From the State Laboratory of Molecular Biology,
Institute of Biochemistry and Cell Biology and § Shanghai
Research Center of Life Sciences, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai, China 200031
Received for publication, December 27, 2000
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ABSTRACT |
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Upon differentiation induction of 3T3-L1
preadipocytes by a hormone mixture containing
1-isobutyl-3-methylxanthine, dexamethasone, and insulin, the
preadipocytes undergo ~2 rounds of mitotic clonal expansion, which
just precedes the adipogenic gene expression program and has been
thought to be an essential early step for differentiation initiation.
By inducing 3T3-L1 preadipocytes with each individual hormone, it was
determined that the mitotic clonal expansion was induced only by
insulin and not by 1-isobutyl-3-methylxanthine or dexamethasone. Cell
number counting and fluorescence-activated cell-sorting analysis
indicated that a significant fraction of 3T3-L1 preadipocytes
differentiated into adipocytes without mitotic clonal expansion when
induced with the combination of 1-isobutyl-3-methylxanthine and dexamethasone. Furthermore, when normally induced 3T3-L1
preadipocytes were treated with PD98059 (an inhibitor of
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase 1) to block the activation of extracellular signal-regulated
kinase (Erk) 1 and Erk2, the mitotic clonal expansion was blocked, but
adipocyte differentiation was not affected. These observations were
confirmed by bromodeoxyuridine labeling. The differentiated adipocytes
induced with 1-isobutyl-3-methylxanthine and dexamethasone or standard hormone mixture plus PD98059 were not labeled by bromodeoxyuridine. Thus, it is evident that 3T3-L1 preadipocytes could differentiate into
adipocytes without DNA synthesis and mitotic clonal expansion. Our
results also suggested that activation of Erk1 and Erk2 is essential to
but not sufficient for induction of mitotic clonal expansion.
Obesity has become a major health hazard in many countries and has
been indicated as a risk factor for many physiological disorders, such
as diabetes, hypertension, and heart problems. As the major cellular
component in adipose tissue, adipocytes play a key role in obesity. The
excessive growth of adipose tissue in obesity has been suggested as
expansion of adipocytes both in cell size and in cell number (1, 2). To
better understand adipocyte physiology, in vitro cell
models, such as 3T3-L1 and 3T3-F442A, have been used. These model cell
lines provide a useful tool to study the adipocyte differentiation
process (1, 3). 3T3-L1 cells can be induced to differentiate into
mature adipocytes in cell culture (4-6). It is one of the most used
preadipocyte models to study the adipogenesis process. The
preadipocyte differentiation program involves several
stages. Immediately after hormonal stimulation (IGF-I1 or insulin at a
nonphysiologically high concentration, a glucocorticoid, dexamethasone
(DEX), and a cAMP phosphodiesterase inhibitor that increases
intracellular cAMP, 3-isobutyl-1-methylxanthine (MIX)), postconfluent
G0 3T3-L1 preadipocytes reenter a period of the cell
cycle called mitotic clonal expansion. The gene expression program
leading to terminal adipocyte differentiation is initiated during and
after this mitotic clonal expansion period. It has been proposed that
this mitotic clonal expansion might facilitate the DNA remodeling for
the adipogenesis gene expression program (3).
Whereas it is clear that the mitotic clonal expansion phase precedes
the adipogenic gene expression program, whether this cell proliferation
process is an obligatory step along the 3T3-L1 adipocyte
differentiation process is still not clear. Several studies have
indicated that mitotic clonal expansion and 3T3-L1 adipocyte
differentiation are both blocked by the DNA synthesis inhibitor
aphidicolin (7), the antiproliferation reagent rapamycin (8), calpain
inhibitor N-acetyl-leu-leu-norleucinal (9), or tumor
necrosis factor In the present study, we reported that two rounds of the mitotic clonal
expansion cell cycle occurred during 3T3-L1 preadipocyte differentiation induction and that the initiation of clonal expansion required activation of the Erk1 and Erk2 MAP kinases. Of three differentiation induction reagents (MIX, DEX, and a high concentration of insulin in place of IGF-I), only insulin was capable of
inducing mitotic clonal expansion. 3T3-L1 preadipocytes induced with
MIX and DEX (omitting insulin) or induced with the standard hormone mixture plus MEK inhibitor PD98059 were still able to differentiate into adipocytes, but without DNA synthesis and mitotic clonal expansion. Thus, our results suggest that DNA synthesis and mitotic clonal expansion is not an essential step required for the 3T3-L1 preadipocyte differentiation process; rather, it is a separable event
induced by the activated IGF-I receptor tyrosine kinase through a
signal pathway of MAP kinases Erk1 and Erk2.
Materials--
Anti-Erk and anti-p-Erk (against the critical
Tyr residue phosphorylated peptide) antibodies were purchased from
Santa Cruz Biotechnology, Inc. Horseradish peroxidase-conjugated
secondary antibody, BrdUrd, DAPI, sodium orthovanadate,
dexamethasone, 3-isobutyl-1-methylxanthine, and insulin were purchased
from Sigma. PD98059 was purchased from New England Biolab. Mouse
monoclonal anti-BrdUrd antibody was purchased from Becton Dickinson.
Fluorescein isothiocyanate-conjugated secondary antibody was purchased
from Jackson Laboratories, and DMEM was obtained from Life
Technologies, Inc.
Cell Culture and Differentiation Induction of 3T3-L1
Preadipocytes--
3T3-L1 preadipocytes were cultured in DMEM
supplemented with 10% calf serum and allowed to reach confluence.
Differentiation of 2-day postconfluent preadipocytes (designated as day
0) was initiated with 1 µg/ml insulin, 1 µM DEX, and
0.5 mM MIX in DMEM supplemented with 10% fetal bovine
serum (6, 14). After 48 h (day 2), the culture medium was replaced
with DMEM supplemented with 10% fetal bovine serum and 1 µg/ml
insulin, and the cells were then fed every other day with DMEM
containing 10% fetal bovine serum. Cytoplasmic triglyceride droplets
were visible by day 4, and cells were fully differentiated by day 8.
For stimulation with individual hormone (MIX, DEX, or insulin alone) or
a two-hormone combination (MIX plus DEX, MIX plus insulin, or DEX plus
insulin), 2-day postconfluent preadipocytes were induced by hormone(s)
at the above-mentioned concentration for 48 h. Cells were
trypsinized from culture dishes, and the cell numbers were counted
using a hemocytometer plate. For cells carried to final
differentiation, the induction culture medium was replaced after
48 h with DMEM containing 10% fetal bovine serum supplemented
with or without 1 µg/ml insulin as described in the figure legends,
and then the normal cell feeding protocol was followed until day 8.
For PD98059 treatment, 20 µM PD98059 was added to the
cells with the differentiation induction hormone mixture on day 0. The cells were then cultured following the standard differentiation induction protocol with a supplement of 20 µM PD98059
until day 4.
FACS Analysis, Cell Counting, and Oil-Red-O Staining--
3T3-L1
cells (6-cm plate) were trypsinized from the culture dishes and
collected by centrifugation. An aliquot was subjected to cell counting
using a hemocytometer plate. The cells were then fixed in 70% ethanol,
pelleted, and treated with 1 mg/ml RNase A for 30 min at 37 °C.
After staining with 20 µg/ml propidium iodide, the DNA content in
cells was determined by FACS analysis. For Oil-Red-O staining, 3T3-L1
adipocyte monolayers (usually on day 8) were washed three times with
phosphate-buffered saline (PBS) and then fixed for 2 min with 3.7%
formaldehyde in PBS. Oil-Red-O (0.5%) in isopropanol was diluted with
1.5 volumes of water, filtered, and added to the fixed cell monolayers
for 1 h at room temperature. Cell monolayers were then washed with
water, and the stained triglyceride droplets in the cells were
visualized and photographed.
Preparation of Cellular Extracts, SDS-Polyacrylamide Gel
Electrophoresis, and Western Blotting--
For protein analysis, cell
monolayers from cells treated as described in the figure legends were
washed three times with cold PBS. Cells were then lysed directly in
boiling 1× Laemmli SDS sample buffer (15) containing 20 mM
dithiothreitol. The cell lysate was then heated at 100 °C for 5 min.
For Western blotting, cell extracts (usually containing ~15 µg of
protein) were subjected to 12.5% SDS-polyacrylamide gel
electrophoresis and then transferred to Immobilon-P membrane
(Millipore). After blocking with 2% nonfat dried milk in 1× TTBS
(Tween/Tris-buffered saline) containing 25 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.05% Tween, and 0.001% thimerosal for
2 h at room temperature, membranes were incubated with primary antibody for 2 h at room temperature, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 45 min. Target
proteins were visualized by enhanced chemiluminescence.
BrdUrd Labeling for DNA Synthesis--
Cells were cultured on
coverslips until 2-day postconfluence and induced to
differentiate with different induction conditions as described in the
figure legends. During the S phase of the cell cycle, 30 µg/ml BrdUrd
was pulsed for 2 h (from the 16th to the
18th h after the induction) to label DNA synthesis. The
coverslips were fixed in 70% ethanol for 30 min and then stored in
70% ethanol at 4 °C for immunofluorescence analysis. For cells
carried to final differentiation, the BrdUrd labeling medium was
replaced with preconditioned medium, which was obtained from a parallel cell culture dish treated with the same induction condition. After 48 h, the medium was replaced with DMEM containing 10% fetal
bovine serum with or without a supplement of 1 µg/ml insulin as
described in the figure legends. By day 8, cells were fixed with 70%
ethanol and stored in the same ethanol solution at 4 °C for
immunofluorescence analysis.
BrdUrd labeling was also conducted on day 3 3T3-L1 cells to reveal the
effect of the insulin supplementation in the medium. At the
16th h after the medium change, 30 µg/ml BrdUrd was
pulsed for 2 h, and the labeling medium was replaced with
preconditioned medium obtained from a cell culture dish fed in
parallel. On day 4, the cells were fed with normal medium every other
day until day 8.
Immunofluorescence--
Ethanol-fixed coverslips were incubated
in 100% methanol for 10 min at room temperature. The coverslips were
then treated with 1.5 M HCl for 30 min, blocked with 0.5%
Tween 20 in PBS solution for 5 min, and incubated with anti-BrdUrd
primary antibody for 1 h at room temperature. After washing,
fluorescein isothiocyanate-conjugated secondary antibody in a Tween-PBS
solution containing 0.1 µg/ml DAPI was added to the coverslips for
1 h at room temperature. After washing coverslips with Tween-PBS
twice for 5 min to remove the secondary antibody, the coverslips were
mounted for immunofluorescence microscope analysis (AXIOSKOP 20; Zeiss).
Analysis of Erk1 and Erk2--
To minimize the effect of medium
change on the activation of Erk1 and Erk2, 3T3-L1 preadipocytes were
fed with DMEM plus 10% FBS 1 day before the differentiation induction.
On day 0, the differentiation inducers were added directly to the
culture medium without changing the medium. The remaining steps
followed the standard differentiation protocol. In this modified
protocol, adipocyte differentiation and mitotic clonal expansion
progressed normally, without any detectable changes. For analysis of
Erk activation, postconfluent 3T3-L1 preadipocytes were fed with DMEM containing 10% FBS. The next day, 20 µM PD98059 or 25 µM sodium orthovanadate was added to the culture medium
for 1 h, and then a mixture of MIX, DEX, and insulin was added to
the medium to initiate the differentiation process. At the indicated
time point (time of the addition of MDI is designated as 0 min), cells
were harvested by lysing the cells directly in 1× boiling Laemmli SDS sample buffer with 20 mM dithiothreitol. Total Erk protein
and the activated tyrosine-phosphorylated Erk were detected with
antibodies against Erk or tyrosine-phosphorylated Erk, respectively, on
Western blot.
For insulin-induced Erk activation, after the medium change (24 h) and
pretreatment with PD98059 or vanadate (1 h pretreatment), insulin was
added to the cells to initiate the mitotic clonal expansion process. At
each time point, the cells were harvested as described above.
Two rounds of mitotic clonal expansion were observed during 3T3-L1
preadipocyte differentiation induction and induced only by insulin. It
was observed that after 3T3-L1 preadipocytes differentiated into
adipocytes, the cell numbers were usually increased by 3-4-fold (Fig.
1A). This is consistent with
our previous results and results reported by other researchers (9-12).
To ascertain how many cell cycles occurred during the clonal
expansion period, we took advantage of the fact that postconfluent
3T3-L1 preadipocytes enter mitotic clonal expansion synchronously after
the differentiation induction. By cell number counting and FACS
analysis of the cellular DNA content, two rounds of cell cycle were
observed, and the cell number increase was matched by the DNA content
analysis, especially for the first cell cycle, which is well
synchronized (Fig. 1, B and C). Complete DNA
synthesis and transition from diploid to tetraploid were observed (Fig.
1C). Thus, the clonal expansion was full cell cycle mitosis.
Because differentiation induction is initiated with three hormones
working through three signal pathways (MIX on cAMP, DEX on
glucocorticoid receptor, and insulin on IGF-I receptor tyrosine
kinase), it is important to know whether the mitotic clonal expansion
is the result of the combined effects of three hormones or the result
of one of the hormones. By inducing 3T3-L1 cells with individual
hormone or a different combination of two hormones on day 0 and
counting the cell number after the completion of the first cell cycle,
it was clear that once insulin was present, the cell number was
increased. Even with insulin alone, mitosis still occurred (Fig.
1D). This insulin-activated mitosis was confirmed by
analysis of DNA synthesis with BrdUrd labeling. The incorporation of
BrdUrd in cells exposed to insulin or to a hormone mixture containing
insulin was significantly higher than that in cells exposed to other
hormones (Fig. 1E). Because insulin is working through the
IGF-I receptor in 3T3-L1 preadipocytes (16, 17), mitotic clonal
expansion is induced by the activation of IGF-I receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(10). These results supported the view that mitotic
growth is a necessary step in the adipocyte differentiation process.
However, in our previous studies, we have found that 3T3-L1 adipocyte
differentiation can be blocked with the protein tyrosine phosphatase
inhibitor vanadate, whereas mitotic clonal expansion is not affected
(11, 12). Vanadate inhibits the differentiation induction signal from
IGF-I receptor tyrosine kinase by blocking the turnover of
tyrosine-phosphorylated c-Crk (12). Further investigation indicates
that the effect of vanadate on blocking 3T3-L1 preadipocyte
differentiation occurs at a very early stage of differentiation
induction, before the initiation of mitotic clonal expansion (12).
These results suggest that the activated IGF-I receptor tyrosine kinase
induces 3T3-L1 adipocyte differentiation and mitotic clonal expansion
by two separate signal transduction pathways. In addition, primary
preadipocytes from human adipose tissue enter the differentiation
process without mitotic clonal expansion (13). These observations
provide an impetus for us to investigate whether mitotic clonal
expansion is an essential step during 3T3-L1 the preadipocyte
differentiation induction process or a parallel event induced by the
differentiation induction hormones.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Mitotic clonal expansion during the 3T3-L1
preadipocyte differentiation induction process. 3T3-L1
preadipocyte differentiation was induced with the standard induction
protocol. The cell number was determined at different times
(A, days; B, hours) after the induction of
differentiation. C, FACS analysis for DNA content of 3T3-L1
preadipocytes during mitotic clonal expansion after the differentiation
induction. 0 hr, 14 hr, 18 hr, and so forth indicate the
time points after the differentiation initiation at which cells were
harvested. D, cell number counting after the first round of
cell division during the mitotic clonal expansion period. Cells were
induced with different combinations of hormone(s). At ~40 h after the
induction, the cell numbers were counted. Day 0, cells
before the induction; M, induction with MIX alone;
D, induction with DEX; I, induction with insulin;
MDI, induction with a combination of MIX, DEX, and insulin;
MI, induction with MIX and insulin; MD, induction
with MIX and DEX; DI, induction with DEX and insulin;
FBS, control cells fed with fetal bovine serum medium
without any inducers. E, BrdUrd labeling of DNA synthesis
during mitotic clonal expansion. BrdUrd was pulsed to the cells at S
phase (from the 16th to the 18th h after the
induction). The induction conditions were the same as those described
in D.
In the Absence of Insulin, MIX and DEX Could Induce Significant
3T3-L1 Adipocyte Differentiation without the Mitotic Clonal
Expansion--
Because insulin is the sole factor to induce mitotic
clonal expansion, we attempted to induce 3T3-L1 preadipocyte
differentiation with MIX and DEX. As shown in Fig.
2A, induction of preadipocytes with MIX, DEX, and insulin led to an almost doubled cell number by day
2, whereas induction with MIX and DEX caused no cell number increase.
After the change of medium on day 2, MIX and DEX-induced cells fed with
insulin-supplemented medium started mitosis. By day 4, the cell number
increased and was close to that induced with the standard
differentiation protocol (MDI for 48 h plus insulin for an
additional 48 h). In contrast, MIX and DEX-induced cells fed with
plain medium remained in the quiescent state, and no cell number
increase was detected. FACS analysis confirmed the results of cell
number counting (Fig. 2B). Only in MIX and DEX-induced cells
fed with insulin-supplemented medium was DNA synthesis observed. Thus,
induction with MIX and DEX (without insulin) in the first 2 days
followed by normal insulin-supplemented medium for an additional 2 days
delayed the cells entrance into mitotic clonal expansion, whereas
induction only with MIX and DEX for 2 days resulted in the cells
staying in the quiescent state without entering into clonal
expansion.
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Adipocyte differentiation under these induction conditions was analyzed by the accumulation of cytoplasmic triglyceride and the expression of adipocyte marker protein aP2 (Fig. 2, C and D). As shown by Oil-Red-O staining (Fig. 2C), 3T3-L1 preadipocytes induced with MIX and DEX for 2 days and then with insulin for an additional 2 days differentiated into adipocytes normally, although the clonal expansion was delayed by 2 days compared with standard condition-induced 3T3-L1 preadipocytes. Thus, delaying the mitotic clonal expansion did not have a detectable effect on the 3T3-L1 cell differentiation. Furthermore, when 3T3-L1 preadipocytes were induced without insulin for the entire differentiation process (MIX and DEX for the first 2 days and then medium without an insulin supplement), a significant fraction of the preadipocytes differentiated into adipocytes, even though no mitotic clonal expansion occurred; and the differentiated adipocytes were evenly distributed in the entire cell monolayer. The expression of adipocyte marker protein aP2 confirmed the results of Oil-Red-O staining. In all three cases (standard induction condition: MDI for 2 days and insulin for 2 days; clonal expansion delayed condition: MD for 2 days and insulin for 2 days; no clonal expansion: MD for 2 days), expression of aP2 protein started on day 3, and its expression was further increased in fully differentiated adipocytes (Fig. 2D). These results indicated that the differentiation of 3T3-L1 preadipocytes into adipocytes was not related to their mitotic clonal expansion.
DNA Synthesis Is Not Required for 3T3-L1 Preadipocyte
Differentiation--
The results of FACS analysis and cell number
counting indicated that a significant fraction of 3T3-L1 preadipocytes
differentiated into adipocytes without undergoing mitotic clonal
expansion when induced with MIX and DEX. To confirm these results,
BrdUrd was used to label DNA synthesis. Therefore, if adipocyte
differentiation requires mitotic clonal expansion, then most of the
differentiated adipocytes will be labeled with BrdUrd. When BrdUrd was
added to the cells during the S phase (from the 16th to 18th h after the hormonal stimulation), most MDI-induced cells were labeled by
BrdUrd as shown in Fig. 3, A
and B. In contrast, only a small percentage of cells induced
with MIX and DEX were labeled with BrdUrd, and it was the same
as the percentage of BrdUrd-labeled control cells fed with DMEM and
10% FBS. Although the adipocyte differentiation in MIX and DEX-induced
cells was not as complete as that in MDI-induced cells (37%
versus 70% of the cells containing visible triglyceride
droplets), only around 15% of differentiated adipocytes with MIX and
DEX induction were labeled by BrdUrd. This ratio of BrdUrd
incorporation in differentiated adipocytes was in the same range of
BrdUrd incorporation in the total cell population. If mitotic clonal
expansion were a required step for adipocyte differentiation, we would
expect a much higher BrdUrd incorporation ratio in adipocytes than in
total cells. Thus, the MIX and DEX-induced adipocytes did not undergo
DNA synthesis and hence mitosis.
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In the standard differentiation induction protocol, after the initial hormone induction the induction medium was replaced with medium supplemented with insulin. Because of the mitogenic effect of insulin on clonal expansion (Fig. 1), MIX and DEX-induced cells would have a delayed mitotic clonal expansion if they were fed with insulin-supplemented medium. Thus, the BrdUrd labeling experiment was also conducted on day 3 cells, i.e. after the medium change. The result shown in Fig. 3C indicated that the basal level of BrdUrd labeling was observed in cells induced with MIX and DEX, which was replaced with a no-insulin medium. However, when replaced with insulin-supplemented medium, a significant amount of cells induced with MIX and DEX were labeled by BrdUrd, indicating a delayed mitotic clonal expansion. However, the adipocyte differentiation was normal. Thus, the results from cell counting, FACS analysis, and BrdUrd labeling were very consistent with each other. They provided compelling evidence that 3T3-L1 preadipocytes could differentiate into adipocytes without DNA synthesis and mitotic clonal expansion.
MEK-1 Inhibitor PD98059 Blocked Mitotic Clonal Expansion without
Affecting Adipocyte Differentiation--
Due to the lack of exposure
to insulin, 3T3-L1 preadipocyte differentiation induced with MIX and
DEX was only about half of the normally induced differentiation. To
analyze the role of mitotic clonal expansion in the normally induced
3T3-L1 preadipocyte differentiation process, MEK-1 inhibitor PD98059
(18-20) was used to block the activation of MAP kinases Erk1 and Erk2.
It has been reported that PD98059 blocks the activation of Erk1 and
Erk2 MAP kinases by IGF-I in 3T3-L1 preadipocytes and that it also
blocks the proliferation of 3T3-L1 preadipocytes (21). In addition, it
has no effect on the adipocyte differentiation (21, 22). By adding 20 µM PD98059 during the differentiation induction process
(from day 0 to day 4), PD98059 almost completely blocked mitotic clonal expansion (Fig. 4A). In the
presence of PD98059, the cell number was only slightly increased as
compared with noninduced cells. The result of FACS analysis (Fig.
4B) was consistent with the cell number counting. In
MDI-induced cells treated with PD98059, only a small fraction of cells
had DNA synthesis during the clonal expansion period, whereas in
control MDI-induced cells, most of the cells had DNA synthesis. Thus,
it is clear that PD98059 treatment inhibits mitotic clonal
expansion.
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Although PD98059 blocked mitotic clonal expansion, it had no effect on adipocyte differentiation. As shown in Fig. 4C, in the presence of PD98059, 3T3-L1 preadipocytes differentiated into adipocytes without any difference from adipocytes induced by standard protocol. Results of BrdUrd labeling confirmed the observation that PD98059 blocked mitotic clonal expansion but not adipocyte differentiation (Fig. 4, D and E). The total adipocyte differentiation in the presence or absence of PD98059 was very similar (both around 80% of the cells containing visible triglyceride droplets). However, only 20% of the cells were labeled by BrdUrd in the presence of PD98059, whereas >70% of the cells were labeled by BrdUrd in the standard induced control cells. In differentiated adipocytes, close to 80% of the normally induced adipocytes were labeled by BrdUrd, whereas less than 20% of the adipocytes were labeled by BrdUrd in PD98059-treated cells. Thus, In the presence of PD98059, most differentiated adipocytes had no DNA synthesis and mitosis.
Activation of MAP Kinase Erk1 and ERk2 Is Essential but Not
Sufficient for Mitotic Clonal Expansion--
During differentiation
induction, 3T3-L1 cells started to enter S phase 14 h after the
addition of differentiation inducers (Fig. 1C). Thus, the
activation of Erk1 and Erk2 by MEK-1 during the first 14 h was
investigated. As shown in Fig.
5A, in MDI-induced cells, Erk1
and Erk2 were significantly activated (as shown by the increase in the
phosphorylated form of Erk1 and Erk2), whereas the addition of PD98059
to MDI-induced cells greatly diminished the phosphorylation of Erk1 and
Erk2. In our previous studies (12), the protein tyrosine phosphatase
inhibitor vanadate blocked 3T3-L1 adipocyte differentiation without
affecting mitotic clonal expansion. Thus, the effect of vanadate on the
activation of Erk1 and Erk2 was also investigated. It was clear that
vanadate did not affect the MDI-induced activation of Erk1 and
Erk2.
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Of MIX, DEX, and insulin, only insulin, acting through the IGF-I
receptor (16, 17), was capable of inducing mitotic clonal expansion
(Fig. 1). The results of study of the activation of Erk1 and Erk2 by
insulin showed a pattern similar to their activation by MDI (Fig.
5B). PD98059 treatment also blocked the activation of Erk1
and Erk2 by insulin, whereas vanadate treatment did not interfere with
insulin-activated Erk1 and Erk2 phosphorylation and even slightly
increased the phosphorylation of Erk1 and Erk2. MIX alone could also
increase the phosphorylation of Erk1 and Erk2, but it caused no mitosis
(results not shown). Thus, activation of Erk1 and Erk2 is not
sufficient to promote growth-arrested postconfluent 3T3-L1
preadipocytes to enter mitotic clonal expansion during differentiation
induction. Treatment with DEX alone did not activate Erk1 and Erk2
(results not shown).
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DISCUSSION |
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Mitotic clonal expansion during differentiation induction is a rather unique process for adipocyte differentiation. However, its role in the adipocyte differentiation process has not been fully understood. In the debate over whether this cell proliferation is a required step along the differentiation process or a parallel event activated by differentiation inducers, our studies provided evidence for the latter. As indicated in our previous studies with vanadate, which selectively blocks adipocyte differentiation but not mitotic clonal expansion, mitotic clonal expansion is not likely to be an obligatory step in the adipocyte differentiation process because differentiation is blocked at the event(s) preceding mitotic clonal expansion, which, on the other hand, is not inhibited (12).
In the present study, we further analyzed the relationship between 3T3-L1 adipocyte differentiation induction and mitotic clonal expansion during differentiation induction. By cell number counting and FACS analysis, it was found that two rounds of cell division occurred in the clonal expansion period: (a) a well-synchronized first round of the cell cycle; and (b) a not very synchronized second round of the cell cycle (Fig. 1, A-C). By delaying or omitting the addition of insulin during differentiation induction, mitotic clonal expansion could be delayed or completely prevented (Fig. 2, A and B). However, there was still normal adipocyte differentiation under these conditions (Figs. 2 and 3). Thus, mitotic clonal expansion and adipocyte differentiation are two separable events. Importantly, the identification of insulin as the hormone responsible for inducing mitotic clonal expansion (Fig. 1, D and E) indicated that the IGF-I receptor was the important factor responsible for activation of mitotic clonal expansion because insulin acts through the IGF-I receptor in 3T3-L1 cell differentiation induction (12, 16, 17). During the induction of adipocyte differentiation, IGF-I receptor tyrosine kinase signaling was very important. Without the addition of insulin in differentiation induction, 3T3-L1 adipocyte differentiation was only about half of the differentiation induced with insulin (Fig. 3). The small amount of hormones, such as insulin and IGF-I, present in the culture medium (containing fetal bovine serum) might also contribute some of this differentiation. Thus, the identification of insulin as the inducer for mitotic clonal expansion indicated that the IGF-I receptor was the important signal initiator for both mitotic clonal expansion and adipocyte differentiation. Based on our previous (12) and present results, it is likely that the IGF-I receptor activates two signal pathways, which lead to the mitotic clonal expansion and adipocyte differentiation separately.
It has been reported that in subconfluent 3T3-L1 preadipocytes,
PD98059, a MEK-1 inhibitor, blocks thymidine incorporation and
accelerates adipocyte differentiation (21). Our results with PD98059
treatment in postconfluent 3T3-L1 preadipocyte differentiation induction indicated that PD98059 blocked mitotic clonal expansion during differentiation induction, whereas adipocyte differentiation was
not affected (Fig. 4). This is just the opposite of the effect of
vanadate, which blocks adipocyte differentiation without affecting mitotic clonal expansion (11, 12). Vanadate blocks adipocyte differentiation by inhibiting the turnover of IGF-I receptor tyrosine kinase phosphorylated c-Crk, therefore blocking the c-Crk-mediated differentiation induction signal (12). On the other hand, PD98059 blocked mitotic clonal expansion by inhibiting the activation of MAP
kinase (Erk1 and Erk2) by MEK-1 (Fig. 5). Because the activation of
Erk1 and Erk2 is important for cell proliferation, inhibition of Erk1
and Erk2 will inevitably affect cell division. Our observation that
PD98059 did not affect adipocyte differentiation is consistent with
reports by other investigators (21, 22). In fact, there is evidence
that Erk1 and Erk2 MAP kinase activation is not necessary for but
antagonizes 3T3-L1 adipocyte differentiation (22). As expected,
vanadate (a tyrosine phosphatase inhibitor), which did not affect
mitotic clonal expansion but blocked adipocyte differentiation, had no
effect on the activation of Erk1 and Erk2 (Fig. 5). Taken together,
these results indicate that the IGF-I receptor tyrosine kinase
activated by the high concentration of insulin stimulates at least two
signal pathways. One pathway leads to the activation of Erk1 and Erk2,
which is essential for mitotic clonal expansion, whereas the other
pathway, through the adapter molecule c-Crk that is inhibited by
vanadate, leads to the adipocyte differentiation.
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FOOTNOTES |
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* This work was supported by Research Grants 39825107 (to K. L.) and 39825115 (to J. W.) from the Chinese National Nature Sciences Foundation, Research Grant G1999053901 (to J. W.) from the Major State Basic Research Program of China, Grant 98XD14015 (to J. W.) from the Science and Technology Commission of Shanghai Municipality, Research Grant 9910 (to K. L. and J. W.) from the Shanghai-Unilever Research and Development Foundation, and grants (to K. L. and J. W.) from the Chinese Academy of Sciences.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 may be addressed: Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Rd., Shanghai, China 200031. Tel.: 86-21-64374430, ext. 5299 or 5298; Fax: 86-21-64338357; E-mail: kliao@sunm.shcnc.ac.cn (K. L.) or wujr@sunm.shcnc.ac.cn (J. W.).
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M011729200
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ABBREVIATIONS |
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The abbreviations used are: IGF-I, insulin-like growth factor I; MIX, 3-isobutyl-1-methylxanthine; DEX, dexamethasone; MDI, 3-isobutyl-1-methylxanthine, dexamethasone, and insulin; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; DAPI, 4',6'-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Erk, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MD, 3-isobutyl-1-methylxanthine and dexamethasone.
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