(Received for publication, May 9, 1997, and in revised form, June 20, 1997)
From the Division of Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Terminal differentiation of stem cells is
characterized by cessation of cell proliferation as well as changes in
cell morphology associated with the differentiated state. For adipocyte
differentiation, independent lines of evidence show that the
transcription factors peroxisome proliferator activated receptor (PPAR
) and CCAAT/enhancer-binding protein
(C/EBP
) as well as
the tumor suppressor retinoblastoma (Rb) protein are essential. How
these proteins promote adipocyte conversion and how they function
cooperatively during the differentiation process remain unclear. We
have used retinoic acid (RA) inhibition of adipogenesis to investigate
these issues. RA blocked adipogenesis of 3T3-L1 cells induced to
differentiate by ectopic expression of PPAR
and C/EBP
independently or together. However, under these circumstances RA was
only effective at preventing adipogenesis when added prior to
confluence, suggesting that factors involved in regulation of the cell
cycle might play a role in establishing the commitment state of
adipogenesis that is insensitive to RA. During differentiation of wild
type 3T3 L1 preadipocytes, we found that Rb protein is
hyperphosphorylated early in adipogenesis, corresponding to previously
quiescent cells re-entering the cell cycle, and later becomes
hypophosphorylated. The data suggest that, together with the
coexpression of PPAR
and C/EBP
, permanent exit from the cell
cycle establishes the irreversible commitment to adipocyte
differentiation.
The molecular mechanisms relating to cell proliferation and cell
differentiation are inadequately understood. Adipocyte conversion provides an excellent model system to study terminal differentiation. In the case of 3T3-L1 cells, differentiation is induced upon exposure of cells to a mixture of hormonal stimulants including dexamethasone, isobutylmethylxanthine, insulin, and fetal calf serum (1, 2). These
pharmacological stimuli, or alternatives such as thiazolidinediones or
other activators of peroxisome proliferator activated receptors (PPARs)1 (3-6), are
necessary for adipocyte differentiation of 3T3-L1 cells. During
adipocyte conversion, a variety of transcription factors are induced,
including C/EBP, PPAR
, and C/EBP
(reviewed in Ref. 7).
Enforced expression of PPAR
(8), C/EBP
(9-11), or C/EBP
(10,
12, 13) stimulates adipogenesis in NIH 3T3 fibroblasts, suggesting the
essential roles of these transcription factors in regulating
adipogenesis. Furthermore, combined expression of PPAR
and C/EBP
has synergistic effects on promoting fat cell conversion in myoblasts
(14). Therefore, it is likely that PPAR
and C/EBP
function
cooperatively to establish terminal adipocyte differentiation.
In addition to the expression of differentiated marker genes, terminal
differentiation is characterized by permanent withdrawal of cells from
the cell cycle. One protein that is involved in cell cycle progression
is the retinoblastoma susceptibility gene product, Rb (15).
Hypophosphorylation of Rb inhibits cell cycle progression, and this
inhibitory effect of Rb is lost upon phosphorylation of the protein
(16, 17). The involvement of Rb in adipocyte differentiation is
suggested by the observation that ectopic expression of protein kinase
C in quiescent NIH 3T3 cells induces hypophosphorylation of Rb and
promotes adipogenesis (18), whereas Rb binding by SV40 large T antigen
interferes with adipogenic differentiation (19). Moreover,
Rb
/
mouse embryonic lung fibroblasts failed to undergo
adipocyte differentiation under appropriate conditions, and ectopic
expression of Rb restored the adipogenic phenotype of the
Rb
/
cells (20). Together, these observations suggest an
essential role of Rb in adipocyte differentiation.
To explore early events in adipogenesis, we have used retinoic acid
(RA), which normally inhibits adipocyte differentiation of 3T3-L1 cells
(21-23). Liganded RAR blocks C/EBP-stimulated gene transcription, and
RA also prevents adipogenesis due to ectopic expression of C/EBP or
-
(13). However, during normal adipogenesis RA exerts its inhibitory
function only when added in the first 24-48 h after exposure to
differentiating stimuli that are applied postconfluence. The inhibitory
function of RA is mediated by RA receptors (RARs), which are
down-regulated early in adipocyte differentiation (23). However,
ectopic expression of RAR in 3T3 L1 cells only extends the period
during which RA is effective in preventing adipogenesis by an
additional 24-48 h. Interestingly, at this time, although PPAR
is
already induced, the level of PPAR
protein is diminished upon RA
treatment. This could be explained by the fact that RA/RAR inhibits
C/EBP function, which may be responsible for maintaining the level of
PPAR during differentiation (23).
We first examined the effect of RA on adipocyte differentiation due to
incubation of wild type 3T3-L1 cells with the PPAR ligand BRL49653,
as well as in 3T3-L1 cells that ectopically express PPAR
. RA
inhibited PPAR
activator-mediated fat cell conversion, suggesting
that RA blocks adipogenesis due to endogenous PPAR
. The inhibitory
effects of RA were also dominant over ectopic co-expression of both
PPAR
and C/EBP
when the cells were maintained in RA at the time
of gene transduction and thereafter. However, if added after gene
transduction but rather at the time of confluency, RA was no longer
effective at blocking differentiation of 3T3-L1 cells that ectopically
expressed these adipogenic transcription factors. Thus, co-expression
of PPAR
and C/EBP
in cells was not sufficient to commit cells to
undergo differentiation in the presence of RA until the cells became
postconfluent. Since confluency is associated with withdrawal from the
cell cycle, we hypothesized that the stage of irreversible commitment
to adipocyte differentiation required exit from the cell cycle as well
as the co-expression of both PPAR
and C/EBP
. Indeed, during
adipocyte differentiation of wild type 3T3-L1 cells, confluent cells
undergo clonal expansion followed by permanent withdrawal from the cell
cycle that occurs at about the time RA loses effectiveness in
preventing differentiation. During this process Rb protein shifts from
a highly phosphorylated state to its hypophosphorylated form. We
conclude that the state of RA-resistant commitment to adipocyte
differentiation involves not only expression of PPAR
and C/EBP
but also hypophosphorylation of Rb and withdrawal from the cell
cycle.
3T3-L1 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were cultured in growth medium (GM) containing 10% iron-enriched fetal bovine serum in Dulbecco's modified Eagle's medium. For standard adipocyte differentiation, 2 days after cells reached confluency (referred as day 0), cells were exposed to differentiation medium (DM) containing 10% fetal bovine serum, 10 µg/ml of insulin, 1 µM dexamethasone, and 0.5 µM isobutylmethylxanthine, for 48 h. Cells then were maintained in postdifferentiation medium containing 10% fetal bovine serum, and 10 µg/ml of insulin. RA was dissolved in ethanol and used at a concentration of 10 µM. For BRL49653-induced adipogenesis, cells were maintained in GM. 1 µM BRL49653 was added to cells on day 0. Cells were exposed to BRL49653 constantly for 7-10 days until fat cells were seen. For experiments involving RA, retrovirally infected cells were studied in the following two protocols: 1) cells were maintained in the constant presence of 10 µM RA from the time of infection and then exposed to the presence or absence of 1 µM BRL49653 at day 0, or 2) cells were grown in the absence of RA until day 0 and then exposed to RA and various conditions. The first of these two conditions corresponds to that described previously (13).
Construction of Plasmids and Retroviral InfectionpLXSN-PPAR2 was generated by insertion of a
1.7-kilobase pair SalI fragment of mouse PPAR
2 cDNA
into the XhoI site of pLXSN (neor) (24).
pTS13-C/EBP
was generated by insertion of a 1.26 kilobase pair
EcoRI-BamHI fragment of C/EBP
into the
BamHI site of the TS13 vector (HgmBr). Standard
calcium phosphate-DNA transfections were performed. To generate
retrovirus-producing packaging cells, 293T cells were transfected with
7.5 µg of plasmid DNA and viral gag and pol
plasmids (25). 48 h post-transfection, filtered viral supernatants
from the ecotropic packaging cell line were used to infect 3T3 L1
cells. Two days after infection, cells were selected in G418 (400 µg/ml; Life Technologies, Inc.) or hygromycin B (200 µg/ml) for
10-14 days. For double infection, pLXSN-PPAR
cells were infected
with virus containing TS13-C/EBP
for 48 h. Cells then were
selected in G418 plus hygromycin growth medium.
Dishes were washed three times with phosphate-buffered saline, fixed by 10% formalin in phosphate buffer for 1 h at room temperature. After fixation, cells were washed once with phosphate-buffered saline and stained with a filtered oil red O stock solution (0.5 g of oil red O (Sigma) in 100 ml of isopropyl alcohol) for 15 min at room temperature. Cells then were washed twice with water for 15 min each and visualized.
Western (Immunoblot) Analysis3T3-L1 cells were lysed in
cell lysate buffer (500 µl for the 10-cm dish and 150 µl for the
60-mm dish), and cells were incubated on ice for 30 min, followed by
centrifugation at 17,000 rpm at 4 °C for 30 min. Supernatant was
collected, and protein concentration was determined by Bio-Rad protein
assay. 60-100 µg of protein was subjected to 10% polyacrylamide gel
electrophoresis. Proteins were then transferred to nitrocellulose
membrane, and Ponceau-S (Sigma) staining was performed to verify equal
loading/transfer. The membrane was incubated first with primary
antibody (anti-PPAR, 1:1500 (23); anti-C/EBP
, 1:300 (Santa Cruz
Biotechnology, Inc.)) for 2 h, followed by secondary antibody
(horseradish peroxidase-conjugated) for 1 h. Blots were developed
using ECL chemiluminescence detection reagent (Amersham Life Sciences,
Inc.) and visualized by exposure to autoradiography film.
Cells were incubated with BrdUrd (Amersham Corp.) for 24 h and then trypsinized, washed twice in phosphate-buffered saline, and fixed with 70% ethanol on ice for 30 min or longer. Fixed cells were incubated with anti-BrdUrd and then fluorescein isothiocyanate-conjugated secondary antibody. BrdUrd-positive cells were sorted by flow cytometry.
[3H]Thymidine Incorporation AssayCells were incubated with 1 µCi/ml of [3H]thymidine for 24 h and then harvested, washed, and resuspended in 0.5 ml of 0.3 N NaOH and incubated on ice for 30 min prior to the addition of 0.5 ml of 20% trichloroacetic acid. Precipitated DNA was filtered through a Whatman GFC filter, washed, dried, and counted in a liquid scintillation counter.
During DM-induced 3T3-L1 differentiation, RA is only
effective in preventing adipogenesis when added during the first 24-48 h (day 0 and day 1), when PPAR has not yet been induced (7). Indeed,
under circumstances when PPAR
is already expressed, RA can lead to
the loss of expression of PPAR
, consistent with its ability to block
the transcriptional activity of C/EBP
, which induces PPAR
during
adipogenesis (12, 23). To further the use of RA as a tool for
understanding the stages of adipocyte differentiation, we first tested
the ability of RA to affect the adipogenesis of 3T3-L1 cells that is
induced by the PPAR
2 activator, BRL49653. BRL49653 induces
adipogenesis of post-confluent 3T3-L1 cells, typically causing 20-70%
of cells to differentiate into adipocytes within 7-10 days (26). This
is presumably caused by the activation of an endogenous low level of
PPAR
in the preadipocytes (23) and subsequently the activation of
the differentiation program and fat cell conversion. These results were
confirmed in Fig. 1 (column 1,
row 2), where fat cell differentiation is indicated by oil
red O staining. RA was able to inhibit BRL49653-induced differentiation
(Fig. 1, column 1, row 3). Fig.
2 shows that BRL49653 treatment induced
both PPAR
and C/EBP
and that, consistent with the oil red O
staining, this induction was blocked by RA. Note that adipocytes
express two forms of C/EBP
, referred to in this paper as
C/EBP
-LAP and C/EBP
-LIP using nomenclature derived from similar
alternative translation products for C/EBP
(27).
RA Inhibits PPAR
Since RA blocked
PPAR induction by BRL49653, we next examined whether expression of
PPAR
would be sufficient to establish insensitivity to RA by testing
the efficacy of RA on cells that are ectopically expressing PPAR
2
(designated as L1-PPAR
2). Fig. 3A shows that these cells
constitutively express PPAR
2 protein. In a typical experiment,
approximately 20-50% of L1-PPAR
2 cells spontaneously
differentiated into fat cells in the absence of any adipogenic stimuli
in 7-10 days postconfluency, due either to a low level of endogenous
ligand or constitutive activity of the ectopic PPAR
(see Ref. 28 and
Fig. 1, column 2, row 1). The addition of the
PPAR
ligand BRL49563 accelerated and enhanced the differentiation
process, with nearly 100% of cells differentiating into fat cells.
This is shown by oil red O staining in Fig. 1 (column 2,
row 2). In addition, Fig. 3 shows that the adipocyte markers
C/EBP
-LAP and C/EBP
-LIP were markedly induced (lane 5). However, when maintained in the presence of RA, cells that were grown in GM with or without the PPAR
ligand BRL49653 did not
convert into fat cells, documented both by oil red O staining (Fig. 1,
column 2, row 4) and C/EBP
expression (Fig. 3,
lanes 6 and 7). The presence of RA had no effect
on the ectopic expression of PPAR
(Fig. 3, compare lanes
6 and 7 to lanes 4 and 5). These results suggest that RA is able to block adipogenesis induced by
ectopic PPAR
protein expression, and expression of PPAR
is not
able to override the inhibitory effect of RA on adipogenesis.
RA Is Ineffective When Added to Postconfluent Cells That Ectopically Express PPAR
Above we showed that RA
blocks the function of PPAR to induce fat cell conversion,
suggesting that additional factor(s) may be necessary to be insensitive
to RA. Previously we have shown that adipogenesis in cells that
ectopically express either C/EBP
or C/EBP
is also blocked when
cells are maintained in the presence of RA from the onset of ectopic
gene expression (Fig. 1 and Ref. 13). This was confirmed in Fig. 1
(column 3, row 3, where the ectopic
C/EBP
-expressing cells are referred to as L1-C/EBP
cells). Interestingly, when RA was added not at the onset of ectopic expression of either PPAR
or C/EBP
, but rather at a later time when cells were confluent, it was no longer effective at blocking differentiation (Fig. 1, row 5). Constitutive expression of C/EBP
in the
L1-C/EBP
cells is demonstrated in Fig.
4 (lanes 5 and 7).
The vector used to ectopically express C/EBP
does not express the
smaller translation product of C/EBP
, called C/EBP
-LIP (23).
Thus, C/EBP
-LIP expression serves as a marker of adipogenesis in
these experiments (29). Note that C/EBP
-LIP was undetectable in the
L1-C/EBP
cells at day 0 (Fig. 4, lane 5), consistent with
their lack of adipocyte phenotype. In contrast, continued incubation of
the L1-C/EBP
cells for 9 days postconfluency did induce adipogenesis (Fig. 1 (column 3, row 1) and Fig. 4 (note
C/EBP-LIP expression in lane 6)). Consistent with the cell
morphology shown in Fig. 1, this adipose conversion of L1-C/EBP
cells was blocked when cells were grown in media containing RA (Fig. 1,
column 3, row 3; Fig. 4, lane 8).
PPAR
Note that while
allowing the cells to reach confluency prior to the addition of RA
appeared to prevent the effects of RA on the ectopic C/EBP- and
PPAR
-expressing cells, RA normally prevents adipocyte
differentiation of wild type 3T3-L1 cells. Those cells however, do not
express PPAR
or C/EBP
until they have begun to differentiate.
Thus, the state of refractoriness to RA seems to require both
confluency and expression of PPAR
and C/EBP
. Indeed, during
normal 3T3-L1 differentiation, RA loses effectiveness at times when
PPAR
and C/EBP
are simultaneously expressed in the cells (23).
Since C/EBP
(13) and C/EBP
induce PPAR
(12, 13), we considered
the possibility that PPAR
and C/EBP
may induce each other,
leading after some time to a state that is refractory to RA inhibition
of adipogenesis. To determine whether PPAR
is able to activate
C/EBP
gene expression, L1-PPAR
cells were collected prior to
differentiation and subjected to Western analysis. Fig. 3 shows that a
low but detectable level of C/EBP
protein was present in day 0 L1-PPAR
preadipocytes (lane 3). This is notably different
from wild type 3T3-L1 cells, which do not express C/EBP
on day 0 (e.g. lane 1). Thus, ectopic PPAR
expression induced a
low level of expression of C/EBP
. The C/EBP
expression was even
greater on day 7, consistent with the adipocyte phenotype of these
cells. Interestingly, RA blocked expression of C/EBP
despite
continued expression of the ectopic PPAR
, suggesting that RA
inhibits PPAR
induction of C/EBP
. This would be consistent with
the results of others indicating that liganded RAR can interfere with
PPAR-mediated gene transcription (31).
In a reciprocal experiment we examined the expression of endogenous
PPAR in L1-C/EBP
cells. Fig. 4 shows that endogenous PPAR
was
expressed in the day 0 L1-C/EBP
preadipocytes (lane 7),
whereas expression of PPAR
was undetectable in day 0 control preadipocytes (lane 1). Expression of PPAR
in the
L1-C/EBP
cells was abolished by treatment with RA from the time of
C/EBP
expression (lanes 7 and 8), consistent
with the ability of RA to inhibit transcriptional activation by
C/EBP
(13).
The above results demonstrate that PPAR
and C/EBP
can activate each other's expression. Therefore, it is
likely that the inability of RA to prevent fat cell conversion in
L1-PPAR
or C/EBP
cells when added postconfluency is due to the
coexpression of PPAR
and C/EBP
. We next tested whether the
requirement for confluency could be overcome by forcing cells to
express both PPAR
and C/EBP
at higher levels. For these
experiments, we doubly infected 3T3 L1 cells with LXSN-PPAR
2
(neor) and TS13-C/EBP
(hygromycinr). These
cells grew slowly, perhaps related to growth suppressive properties of
C/EBP
in other cell lines (30); cells that express only ectopic
C/EBP
grew somewhat slowly but closer to normal than those
ectopically expressing both PPAR
and C/EBP
, suggesting that
PPAR
provided a second growth-inhibitory signal in 3T3-L1 cells. In any case, the cells co-expressing C/EBP
and PPAR
spontaneously differentiated into adipocytes during 10 days of
selection, even prior to confluency. However, when cells were selected
and maintained in the presence of RA, they were able to grow to
confluence at normal rates, and no fat cells were observed in the
absence or presence of BRL49653 (Fig. 1, column 4, row
1). The ability of RA to prevent differentiation of cells that
ectopically express both PPAR
and C/EBP
suggested that
concomitant expression of these two proteins was not sufficient to
establish a commitment state to adipogenesis that is no longer
responsive to RA.
We demonstrated that RA is able to
inhibit 3T3-L1 adipogenesis due to C/EBP and PPAR
, alone or in
combination, provided that RA is added at the time of gene
transduction. In contrast, RA did not have an appreciable effect when
added to cells postconfluency. Since confluency is associated with
withdrawal of cells from the cell cycle and since cell proliferation
and differentiation are often mutually exclusive events, we
hypothesized that both cell cycle arrest and the expression of
adipogenic genes are necessary for the commitment to fat cell
differentiation. It is known that standard differentiation medium
induces mitosis of quiescent 3T3-L1 cells prior to cell cycle
withdrawal and completion of differentiation along adipogenic lineage.
Although RA blocks differentiation, it does not prevent the mitosis and
resultant increase in cell number due to the adipogenic stimulation
(22). Fig. 5A shows the time
course of the mitotic response to adipogenic stimulation in the absence
and in the presence of RA, using BrdUrd incorporation as a measure of
DNA synthesis. In both cases, a major increase in BrdUrd incorporation
occurs on days 1 and 2 following adipogenic stimulation. Thus, the
cells become postmitotic on day 3 and beyond. These data indicate that,
although RA prevented adipocyte differentiation, it did not block the
clonal expansion that occurs following adipogenic stimulation, although
it appears to be quantitatively reduced. Note that in the presence or
absence of RA, the adipogenically stimulated cells return to a
quiescent state characterized by few mitotic events. However, these
states are fundamentally different as shown in Fig. 5B. The
RA-treated cells are quiescent due to contact inhibition, because
reseeding them at low density allows them to re-enter the cell cycle.
In contrast, Fig. 5B shows that adipocytes differentiated by
the standard protocol in the absence of RA are permanently postmitotic
and do not divide after reseeding at low density. These results suggest
that after the clonal expansion that occurs in the early phase of
adipogenesis, cells permanently exit from the cell cycle.
The Phosphorylation State of Rb as a Marker of the Commitment to Adipocyte Differentiation
Interestingly, the time after
adipogenic stimulation at which the cells are permanently postmitotic
corresponds to the time at which cells become refractory to the effects
of RA, i.e. between 48 and 72 h after beginning the
differentiation protocol (23). This further suggested the role of cell
cycle events in adipocyte differentiation. The Rb protein plays a major
regulatory role in the withdrawal of cells from the cell cycle, and
recently it was shown that Rb is required for adipogenic
differentiation in a less well characterized model involving embryonic
lung fibroblasts (20). We therefore examined expression and
phosphorylation of Rb protein in 3T3-L1 cells before and during
differentiation in the presence and absence of RA. The immunoblot in
Fig. 6 shows that confluent, quiescent
3T3-L1 cells (day 0) contained both hyperphosphorylated and
hypophosphorylated forms of Rb protein. The total amount of Rb protein
was relatively constant during differentiation. Adipogenic stimulation
led to an increase in hyperphosphorylation of Rb during the first 2 days, consistent with the process of clonal expansion that would be
aided by hyperphosphorylation and, thereby, inactivation of Rb. As
adipogenesis proceeded, as confirmed by expression of PPAR and
C/EBP
, the Rb protein began to shift to its hypophosphorylated form
(Fig. 6A, lane 3), corresponding to cells exiting
the cell cycle. By day 7, when nearly 100% adipocyte conversion was
achieved, Rb protein was almost entirely hypophosphorylated. The stage
of RA insensitivity corresponds to the time of withdrawal from the cell
cycle in addition to the expression of the adipogenic factors PPAR
and C/EBP
(day 3).
Fig. 6B shows that the presence of RA at the time of
adipogenic stimulation altered the phosphorylation state of Rb. The
degree of hyperphosphorylation on day 1 was reduced, consistent with the reduced percentage of cells that underwent clonal expansion (Fig.
5A). Furthermore, in the presence of RA, Rb was present in
both hyper- and hypophosphorylated forms on all days after exposure to
adipogenic stimulation. Consistent with the lack of adipogenesis, there
was no induction of PPAR or C/EBP
in the presence of RA (Fig.
6B). These data suggested a correlation between terminal
differentiation and the hypophosphorylation of Rb protein. Together
with our earlier demonstration of the role of confluency in determining
RA insensitivity of cells that ectopically express both PPAR
and
C/EBP
, we conclude that irreversible commitment to adipogenic
differentiation requires both the expression of the adipogenic
transcription factors and the cell cycle arrest associated with
hypophosphorylation of Rb.
We have used the ability of RA to inhibit adipogenesis as a tool
to explore the molecular events that occur during the adipogenic differentiation process. In particular, we were interested in pursuing
the observation that at early times after exposure of confluent 3T3-L1
cells to adipogenic stimulation, the cells become committed to the
differentiation pathway and no longer respond to RA. RA was able to
block BRL49653-mediated differentiation in wild type 3T3-L1 cells, as
well as adipogenesis due to ectopic expression of C/EBP, PPAR
, or
both, even in the presence of the PPAR
ligand BRL49653. These
results strongly suggest that the insensitivity to RA inhibition that
occurs during normal differentiation is not due solely to the
concomitant expression of PPAR
and C/EBP
. However, we have noted
that allowing C/EBP
- or PPAR
-expressing cells to reach confluence
reproduces the committed, RA-irreversible state that occurs after about
48 h during wild type 3T3-L1 cell differentiation. Consistent with
this notion, we found that during standard 3T3-L1 cell differentiation,
at the point at which cells normally become refractory to the effects
of RA, cells are not only expressing PPAR
and C/EBP
but are
exiting the cell cycle following clonal expansion.
The ability of RA to block PPAR-induced adipocyte differentiation
suggests that liganded endogenous RAR can interfere with the function
of PPAR
in addition to its ability to block transcriptional activity
of C/EBPs (13). Indeed, the ability of liganded RAR to block PPAR
transactivation can be demonstrated in transfected cells (Ref. 31 and
data not shown). However, during normal adipogenesis, RA is effective
at times prior to expression of PPAR
, suggesting that its natural
target is C/EBP
-mediated transcription (13).
We have now shown that RA blocks adipocyte differentiation of confluent
3T3-L1 cells when added prior to expression of adipogenic transcription
factors and also prevents adipogenesis of cells that constitutively
express C/EBP and PPAR
when added prior to confluency. Thus,
excluding the situation where RAR is limiting (23), preadipocytes enter
a state of commitment to differentiation that is not reversible by RA
when PPAR
and C/EBP
are expressed and growth cessation has
occurred. In the absence of RA, cells that ectopically express PPAR
and C/EBP
undergo adipocyte differentiation before cells reach
confluency, indicating that cell-cell contact may not be necessary for
adipogenesis to occur. However, these cells grow very slowly,
suggesting that expression of adipogenic genes may contribute to the
cessation of cell growth that is also required for cells to undergo
adipogenic differentiation.
Both PPAR and C/EBP
activate several adipocyte-specific genes.
Coexpression of PPAR
and C/EBP
has been shown to have a synergistic effect and strongly promote fat cell differentiation of
fibroblastic (32) and myogenic cell lines (14). C/EBP
has known
antimitotic effects, suggesting that its role in promoting differentiation is at least partially related to suppression of cell
growth (30). Expression of C/EBP
in 3T3-L1 cells reduced their
growth rate, and cells that ectopically expressed both PPAR
and
C/EBP
grew extremely slowly and underwent adipocyte conversion even
before they reached confluence. However, we found that co-expression of
these two proteins was not sufficient to commit cells to undergo differentiation in a manner that was irreversible by RA unless the
cells were growth-arrested. These observations suggest that growth
arrest played a positive role, along with the specific differentiation
factors, in determining whether cells proliferate or differentiate.
During the normal differentiation process, we found that Rb protein is first hyperphosphorylated and then hypophosphorylated. This correlated well with the cell cycle status of the cells both during and after clonal expansion. While this work was in progress, another group also showed that Rb protein levels were stable throughout adipogenesis but reported that the Rb protein was hypophosphorylated throughout the differentiation process (33). The discrepancy appears to be due to the use of different Rb antibodies, and using the same antibody as we used, those investigators have more recently found changes in Rb phosphorylation similar to those reported here.2 The role of Rb in adipogenesis has been predicted by other studies. In addition to being required for the adipogenesis of mouse lung fibroblasts (20), Rb is necessary for myoblastic differentiation (35). Indeed, there is evidence that RB interacts with adipogenic C/EBP proteins (34) as well as with the myogenic differentiation factor Myo D (35). However, the precise role of Rb in differentiation should be further pursued.
It is unlikely that cell cycle arrest is merely a downstream effect of
adipogenic differentiation factors because expression of C/EBP and
PPAR complement but cannot replace the important effects of cell
growth arrest on the ability of 3T3-L1 cells to irreversibly
commit to adipocyte differentiation. Taken together, our results
suggest that cell cycle arrest is as important as the expression of
adipogenic genes in promoting cell differentiation and the irreversible
commitment of preadipocytes to this process.
We thank E. Huang for comments on the manuscript.