9%, 30:0.32; and phosphotyrosine: 8%, 30:0.8.
After transferring to nitrocellulose, immunoblots were blocked in 3%
(w/v) bovine serum albumin and probed with anti-MAP kinase (1:1000),
anti-p70S6k (1:1000), or anti-phosphotyrosine (1:1000)
antibodies for 3 h followed by horseradish peroxidase-conjugated
anti-IgG (1:10000) for 45 min. Immunoreactive bands were visualized by
chemiluminescence using the ECL (Amersham) system.
Immunoprecipitation--
JAK-2 and STAT-5 were detected after
their immunoprecipitation and the subsequent immunoblotting of the
immunoprecipitates with anti-phosphotyrosine antibodies (PY99; Santa
Cruz) as described previously (11).
Oligodeoxynucleotide (ODN) Treatment of Cells--
The design,
synthesis, and purification of MAP kinase phosphorothioate ODNs have
been described previously (14). The antisense ODN to JAK-2 had the
sequence 5'-GCTTGTGAGAAAGC-3' that is complementary to nucleotides
1902-1915 in murine JAK-2. The complementary sense ODN had the
sequence 5'-GCTTTCTCACAAGC-3'. The JAK-2 control ODN had the scrambled
sequence 5'-TCAGGCAATGTGAG-3'. The antisense ODN to STAT-5 had the
sequence 5'-AGCCCGCCAT-3', and the complementary sense ODN had the
sequence 5'-ATGGCGGGCT-3'. The scrambled STAT-5 control ODN had the
sequence 5'-CACCTGACAC-3'. Cells in 22-mm-diameter wells were grown to
80-90% confluence before treatment. Appropriate dilutions of ODNs
in 100 µl of DMEM were preincubated at room temperature for 30 min
with 100 µl of Lipofectin (200 µg/ml). Monolayers were washed three
times with 2 ml of DMEM, and the ODNs were added to the cells together
with an additional 200 µl of DMEM. The final concentration of ODN was
5 µM. Cells were incubated for 8 h at 37 °C.
After this time, the medium was removed, and the incubation was
continued for an additional 40 h using fresh medium containing
10% heat-inactivated calf serum and 5 µM ODN but no
Lipofectin. The medium was then removed, and cells were either
harvested immediately or washed and subjected to the standard differentiation procedure in DDM containing 5 µM ODN, as
described above. The differentiation medium was replenished at 2-day
intervals, and the ODN was present throughout the differentiation period.
Microscopy--
Cells were photographed under Phase Contrast
Optics (×24 magnification) using an Olympus IMT3 inverted microscope.
-Glycerol-3-Phosphate Dehydrogenase (GPDH) and DNA Assays and
Expression of Results--
GPDH activity was measured by the method of
Wise and Green (15). In accordance with others (16), we found that the
amount of protein per cell was increased by a factor of
2.3 in
adipocytes compared with fibroblasts. GPDH activities are therefore
expressed relative to DNA content rather than the amount of cellular
protein. The DNA content of cellular homogenates was determined
fluorometrically (17). The enzyme activity per plate of cells was
expressed as the number of micromoles of NADH oxidized/min (unit)/mg
DNA. Statistical significance was assessed by Student's t test.
 |
RESULTS |
Previous work has shown that differentiation of 3T3-F442A
preadipocytes can be induced in a serum-free medium (6, 13). GH,
insulin, and EGF are essential components of this medium, with other
factors exerting modulatory influences. In order to study GH action
independently, we developed a two-step protocol (described under
"Experimental Procedures") for differentiation of 3T3-F442A
preadipocytes. Cells were grown to confluence in the presence of serum
depleted of GH and then primed for 48 h with or without GH. Cells
were then exposed to EGF, T3, and insulin (ETI) to induce
terminal differentiation that was quantitatively assessed by measuring
the cellular activity of the adipocyte-specific marker GPDH and
qualitatively assessed morphologically (Fig.
1). These data show that cells must be
primed with GH before the induction of terminal differentiation by ETI
and that terminal differentiation is GH-independent. Because IGF-1 is
known to mediate many of the biological effects of GH (18), we tested
whether IGF-1 alone could prime cells for differentiation. Fig. 1 shows
that, although pretreatment of cells with IGF-1 promoted the subsequent
terminal differentiation to a degree, the magnitude of this effect was much less than that induced by GH. This indicates that the priming effect of GH is primarily due to direct actions of the hormone.

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Fig. 1.
Growth hormone priming is essential for
terminal differentiation of 3T3-F442A preadipocytes. Cells were
grown to confluence in GH-depleted medium. The cells were then washed
and treated for 48 h in standard serum-free medium alone
(con), in standard medium plus 100 nM IGF-1
(IGF-1), or in standard medium plus 2 nM GH
(GH). Cells were then washed and incubated in standard
medium containing insulin, EGF, and T3 for 8 days before
photography. Pictures representative of three independent experiments
are shown. In parallel, lysates were prepared from cells treated as
described above, and GPDH activity was determined as described under
"Experimental Procedures." Data represent the means ± S.D.
from three independent experiments.
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Cellular Depletion of JAK-2 Blocks GH-dependent
Differentiation--
Activation of the non-receptor tyrosine kinase
JAK-2 is considered to be essential for GH-dependent signal
transduction (19-21). To test whether JAK-2 is necessary for
GH-induced priming of preadipocytes, we used an antisense ODN to
deplete JAK-2 from the cells. Preadipocytes, which had been grown to
confluence in GH-depleted medium to ensure that subsequent
differentiation took place under GH-dependent conditions,
were treated with the JAK-2 antisense ODN before the induction of
differentiation. The extent of differentiation was then assessed by
measurement of GPDH activity. These experiments revealed that depletion
of cellular JAK-2 almost completely blocked the subsequent adipogenic
differentiation of the cells assessed by GPDH activity (Fig.
2). In contrast, preincubation of cells with the corresponding sense ODN failed to significantly affect the
extent of differentiation (Fig. 2). Immunoblotting of JAK-2 immunoprecipitates with anti-phosphotyrosine antibodies after the
antisense treatment confirmed an almost complete depletion of
GH-activable JAK-2 from the cells (Fig. 2). These experiments indicate
that JAK-2 is essential for GH-dependent
differentiation of 3T3-F442A preadipocytes.

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Fig. 2.
Effect of JAK-2 antisense
oligodeoxynucleotides on GH-dependent differentiation of
3T3-F442A cells. Cells were grown in medium containing GH-depleted
calf serum before treatment with Lipofectin alone
(Lipofectin), Lipofectin plus sense (sense),
Lipofectin plus JAK-2 antisense (JAK2 antisense), or
Lipofectin plus scrambled (scrambled) oligonucleotides.
A, cells were stimulated for 10 min with GH (2 nM), and cell lysates were prepared and immunoprecipitated
with anti-JAK-2 antibodies. The immunoprecipitates were then blotted
with antiphosphotyrosine antibodies. B, after antisense
treatment, cells were incubated in the defined differentiation medium
(see "Experimental Procedures"), and GPDH activity was measured
after 8 days. Results are expressed as the means ± S.E. relative
to GPDH activity in control cultures treated with Lipofectin alone
(100%) from three independent experiments. The mean control GPDH
activity was 291 ± 51 units/mg DNA (n = 3).
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Role of MAP Kinases in the Differentiation of 3T3-F442A
Cells--
To determine the nature of the signals downstream of JAK-2
that are involved in the promotion of differentiation by GH, we first
analyzed the role of the MAP kinase pathway. The addition of GH to
cells grown to confluence in GH-deficient medium induced a small
activation of p42MAPK, as assessed by decreased
electrophoretic mobility, that was transient and returned to basal
levels after around 30 min (Fig. 3). A
similar response was observed for activation of p44MAPK
(data not shown). These data confirm that MAP kinases are activated during GH priming under these cell culture conditions. To test whether
activation of these kinases is required for GH-dependent priming, we made use of the inhibitor PD 098059, which specifically inhibits the activation of mitogen-activated kinase and
extracellular-regulated kinase kinase (MEK), the immediate upstream
activator of p42MAPK and p44MAPK (22).
Incubation of cells with PD 098059 during the 48-h priming period
completely blocked the activation of MAP kinase by GH as assessed
electrophoretic mobility (Fig. 4).
Despite this blockade of MAP kinase activation, PD 098059 failed to
inhibit the ability of GH to prime cells (Fig. 4). This was not due to
a loss of PD 098059 activity during the 48-h priming period because the
inhibitor retained its ability to block the activation of MAP kinase by GH after a 48-h preincubation (Fig. 4).

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Fig. 3.
Time course for activation of
p42MAPK by GH and by EGF/T3/insulin. Cells
were treated for the indicated times with 2 nM GH
(top panel) or with a combination of EGF (100 ng/ml),
T3 (100 pg/ml), and insulin (1.8 µM)
(E/T/I, bottom panel). Lysates were prepared and
immunoblotted with anti-p42MAPK antibodies as described
under "Experimental Procedures." Cells treated with EGF,
T3, and insulin had undergone priming in the presence of GH
for 48 h. The relative positions of the hypophosphorylated
(lower arrow) and hyperphosphorylated (upper
arrow) MAP kinase are indicated.
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Fig. 4.
Effect of PD 098059 on GH priming of
3T3-F442A cells. A, cells were treated as follows: no
additions, lane 1; GH (2 nM, 10 min), lane
2; PD 098059 (50 µM, 1 h) and then GH,
lane 3; or PD 098059 (50 µM, 48 h) and
then GH, lane 4. Lysates were prepared and immunoblotted for
p42MAPK as described in under "Experimental
Procedures." B, cells were grown to confluence in the
presence of GH-depleted calf serum. The medium was removed, and cells
were primed for 2 days in standard serum-free medium along with GH (2 nM) or PD 098059 (50 µM) as indicated. This
medium was then replaced with standard medium containing EGF,
T3, and insulin, and GPDH activity was measured 8 days
later. For further details, see "Experimental Procedures." Results
are expressed as the means ± S.E. relative to GPDH activity
measured in control cultures primed with GH in the absence of PD 098059 (100%) from three independent experiments. The mean control GPDH
activity was 204 ± 19 units/mg DNA (n = 3).
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The above results suggested that the activation of MAP kinases by GH
was not essential for the priming of cells by GH. However, because
previous studies (14) with another preadipocyte cell line have shown a
requirement of MAP kinases for adipogenic differentiation in the
presence of serum, we tested whether they played any role in the
differentiation of 3T3-F442A preadipocytes under serum-free conditions.
Using our two-stage differentiation procedure, we first showed that the
addition of ETI to primed cells resulted in a strong activation of
p42MAPK, indicated by the conversion of essentially all of
the cellular p42MAPK to the more slowly migrating
hyperphosphorylated form (Fig. 3). Moreover, this activation persisted
above basal levels for at least 24 h (Fig. 3). The addition of PD
098059 at concentrations of up to 100 µM failed to block
the potent activation of MAP kinase by ETI (data not shown; see Ref.
22), thus precluding its use in testing the requirement for MAP kinases
solely during terminal differentiation. We therefore made use of an
antisense ODN to p42MAPK and p44MAPK to deplete
these kinases from the cells. Treatment of cells with 5 µM antisense EAS1 for 48 h caused a dramatic
(>90%) depletion of cellular p44MAPK when compared with
cells treated with the transfection agent alone (Fig.
5). The p42MAPK isoform of
MAP kinase exhibited an even greater sensitivity to depletion with
antisense EAS1 because levels of the protein were undetectable (Fig. 5)
even with long exposures of the immunoblot (data not shown). Incubation
of cells with sense or scrambled ODNs did not significantly affect the
expression of p42MAPK or p44MAPK (Fig. 5). To
test the requirement for p42MAPK and p44MAPK in
the differentiation process, cells were transfected with 5 µM antisense EAS1 or control ODNs and then subjected to
differentiation using the defined differentiation medium (see
"Experimental Procedures"). EAS1 caused a striking reduction in the
extent of differentiation, as judged by lipid accumulation (data not
shown), and a corresponding 98 ± 0.02% (p < 0.001; n = 3) reduction in GPDH activity (Fig. 5).
Incubation with sense or scrambled ODNs did not significantly affect
the extent of differentiation (Fig. 5). Therefore, the specific
depletion of cellular MAP kinase blocks the transmission of
differentiative stimuli in 3T3-F442A cells under serum-free conditions.
The combined results of using antisense ODNs and the PD 098059 inhibitor strongly support the conclusion that activation of MAP
kinases is not required for GH priming of confluent cells but that MAP
kinases are required for the induction of terminal differentiation
events by EGF, T3, and insulin.

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Fig. 5.
Effect of p42MAPK and
p44MAPK depletion on terminal differentiation.
Confluent cells were treated with antisense EAS1 (5 µM),
scrambled oligonucleotides (5 µM), or sense
oligonucleotides (5 µM) for 48 h with Lipofectin
present for the first 8 h as described under "Experimental
Procedures." A, the level of p42MAPK was
assessed by Western blotting with specific antisera as described under
"Experimental Procedures." Results are representative of three
experiments. B, cells were grown to confluence and treated
with antisense EAS1 or control oligonucleotides. Cells were then
transferred to the DDM in the continued presence of oligonucleotides.
After 10 days, cells were harvested and processed for the assay of GPDH
activity. Activity obtained in cultures differentiated with DDM medium
alone was 180 ± 24 units/mg DNA (n = 3), and all
values are expressed as a percentage of this activity. Results are the
means ± S.E. for three observations.
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The Role of the p70s6k Pathway in
Differentiation--
We next examined the requirement of the
p70s6k signaling pathway for GH-dependent
differentiation of 3T3-F442A cells under our defined serum-free
conditions. GH induced an activation of p70s6k, as assessed
by reduced electrophoretic mobility, that was sustained for at least
5 h (Fig. 6). To test the
requirement for p70s6k activation, we used rapamycin, which
blocks activation of p70s6k by all known agents (23).
Rapamycin completely inhibited the activation of p70s6k by
GH (Fig. 7) but failed to affect the
subsequent terminal differentiation as assessed by GPDH activity (Fig.
7), indicating that p70s6k does not mediate the priming
action of GH. Control experiments confirmed that rapamycin retained its
ability to block p70s6k activation at the end of the 48-h
incubation period (Fig. 7). Because an obligatory role for the
p70S6k pathway in the differentiation of 3T3-L1 cells in
the presence of serum and insulin has been reported (24), we wished to
establish whether it was similarly required for the overall process of
differentiation under serum-free conditions. The activation of
p70s6k by ETI was strong and was sustained above basal
levels for 5-12 h (Fig. 6). The inclusion of rapamycin during terminal
differentiation induced by ETI was found to inhibit adipogenic
conversion by 58.7 ± 1.1% (p < 0.01;
n = 3; Fig. 7). Thus, the p70s6k signaling
pathway contributes significantly to the action of ETI during terminal
differentiation but is not required for GH to prime cells for
differentiation.

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Fig. 6.
Time course for activation of
p70s6k by GH and by EGF/T3/insulin. Cells
were treated for the indicated times with 2 nM GH
(top panel) or with a combination of EGF (100 ng/ml),
T3 (100 pg/ml), and insulin (1.8 µM)
(E/T/I, lower panel). Lysates were prepared and
immunoblotted with anti-p70s6k antibodies as described
under "Experimental Procedures." Cells treated with EGF,
T3, and insulin had undergone priming in the presence of GH
for 48 h. The relative positions of the hyperphosphorylated
(upper arrow) and hypophosphorylated (lower
arrow) forms of p70s6k are indicated.
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Fig. 7.
Effect of rapamycin on the priming and
terminal differentiation of 3T3-F442A cells. A, confluent
cells were treated as follows: no additions, lane 1; GH (2 nM, 20 min), lane 2; rapamycin (20 nM, 20 min) and then GH, lane 3; ETI, lane
4; rapamycin (20 min) and then ETI, lane 5; rapamycin
(48 h) and then GH, lane 6. Lysates were then prepared and
immunoblotted with anti-p70s6k antibodies as described
under "Experimental Procedures." B, cells were grown to
confluence in the presence of GH-depleted calf serum. The medium was
removed, and cells were primed for 2 days in standard serum-free medium
containing 2 nM GH. After this, the medium was replaced
with medium containing EGF, T3, and insulin. Rapamycin (20 nM) was not added (NO ADDITION) or was added to
cells during priming (GH PRIMING) or during terminal
differentiation (TERMINAL DIFFN.). GPDH activities are the
means ± S.E. for three observations and are expressed relative to
activity measured in control (NO ADDITION) cultures (100%).
Control GPDH activity was 213 ± 21 units/mg DNA
(n = 3). * signifies that the value differs
significantly from that for cells differentiated in the absence of
rapamycin (NO ADDITION); p < 0.01.
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The Role of the STAT Pathway in Differentiation--
The above
data indicate that the ability of JAK-2 antisense ODNs to block
GH-dependent differentiation is not due to the subsequent blocking of the MAP kinase or p70s6k pathways. In addition
to activation of these pathways, JAK-2 activation leads to the
phosphorylation of STAT transcription factors (see Ref. 10). Treatment
of cells with JAK-2 antisense ODNs but not sense or scrambled ODNs
almost completely prevented GH-stimulated STAT-5 tyrosine
phosphorylation (data not shown). To investigate whether STAT-5 was
required for GH-dependent differentiation, we used an
antisense ODN specifically to deplete STAT-5 from cells. Preincubation
of 3T3-F442A preadipocytes with STAT-5 antisense ODNs caused a complete
and specific depletion of cellular STAT-5a protein (Fig.
8), whereas the expression of STAT-1 and
STAT-3 were unaffected (Fig. 8). These cells also contain STAT-5b, and the antisense treatment also resulted in a complete depletion of this
isoform (data not shown). Treatment of cells with the corresponding
sense or scrambled ODNs failed to affect the level of STAT-5a (Fig. 8)
or STAT-5b (data not shown) protein expression. The requirement of
STAT-5 proteins to support GH-dependent differentiation under serum-free conditions was then evaluated. Cells were transfected with 5 µM antisense STAT-5 or control ODNs before the
induction of differentiation using the defined differentiation medium.
Antisense STAT-5 ODNs caused a dramatic reduction (>99%) in the
extent of differentiation, as judged GPDH activity (Fig.
8B). Incubation with sense or scrambled ODNs did not
significantly affect the extent of differentiation. These results
strongly implicate the JAK-STAT signaling pathway in transducing
GH-dependent signals during the initiation of 3T3-F442A
preadipocyte differentiation.

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Fig. 8.
Effect of STAT-5 depletion on terminal
differentiation. Confluent cells were treated with antisense
STAT-5 (10 µM), scrambled oligonucleotides (10 µM), or sense oligonucleotides (10 µM) for
48 h with Lipofectin present for the first 8 h as described
under "Experimental Procedures." A, the levels of
STAT-1, STAT-3, and STAT-5a were assessed by Western blotting with
specific antisera as described under "Experimental Procedures."
Results are representative of three experiments. B, cells
were grown to confluence in medium depleted of endogenous GH and then
treated with antisense STAT-5 or control oligonucleotides. Cells were
then transferred to the DDM in the continued presence of
oligonucleotides. After 10 days, cells were harvested and processed for
the assay of GPDH activity. Activity obtained in cultures
differentiated with DDM alone was 192 ± 36 units/mg DNA
(n = 3), and all values are expressed as a percentage
of this activity. Results are the means ± S.E. for three
observations.
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DISCUSSION |
Although GH is well known to promote the differentiation of
preadipocytes, the relevant intracellular signaling pathways involved have not been clearly identified. Because preadipocyte differentiation requires many extracellular factors individually acting at particular stages, it is difficult to dissect the contribution made by individual agents to the overall process. In this study, we have developed a
serum-free system for the differentiation of 3T3-F442A preadipocytes in
which the GH-dependent aspect of the process can be
separated from the influence of other hormones. Several studies have
indicated that GH acts at an early stage in differentiation by inducing an anti-mitogenic state and sensitizing cells to the actions of factors
such as insulin, which then promote terminal differentiation (3-6).
Accordingly, we show that confluent cells can be primed with GH alone
before the addition of other hormones which then induce terminal
differentiation. Differentiation required treatment of cells with
exogenous GH only if cells had been grown to confluence in medium
containing serum depleted of GH. Thus, if cells are grown in normal
calf serum, the endogenous GH present is sufficient to sensitize cells
at confluence to the action of agents that induce terminal differentiation.
A wide body of evidence indicates that activation of the tyrosine
kinase JAK-2 is critical and perhaps essential to GH action. Notably,
mutant receptors, which do not interact with JAK-2, fail to activate
MAP kinases and to induce gene activation (20, 21). Our JAK-2 antisense
strategy now shows conclusively that JAK-2 is essential for
GH-dependent differentiation of 3T3-F442A preadipocytes. Although tyrosine kinases of the JAK family are generally involved in
signaling by cytokines, some evidence indicates that they also participate in signaling by other classes of growth factor. Of the
other components of our serum-free medium essential for
differentiation, both EGF (25) and insulin (26) have been shown to
activate JAK tyrosine kinases. However, we have been unable to detect
any EGF- or insulin-stimulated tyrosine phosphorylation of JAK-2 in 3T3-F442A preadipocytes or
adipocytes.2 We therefore
believe that JAK-2 is specifically involved in the GH-dependent priming stage of the differentiation process.
To our knowledge, an absolute requirement for JAK kinases in the differentiation of preadipocytes or, indeed, of any other cells has not
previously been reported. In the absence of synthetic JAK-2-specific
inhibitors, the antisense strategy should prove useful in defining the
role played by this kinase in other cellular processes.
Three major signaling pathways, which lie downstream of JAK-2
activation, are the MAP kinase pathway, the p70s6k pathway,
and the STAT pathway (27-33). We have shown here that all three
pathways are activated by GH during the priming of preadipocytes. However, although MAP kinases appear to be essential for the overall process of differentiation, blocking their activation with inhibitor PD
098059 during GH priming did not prevent subsequent differentiation. This indicates that MAP kinases are not involved in
GH-dependent priming of the cells. Our data further show,
for the first time, that MAP kinase activation is required specifically
for terminal differentiation, which was induced in our study by the
combination of EGF, T3, and insulin. The strong and
sustained activation of MAP kinases induced by these factors in
combination may result in nuclear translocation of the MAP
kinases and, subsequently, the phosphorylation of key transcription
factors. Further work will be required to identify the distal gene
targets for the MAP kinase pathway, which lead to terminal differentiation.
Our study also shows that, although GH induces a sustained activation
of p70s6k, this is not required for priming of the cells.
Rapamycin, which specifically blocks p70s6k activation at
the level of mammalian target of rapamycin (mTOR) (34), reduced the
ability of ETI to support terminal differentiation of GH-primed cells
but had no effect upon the priming action of GH. Therefore, the
involvement of this pathway also appears to be restricted to terminal
differentiation events.
Finally, this study has shown that GH induced the tyrosine
phosphorylation of STAT-5a/5b during the priming of 3T3-F442A
preadipocytes. This did not occur when differentiation was prevented by
treatment of the cells with an antisense ODN to JAK-2. Moreover,
specific depletion of STAT-5a/5b blocked differentiation. Taken
together, these results indicate that JAK-2-dependent
activation of STAT-5a/5b is a necessary event in the promotion of
differentiation by GH.
In conclusion, we have established a system that allows the action of
GH in promoting the differentiation of 3T3-F442A preadipocytes to be
studied in isolation. GH is shown to prime confluent cells before the
addition of factors that then induce terminal differentiation. We have
used this system to show that activation of JAK-2 is necessary for
GH-dependent differentiation. Although activation of JAK-2 leads to downstream activation of both p42/p44 MAP kinase and p70s6k, these signaling pathways are not involved in the
priming action of GH. By contrast, these signaling pathways are both
necessary for terminal differentiation after GH priming. From our data, it appears that other JAK-2-dependent signaling pathways,
including the activation of STAT-5a/5b, regulate this important
biological action of GH.
 |
ACKNOWLEDGEMENT |
We thank Dr D. J. Flint for the supply of
anti-GH antiserum and for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the Biotechnology and Biological
Sciences Research Council.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.
**
To whom correspondence should be addressed: Dept. of Surgery,
School of Biological Sciences, Rm. G38 Stopford Bldg., Oxford Rd.,
Manchester M13 9PT, United Kingdom. Tel.: 0161-275-5496; Fax:
0161-275-5600; E-mail: Neil.Anderson{at}man.ac.uk.
2
N. G. Anderson, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
GH, growth hormone;
MAP, mitogen-activated protein;
STAT, signal transducer and activator
of transcription;
IGF-1, insulin-like growth factor-1;
EGF, epidermal
growth factor;
T3, tri-iodothyronine;
ETI, EGF,
T3, and insulin;
GPDH,
-glycerol-3-phosphate
dehydrogenase;
ODN, oligodeoxynucleotide;
DMEM, Dulbecco's Modified
Eagle's Medium;
DDM, defined differentiation medium (standard
serum-free medium (F12:DMEM, 2:1) plus 10 µg/ml transferrin, 50 µg/ml fetuin, 2.5 mM glutamine, and 1 mg/ml bovine serum
albumin containing 2 nM GH, 1.8 µM insulin,
0.1 ng/ml T3, and 50 ng/ml EGF;
JAK, Janus kinase.
 |
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