From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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Molecular mechanisms coupling growth arrest and
cell differentiation were examined during adipogenesis. Data are
presented that document a cascade expression of members of two
independent families of cyclin-dependent kinase inhibitors
that define distinct states of growth arrest during 3T3-L1 preadipocyte
differentiation. Exit from the cell cycle into a pre-differentiation
state of post-mitotic growth arrest was characterized by significant
increases in p21 and p27. During onset of irreversible growth arrest
associated with terminal differentiation, the level of p21 declined
with a concomitant, dramatic increase in p18 and a sustained level of
p27. The expression of p18 and p21, regulated at the level of protein
and mRNA accumulation, was directly coupled to differentiation. Stable cell lines were engineered to express adipogenic transcription factors to examine the active role of trans-acting elements
in regulating these cell cycle inhibitors. Ectopic expression of peroxisome proliferator-activated receptor (PPAR) Adipocytes of white adipose tissue, as well as myocytes from heart
and skeletal muscle, represent examples of terminal differentiation whereby the expression of a specialized phenotype is marked by cessation of cell proliferation and the accumulation of cells in the
G1 phase of the cell cycle. In mammalian cells, phase
transition is regulated by the phosphorylated states of various
substrates including the retinoblastoma family proteins which mediate S
phase progression (1). These substrates are phosphorylated by a dimer complex comprising a regulatory "cyclin" subunit and a catalytic cyclin-dependent kinase
(cdk).1 Phosphorylating
activity of cyclin/cdk complexes is further modulated by
cyclin-dependent kinase inhibitors (CKIs), which are
grouped into two distinct families based on sequence homology and
targets of inhibition (2). To date, seven CKIs have been identified, including p15INK4b,
p16INK4a, p18INK4c, and
p19INK4d defining the INK4 family, and
p21Cip1, p27Kip1, and
p57Kip2, representing the CIP/KIP family. Recent
reports have demonstrated that CKI expression is up-regulated during
cell differentiation in vitro and in vivo (3, 4),
suggesting that these cell cycle inhibitors may play a universal role
in exit from the cell cycle and/or maintenance of the irreversible
growth arrest which defines terminal differentiation.
Adipocyte differentiation is largely controlled by two families of
transcription factors: the CCAAT/enhancer-binding proteins (C/EBPs) and
peroxisome proliferator-activated receptors (PPARs) (5-7). Members of
the C/EBP family (C/EBP Current models of the molecular process of adipogenesis involve a
cascade expression of C/EBP In this investigation, we demonstrate that induction of differentiation
of 3T3-L1 preadipocytes results in gene expression representing classic
cell cycle progression that switches to adipogenic gene expression
concomitant with exit from the cell cycle. In addition, the data
presented here document a cascade expression of members of two
independent families of CKIs that define distinct states of growth
arrest associated with adipogenesis. Moreover, the expression of p18
and p21 is shown to be regulated during the conversion of non-precursor
fibroblasts into adipocytes by ectopic expression of the adipogenic
transcription factor, PPAR Stable Cell Lines--
The NIH-3T3 cell line ectopically
expressing C/EBP Cell Culture and Induction of Differentiation--
Murine 3T3-L1
preadipocytes and fibroblast cell lines ectopically expressing
adipogenic transcription factors were induced to differentiate into
adipocytes as described previously (21). Briefly, cells were propagated
in DMEM containing 10% calf serum (growth medium). At 2 days
postconfluence, the medium was changed to DMEM containing 10% FBS
supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1.7 µM insulin (MDI;
differentiation medium). After 48 h, cells were maintained in DMEM
containing 10% FBS and 0.4 µM insulin throughout the
remaining time course of experimentation. Maintenance medium was
changed every 48 h until the cells were utilized for
experimentation. Throughout the study, "time 0" refers to
postconfluent cells immediately before chemical induction of differentiation with the addition of MDI to the culture medium. The
term "post-MDI" refers to the time elapsed since the addition of
MDI to the culture medium.
RNA Analysis--
Total RNA was extracted from fibroblast cell
lines with Trizol (Life Technologies, Inc.) according to
manufacturer's instructions with modifications. Briefly, cultured
cells were washed in ice-cold phosphate-buffered saline, lysed with
Trizol reagent, passed through a 21-gauge needle, and gently mixed
(5:1) with chloroform. Following centrifugation, the aqueous phase was
mixed with an equal volume of isopropyl alcohol and centrifuged. The
resulting pellet was dissolved in RNase-free water, mixed with an equal
volume of chloroform/butanol (4:1), vortexed vigorously for 15 s,
and centrifuged. The aqueous phase was collected and RNA precipitated
with sodium acetate/ethanol. Following quantitation, 20 µg of total
RNA was denatured in formamide and electrophoresed through
formaldehyde/agarose gels. The RNA was blotted to Hybond-N nylon
(Amersham Pharmacia Biotech), cross-linked, hybridized, and washed.
Probes were labeled by random priming using the Klenow fragment of DNA
polymerase I (New England Biolabs Inc., Beverly, MA) and
[ Protein Analysis--
Preparation and fractionation of isolated
adipocytes from rat fat pads was performed as described previously
(22). Cultured cells were washed with phosphate-buffered saline, lysed
in Tris/SDS buffer containing Nonidet P-40 and protease inhibitors,
vortexed, and centrifuged. Protein content of the supernatant was
determined using a BCA kit (Pierce) according to manufacturer's
instructions. Following quantitation, proteins were separated by
electrophoresis through SDS-polyacrylamide gels and transferred to
polyvinylidene difluoride membrane (Bio-Rad). Following transfer,
membranes were blocked with milk and probed with the following primary
antibodies: p21 and proliferating cell nuclear antigen (Oncogene); p27
and cyclin D1 (Transduction Laboratories); p18, C/EBP Molecular Events Demonstrating a Switch between Adipocyte Growth
and Differentiation--
Cultured 3T3-L1 preadipocytes induced to
differentiate are documented to undergo an early phase of clonal
expansion, which precedes the acquisition of a fat-laden phenotype.
Heretofore, few investigations have explored potential molecular
mechanisms that may play a role in exit from clonal expansion and/or
maintenance of growth arrest associated with terminal differentiation.
To assess the switch between growth and differentiation, we initially characterized changes in gene expression during cell cycle progression that follows the induction of differentiation. Cells were cultured to 2 days post-confluence and induced to differentiate as described under
"Experimental Procedures." Total RNA was collected every 2 h
for 30 h following a change from growth to differentiation medium
and subjected to Northern analysis. As shown in Fig.
1A, 2-day post-confluent cells
not exposed to chemical inducers (0 h) had entered a state of
density-induced growth arrest, as indicated by the comparison of
histone and cyclin gene expression to subconfluent, proliferating
preadipocytes (PPA). Switching to differentiation medium consisting of
DMEM supplement with 10% FBS and MDI resulted in cell cycle
progression with sequential activation of ornithine decarboxylase
(early G1), cyclin D1 (mid G1), cyclin E (late
G1), cyclin A (late G1/S), histone H2B (S
phase), and cyclin B (G2/M) gene expression. The rapidity
of early gene activation and the peak of histone expression, estimated
at 18-20 h, suggests that reentry of these density-arrested
preadipocytes into the cell cycle occurred immediately following the
change to differentiation medium. The kinetics of cyclin gene
expression presented here are consistent with reported changes in Rb
phosphorylation (24) and E2F-binding complexes (25) determined for
differentiating 3T3-L1 preadipocytes. Based on the additional
observation of immediate early (c-Myc), delayed early (ornithine
decarboxylase), and S-phase (histone H2B) gene activation reported here
and elsewhere (26, 27), it appears that chemical induction of
differentiation of these preadipocytes resulted in synchronous
activation of cell cycle gene expression that begins in the very early
phases of G1, possibly G0, and continues
through to cell division.
Fig. 1A also compares the kinetics of clonal expansion with
adipogenic transcription factor gene expression. Chemical induction of
differentiation resulted in immediate activation of C/EBP A Cascade Expression of Cyclin-dependent Kinase
Inhibitors That Define Distinct States of Growth Arrest during
Adipocyte Differentiation--
It is well accepted that cell cycle
progression is controlled by cyclin/cdk protein kinases where
phosphorylating activity can be modulated by functionally and
structurally distinct CKIs. To assess the involvement of CKIs in
coupling of growth arrest and adipocyte differentiation, the gene
expression of the seven known members of the INK4 and CIP/KIP families
of CKIs was examined by Northern analysis, where it was determined that
terminal differentiation was marked only by elevated levels p18 and p21
mRNA (data not shown). Based on this screen, p18 and p21 were
further examined at the level of protein expression during the time
course that entailed the switch between growth and differentiation.
Albeit modestly regulated at the level of gene expression, p27 was also examined due to numerous reports linking this CKI to density arrest and
the well documented post-transcriptional regulation of its protein
expression (30, 31). To carefully evaluate the kinetics of CKI
expression during exit from clonal expansion and the onset of
irreversible growth arrest, whole cell lysate proteins were harvested
following a change to differentiation medium and subjected to Western
analysis during two independent time courses. The first examined
protein expression every 24 h for 6 days (Fig.
2A) and the second every
6 h during the first 48 h and every 12 h thereafter for
96 h (Fig. 2B). For comparison, total RNA was isolated
from the same experiment depicted in Fig. 2B and subjected
to Northern analysis for histone H2B mRNA expression as a marker of
S-phase progression. Protein expression of C/EBP
Numerous reports have indicated that CKI expression, in particular p21,
can be directly modulated by various mitogens and hormonal agents. To
confirm that the cascade regulation of CKI expression was linked to
molecular processes of differentiation and not simply due to exposure
to the chemical inducers, density-arrested preadipocytes were
chemically induced to differentiate in the presence and absence of
tumor necrosis factor
To further support the pattern of CKI expression following terminal
differentiation, protein expression was examined in native adipocytes
isolated and purified from rat epididymal fats pads as described under
"Experimental Procedures." The rat adipocytes were fractionated
into cytosolic, nuclear, and whole cell lysates to ensure that protein
expression in native adipocytes was not localized and therefore diluted
by compartmental protein normalization. For comparison, whole cell
lysates were isolated from 3T3-L1 cultured adipocytes at 0, 3, and 6 days of differentiation and subjected to Western analysis. As shown in
Fig. 3B, both p18 and p27 proteins were expressed in rat
whole cell lysates (W) to a value equivalent to that
observed for terminally differentiated 3T3-L1 adipocytes (day 6).
Similarly, p21 protein was not observed to any significant amount in
fully differentiated 3T3-L1 adipocytes (day 6) or in any measured
fraction of differentiated rat adipocytes.
A Role for Adipogenic Transcription Factors in Regulating CKI
Expression--
Considerable evidence implicates C/EBPs and PPAR
To investigate the contribution of adipogenic transcription factors in
the regulation of CKI gene expression, we utilized a previously
described NIH3T3 fibroblastic cell line engineered to ectopically
express C/EBP
As accumulation of both p18 and p21 mRNAs correlated with
ligand-activated PPAR
Swiss and Balb/c fibroblasts are often considered preadipocytes
inasmuch as some conversion can be noted when normal, untransfected cells are exposed to chemical inducers of differentiation. To determine
the contribution of PPAR
Both transcriptional and post-transcriptional mechanisms were indicated
in the regulation of p21 and p27 during 3T3-L1 adipocyte differentiation. To determine if adipocyte conversion by PPAR This investigation presents a molecular mechanism coupling growth
arrest and adipocyte differentiation. First, we demonstrate a clear
switch in gene expression mediating the processes of growth and
differentiation and that growth arrest following clonal expansion correlates closely with the expression of adipogenic transcription factors, C/EBP Data presented in this investigation confirm and extend in greater
detail a recent report (33) documenting a cascade expression of members
of two independent families of CKIs during the course of adipogenesis.
As summarized in Fig. 7A, the
protein expression of p27, p21, and p18 defines three unique states of
growth arrest associated with saturation density, post-mitotic growth
arrest, and the onset of terminal differentiation, respectively. The
complexity and timing of expression suggest that multiple CKIs may play
specific and diverse roles in coupling growth arrest and cell
differentiation. It is interesting to note the synchrony of the inverse
relationship between p27 protein and histone gene expression during
clonal expansion. Other reports have indicated that p27 protein
accumulates under quiescent conditions of serum deprivation and density
arrest, decays rapidly with the onset of cell cycle progression,
remains low during subsequent cell cycles and returns to high levels
concomitant with the onset of growth arrest (35). The high levels of
p27 immediately juxtaposed to S-phase presented in this investigation attest to a potential function of this cell cycle inhibitor and to the
synchrony of entry into and exit from the cell cycle associated with
clonal expansion. Although the increase in p27 following clonal
expansion is consistent with establishment of a new saturation density,
the combined expression of p27 with p21 during exit from the cell cycle
and p27 with p18 during terminal differentiation may represent a
synergistic role for multiple CKIs during distinct states of growth
arrest. In this regard, the expression of p21 and p27 simultaneously
may attribute to the synchrony and rapidity of growth arrest following
clonal expansion. The possibility also exists that combined
up-regulation of p21 and p27 following clonal expansion may play a
permissive and/or regulatory role for subsequent adipocyte
differentiation. In support of this notion, the highest degree of
myelomonocytic cell differentiation has been shown to occur independent
of chemical inducers when p21 and p27 were ectopically expressed
together, suggesting that multiple CKI expression may be required for
complete cell differentiation (36). Moreover, the observation that
ectopic expression of p21 or p27, but not of p16, leads to
megakaryocytic differentiation suggests the possibility of CKI
specificity in regulating cell differentiation independent of growth
arrest (37). Therefore, the timing and overlapping nature of CKI
expression during adipogenesis may impart synergistic and specific
functions specific to distinct states of growth arrest and different
stages of adipocyte differentiation.
in non-precursor fibroblastic cell lines resulted in conversion to adipocytes and a
coordinated increase in p18 and p21 mRNA and protein expression in
a PPAR
ligand-associated manner. These data demonstrate a role for
PPAR
in mediating the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors, thereby
providing a molecular mechanism coupling growth arrest and adipocyte differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, C/EBP
, and CEBP
) form heterodimers
and homodimers via a leucine zipper motif with dimers binding to
regulatory elements within target genes via basic DNA binding domains.
Ectopic expression of various C/EBPs has been shown to convert
non-precursor fibroblastic cell lines into fully differentiated
adipocytes (8-10), whereas genetic knockouts in vitro and
in vivo block adipocyte differentiation (11-13). The PPARs
(PPAR
, PPAR
, and PPAR
) define a family of ligand-activated nuclear hormone receptors that heterodimerize with the retinoid X
receptor and bind to specific peroxisome proliferator-responsive elements located within the promoters of target genes. Through utilization of different start sites and alternate splicing, the PPAR
gene gives rise to two isoforms,
1 and
2. Tissue
distribution of PPAR
2 is highly enriched in adipose tissue, and
ectopic expression in various non-precursor cell lines also gives rise
to adipocyte differentiation (14). Although the natural ligand for
PPAR
is still under investigation, a synthetic class of specific
ligands, called thiazolidinediones (TZDs), greatly enhance
transcriptional activity (15).
and C/EBP
, followed by the expression
of C/EBP
and PPAR
, which precede and regulate the expression of
many genes representative of the mature adipocyte. Much of our
understanding of the interplay between these and other trans-acting elements that regulate adipocyte
differentiation has been made possible with the establishment of
preadipocyte cell lines (e.g. 3T3-L1 and 3T3-F442A) that
differentiate from proliferative, fibroblastic-like cells into mature
adipocytes exhibiting nearly identical morphological and biochemical
properties of white adipose tissue (16). After reaching a state of
density-induced growth arrest, preadipocyte cell lines can be induced
to differentiate with exposure to a combination of mitogen and hormonal
agents. Immediately after exposure to these agents, the cells reenter the cell cycle for a limited period of cell proliferation, commonly referred to as clonal expansion. This is followed by the establishment of a unique state of post-mitotic growth arrest, referred to as GD, which has been reported to be permissive for subsequent
differentiation (17). With the onset of differentiation, the cells
enter a third state of growth arrest that is irreversible, where they
are then considered to be terminally differentiated. Although numerous reports have recently emerged concerning the transcriptional regulation of adipocyte specific gene expression, little is known concerning the
molecular events involved in the progression of clonal expansion and
the establishment of distinct states of growth arrest that mark the
progression toward terminal differentiation.
, providing a molecular mechanism coupling
growth arrest and adipocyte differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and C/EBP
under control of a tetracycline
operator was created and described previously (18). Stable cell lines
expressing PPAR
were derived by retroviral infection as described
previously (19). Briefly, packaging cells (BOSC23) were grown in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal
bovine serum (FBS). At approximately 80% confluence, the cells were
transiently transfected with pBabe-derived PPAR
2 expression vector
by calcium phosphate coprecipitation with chloroquine as described
(20). Viral supernatants were collected 48 h after transfection,
filtered, and applied with 4 µg/ml hexadimethrine bromide to
proliferating Swiss and Balb/c fibroblasts for 24-36 h. Medium was
then changed to DMEM containing 10% calf serum. After 48 h, cells
were passaged if necessary and exposed to 2 µg/ml puromycin for
selection. Resistant cells were propagated in puromycin until experimentation.
-32P]dCTP (NEN Life Science Products). Hybridization
to the ribosomal 18 S subunit was used to quantitate equal loading.
, and PPAR
(Santa Cruz); and Glut4 (23). Results were visualized with horseradish peroxidase-conjugated secondary antibodies (Sigma) and enhanced chemiluminescence (Pierce) according to manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pattern of gene expression that demonstrates
the switch between 3T3-L1 preadipocyte growth and differentiation.
A, cell cycle gene expression following induction of
differentiation. Cultured preadipocytes were differentiated as
described under "Experimental Procedures." Total RNA was collected
every 2 h for 30 h following chemical induction of
differentiation, and 20 µg of RNA was examined by Northern analysis.
RNA from subconfluent PPA and fully differentiated adipocytes
(6 day) was included for reference to
proliferation and differentiation, respectively. Phase change denoting
cell cycle progression following chemical induction of differentiation
was approximated and diagrammed above the illustrated data.
B, switch between growth and differentiation gene
expression. Total RNA was collected every 24 h for 9 days
following chemical induction of differentiation, and 20 µg of RNA was
examined by Northern analysis. The switch between growth and
differentiation was approximated and diagrammed above the illustrated
data.
and
C/EBP
, a process that has been shown to occur independent of protein
synthesis and afforded by isobutylmethylxanthine and dexamethasone,
respectively (28). Although it is still uncertain what role, if any,
that C/EBP
and C/EBP
may play in the activation of post-confluent
cell cycle progression, it is clear that cell proliferation kinetically
preceded the expression of C/EBP
and PPAR
. The virtual
absence of cyclin and histone gene expression during late stages of
differentiation (6 day; Fig. 1A) indicates that
preadipocytes ceased proliferating at some point during the process of
adipogenesis. To assess when the switch between growth and
differentiation occurred, RNA was collected every 24 h for 9 days
following the change to differentiation medium and subjected to
Northern analysis. Data presented in Fig. 1B clearly
illustrate the mutually exclusive nature of growth and differentiation
with a clear switch in gene expression associated with these
independent processes occurring approximately 3 days following
induction of differentiation. Of particular interest, the onset of
C/EBP
and PPAR
coincided with the switch in gene expression,
suggesting that these transcription factors may play a role in coupling
growth arrest and adipocyte differentiation. It is also important to note that the same chemical agents responsible for the induction of
differentiation were also responsible for activation of clonal expansion. Thus, the decision to switch between growth and
differentiation pathways, although continually in the presence of
abundant mitogens, is made at the cellular level and not by the
investigator. This is in contrast to other differentiating systems
(e.g. skeletal muscle), where induction of differentiation
typically requires technical manipulations necessary to ensure a
prerequisite state of growth arrest. Considering this and the synchrony
of clonal expansion, we propose, as have others (29), that
differentiation of preadipocyte cell lines provides an excellent model
for mechanistic studies concerning the coupling of growth arrest and
cell differentiation.
and PPAR
was
also examined to document the early onset of differentiation. As shown
in Fig. 2, p27 protein was abundantly expressed in density-arrested
preadipocytes (0 day), decreased transiently during the first 48 h
of differentiation, returned to predifferentiation levels by day 3, and
remained elevated throughout the course of differentiation.
Interestingly, the transient increase in histone mRNA and the
transient decrease in p27 protein correlated in a direct reciprocal
fashion as cells entered and exited the S phase of clonal expansion. In
direct contrast, the protein expression of p21 was significantly
elevated in proliferating (PPA) but not density-arrested preadipocytes
(day 0). Following a change to differentiation medium, p21 transiently
increased during early stages of G1, decreased during S
phase progression, increased again to abundant levels concomitant with
the expression of C/EBP
and PPAR
, and then decayed during later
stages of differentiation (day 6). Although the first peak coincided
with early (G1 phase) cell cycle progression, the second
peak of p21 protein accumulation (72-96 h) clearly occurred as cells
exited the cell cycle as indicated by a significant decrease in the
number of cells entering S phase (i.e. decreased histone
expression). Interestingly, the protein expression of proliferating
cell nuclear antigen, which began to accumulate during early
G1, was maintained during this period, suggesting that the
cells had entered a transient state of growth arrest that was unique
from that observed during density arrest (day 0) or terminal
differentiation (day 6). The expression of p18 appeared to kinetically
succeed exit from the cell cycle and the early onset of adipocyte gene
expression with significant protein levels accumulating only during
later stages of terminal differentiation. Unlike p21 and p27, p18
protein was not significantly expressed in proliferating or
density-arrested preadipocytes.
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Fig. 2.
Cascade expression of p18, p21, and p27
proteins during 3T3-L1 preadipocyte differentiation. A,
cultured preadipocytes were differentiated as described under
"Experimental Procedures." Whole cell lysates were collected every
24 h for 6 days following chemical induction of differentiation.
One hundred µg of protein was examined by Western analysis. Protein
from subconfluent PPA was also included. B, whole cell
lysates were harvested every 6 h during the first 48 h and
every 12 h thereafter for 96 h, following a change to
differentiation medium. Fifty µg of protein was examined by Western
analysis. The 42-kDa band of C/EBP and the 48- and 50-kDa bands
representing PPAR
1 and PPAR
2, respectively, were illustrated. For
reference to S-phase progression, 20 µg of total RNA, collected from
the same experiment, was examined by Northern analysis for histone
mRNA expression (*).
(TNF
). This cytokine has been shown to
completely block the development of the fat-laden phenotype and
associated gene expression when applied to cells during the induction
of preadipocyte differentiation. Whole cell lysates were harvested on
days 3 and 6 following chemical induction in the presence and absence
of TNF
and subjected to Western analysis for CKI expression. Protein
expression of Glut4 was also examined to confirm the state of adipocyte
differentiation. As shown in Fig.
3A, the increase in p21 and
p18 protein expression, observed on days 3 and 6, respectively, was
prevented in the presence of TNF
, suggesting that the expression of
these CKIs was dependent upon adipocyte differentiation and not due to
secondary effects of the mitogen and hormonal agents necessary to
induce differentiation. This conclusion was further supported by the
lack of effect of TNF
on p27 protein, suggesting that the inhibitory
effect on differentiation was not due to toxicity of this potent
cytokine.
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Fig. 3.
Dependence of p18, p21, and p27 protein
expression on adipocyte differentiation. A, 3T3-L1
preadipocytes were differentiated as described under "Experimental
Procedures" in the presence and absence 500 pM TNF .
Whole cell lysates were collected at 3 and 6 days following chemical
induction of differentiation. One hundred µg of protein was examined
by Western analysis. B, protein from cytosolic
(C), nuclear (N), and whole cell lysates
(W) was prepared from differentiated adipocytes isolated
from epididymal fat pads collected from 200-g male Sprague-Dawley rats
as described under "Experimental Procedures." Whole cell lysates
were also isolated from 3T3-L1 cultured adipocytes at 0, 3, and 6 days
of differentiation. Fifty µg of protein was examined by Western
analysis. The 42-kDa band of C/EBP
was illustrated.
as
major transcription factors responsible for development of the mature adipocyte. The next objective was to determine if these adipogenic transcription factors play a role in regulating the cascade expression of CKIs during adipogenesis. Initially, the mRNA accumulation of
p18, p21, and p27 was kinetically compared with the expression of
C/EBP
and PPAR
over the time course of 3T3-L1 preadipocyte differentiation. Total RNA was harvested every 24 h for 6 days following chemical induction and subjected to Northern analysis. As
shown in Fig. 4, the mRNA expression
of p27, highest in density-arrested preadipocytes (0 day), declined
following chemical induction and remained at low levels throughout the
course of differentiation. Although the precipitous decline in
protein that immediately precedes S phase of clonal expansion was
accompanied by a moderate fall in mRNA accumulation (compare Figs.
2 and 4), the return of p27 protein to predifferentiation levels
following cell cycle progression appears to be predominantly
independent of gene expression and likely to occur via
post-transcriptionally controlled pathways. Steady state levels of p21
mRNA gradually increased kinetically with protein expression
(compare Figs. 2 and 4) as cells entered the state of post-mitotic
growth arrest following clonal expansion. Interestingly, the onset of
terminal differentiation was marked by a decrease in p21 protein even
though the mRNA remained elevated, suggesting both transcriptional
and post-transcriptional processes were likely to be involved in p21
regulation. Northern blot analysis of p18 demonstrated a 2.4- and
1.2-kb transcript that began to accumulate coordinately with protein
expression on day 3 and remained elevated throughout differentiation.
Although the kinetics of both p18 mRNAs were similar, the onset of
terminal differentiation was marked by dramatic changes in the 1.2-kb
transcript. Consistent with this observation, others have reported two
p18 mRNAs where changes occurred predominantly in the 1.2-kb
transcript during cell differentiation (32, 33). Collectively, these
data indicate that differentiation-dependent increases in
p18 and p21 protein expression were kinetically accompanied by
coordinate changes in mRNA accumulation. Of particular interest,
the accumulation of p18 and p21 mRNAs coincided with or succeeded
the onset of C/EBP
and PPAR
gene expression.
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Fig. 4.
Correlation of p18, p21, and p27 mRNA
accumulation with the expression of adipogenic transcription factors
during 3T3-L1 preadipocyte differentiation. Cultured preadipocytes
were differentiated for as described under "Experimental
Procedures." Total RNA was collected every 24 h for 6 days
following chemical induction of differentiation, and 20 µg of RNA was
examined by Northern analysis. The 1.2- and 2.4-kb transcripts of p18
were illustrated.
and C/EBP
under the control of a
tetracycline-responsive inducible expression system (18). These cells,
designated "
cells," were propagated in the presence of
tetracycline, which has been shown to repress the ectopic expression of
both C/EBPs. Tetracycline was removed from the growth medium at near
confluence, and at approximately 2 days post-confluence (day 0), the
growth medium was replaced with differentiation medium supplemented
with the TZD, ciglitazone. With the exception of the TZD supplement,
conditions were maintained identical to those used for 3T3-L1
differentiation described under "Experimental Procedures." Total
RNA was collected at 0 and 6 days of differentiation and subjected to
Northern analysis. As depicted in Fig.
5A, ectopic expression of
C/EBP
and C/EBP
, in the presence of MDI and TZD, led to the
expression of PPAR
and adipocyte-specific genes (e.g. adipsin). Concomitant with the onset of adipocyte differentiation was
the accumulation of p18 and p21 mRNAs to values equivalent to those
observed in fully differentiated 3T3-L1 adipocytes. Interestingly, CKI
mRNA accumulation occurred in the absence of C/EBP
, which has
been shown to be repressed in NIH3T3 fibroblasts (9, 10). It is
important to note, however, that the role of C/EBP
as an adipogenic
transcription factor may, in part, be played by the ectopic expression
of C/EBP
and/or C/EBP
. To continue to dissect the molecular
mechanism responsible for CKI mRNA accumulation, these
engineered fibroblasts were cultured under various diagnostic conditions to alter gene expression and exposure to chemical inducers and TZDs. Total RNA was harvested following 6 days of conditional treatment and the results of Northern analysis depicted in Fig. 5B. Exposing the NIH-3T3 fibroblasts, not expressing ectopic
C/EBP
and C/EBP
(i.e. in the presence of
tetracycline), to chemical inducers and TZD (lane
1) resulted in a modest increase in p21 mRNA that was
not observed in cells ectopically expressing the C/EBPs in the absence
of differentiation mixture (lane 2). Although chemical induction of cells expressing the C/EBPs resulted in PPAR
and adipocyte-specific (e.g. adipsin) gene expression
(lane 3), significant p18 and p21 mRNA
accumulation and acquisition of the adipocyte phenotype was observed
only when cells where cultured under conditions leading to PPAR
gene
expression in the presence of an exogenously supplied ligand specific
for PPAR
activation (lane 4).
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Fig. 5.
Inducible ectopic expression of
C/EBP and C/EBP
in
NIH-3T3 fibroblasts induces adipogenesis and accumulation of p18 and
p21 mRNA in a PPAR
ligand-dependent manner. A, NIH-3T3
fibroblasts, ectopically expressing C/EBP
and C/EBP
in a
tetracycline-repressive manner (
cells) were differentiated as
described under "Experimental Procedures." Total RNA was collected
at 0 and 6 days following chemical induction of differentiation, and 20 µg of RNA was examined by Northern analysis. The 1.2-kb transcript of
p18 was illustrated. For comparison, RNA from 3T3-L1 adipocytes
(L1) differentiated for 6 days was included. B,
cells were differentiated for 6 days under various conditions
depicted below the illustrated data. The absence of ectopic C/EBP
and C/EBP
expression (
/
) was accomplished by
supplement of tetracycline (1 µg/ml) to the differentiation medium.
Cells expressing
/
only were cultured for an identical length of
time as other conditions but in the absence of chemical inducers (MDI)
and TZD supplement (ciglitazone; 10 µM). Twenty µg of
total RNA was examined by Northern analysis.
and not C/EBP expression, we continued to the
explore the relationship between this adipogenic transcription factor
and CKI gene expression by utilizing a retroviral system to produce
fibroblastic cell lines ectopically expressing the
2 isoform of
PPAR
. The pBabe-Puro expression vector containing the cDNA for
PPAR
used in this investigation (kindly provided by B. M. Spiegelman) was previously characterized for its efficacy in producing
functionally active protein in NIH-3T3 fibroblasts (34). Parental (V)
and PPAR
(P
) containing vectors were packaged into viruses that
were used to infect Swiss and Balb/c fibroblasts as described under
"Experimental Procedures." Following puromycin selection, the
resulting stable cell lines were grown to confluence and induced to
differentiate with MDI in the presence of the TZD, troglitazone.
Northern analysis of total RNA collected from Swiss (SP
) and Balb/c
(BP
) fibroblasts ectopically expressing PPAR
at 0 and 6 days of
differentiation is depicted in Fig.
6A. Total RNA from
differentiated 3T3-L1 adipocytes was included for comparison. The
larger ectopically expressed transcript (arrow) was easily resolved from the endogenous PPAR
, as illustrated in
lanes 1-4 (Fig. 6A). Following the
standard differentiation protocol including TZD supplement, both
fibroblastic cell lines ectopically expressing PPAR
significantly
displayed gene expression (i.e. Glut4) and morphology
(
90% cells containing lipid droplets; data not shown) indicative of
the mature adipocyte. Interestingly, both cells lines demonstrated a
significant increase in p18 and p21 mRNAs concomitant with the
acquisition of the adipocyte phenotype. It should be noted that, in
contrast to NIH3T3 cells, C/EBP
was not repressed in these
fibroblasts and was regulated in a coordinate fashion with adipocyte
differentiation.
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Fig. 6.
Retroviral expression of
PPAR in Swiss and Balb/c fibroblasts results
in conversion to adipocytes and a coordinate regulation of p18 and p21
at the level of mRNA and protein expression. A,
retroviral infection was used to generate stable cell lines with
ectopic expression of PPAR
2 in Swiss (SP
) and Balb/c
(BP
) fibroblasts as described under "Experimental
Procedures." Following puromycin selection, cells were differentiated
in the presence of troglitazone (10 µM). Total RNA was
collected at 0 and 6 days following chemical induction of
differentiation, and 20 µg of RNA was examined by Northern analysis.
The 1.2-kb transcript of p18 was illustrated. For comparison, RNA from
3T3-L1 adipocytes (L1) differentiated for 6 days was
included. B, Swiss and Balb/c fibroblasts ectopically
expressing PPAR
(P
) or empty vector (V)
were differentiated in the presence or absence of troglitazone (10 µM). Total RNA was collected following 6 days of
differentiation and 20 µg of RNA was examined by Northern analysis.
For comparison, RNA from 3T3-L1 adipocytes (L1)
differentiated for 6 days was included. C, Balb/c
fibroblasts ectopically expressing PPAR
(P
) or empty
vector (V) were differentiated in the presence and absence
of troglitazone (10 µM), respectively. Whole cell lysates
were collected following 6 days of differentiation and 100 µg of
protein was examined by Western analysis. Protein from differentiated
3T3-L1 adipocytes was included for comparison.
above this adipogenic background, fibroblasts ectopically expressing the PPAR
construct (P
) or the
parental vector (V) were differentiated for 6 days in the presence and
absence of troglitazone. Total RNA was collected and the Northern
analysis is depicted in Fig. 6B. Troglitazone supplement to
the differentiation medium applied to Swiss P
cells resulted in a
differential increase in Glut4, p18, and p21 mRNAs (compare
lanes 1 and 2) that was not observed
with TZD supplement to cells expressing the parental vector (compare
lanes 3 and 4). It appeared, however,
that exposing vector cells to the differentiation protocol increased
the background expression of both p18 and p21 independent of TZD
treatment. This level of expression was possibly due to a direct effect
of the chemicals utilized to induce differentiation and/or the 10-20%
adipocyte conversion of Swiss vector cells that occurred following the
differentiation protocol (data not shown). In contrast to Swiss, the
Balb/c fibroblasts expressing PPAR
converted to adipocytes, as
marked by Glut4 expression, independent of TZD supplement (compare
lanes 6 and 7). Consistently, the
mRNA for both p18 and p21 was significantly and equivalently
enhanced in Balb/c P
cells in direct correlation with Glut4
expression and adipocyte differentiation. The increase in mRNA was
most pronounced when comparing P
cells supplemented with
troglitazone (lanes 7) and vector cells
differentiated in the absence of any exogenous PPAR
ligand
(lane 8) where greater than a 30-fold induction
was observed for both p18 and p21 mRNAs. These data support the
notion that the increase in CKI mRNA was not simply due to exposure
to chemical inducers or changes in cell density as both P
and vector cells were exposed to an identical differentiation protocol. The increase in p18 and p21 mRNAs with troglitazone treatment of Balb/c vector cells (compare lanes 8 and 9)
could be attributed to activation of endogenous PPAR
, which
increased under these conditions.
could
also lead to CKI protein expression, Balb/c fibroblasts expressing
PPAR
(BP
) and the empty vector (BV) were induced to differentiate
with MDI in the presence and absence of troglitazone, respectively.
Whole cell lysate proteins were collected over the course of
differentiation, and the results of Western analysis are depicted in
Fig. 6C. Interestingly, both magnitude and kinetics of p18
and p21 protein expression were equivalent to that observed for 3T3-L1
adipocytes with p21 preceding p18 expression by approximately 24 h. The pattern of p27 expression was identical in BP
cells, which
converted to greater than 90% adipocyte morphology, and BV cells,
which remained as fibroblasts demonstrating the independence of p27 on
adipocyte differentiation for protein expression. It was also observed
that cyclin D1 protein increased dramatically in BP
cells, which
expressed abundant levels of p18 and p21, suggesting that adipocyte
differentiation resulted in the accumulation of cells in the
G1 phase of the cell cycle. Although the specific functions
of these CKIs during adipogenesis has yet to be determined, it would
not be unexpected to find that one of their roles is to inhibit cell
proliferation prior to S phase transition during the onset of terminal differentiation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PPAR
. Second, data are presented
documenting a cascade of CKI expression that mark distinct states of
growth arrest associated with adipogenesis. Third, the
differentiation-dependent up-regulation of p18 and p21 is
regulated at the level of mRNA and protein expression when
non-precursor fibroblasts are converted to adipocytes by the expression
of PPAR
in a ligand-associated fashion. Collectively, these data
demonstrate that transcription factors that mediate adipogenesis also
regulate the expression of cell cycle inhibitors providing a molecular
mechanism coupling these processes during exit from the cell cycle and
ensuring the irreversible growth arrest of terminal differentiation.
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Fig. 7.
Schematic model coupling growth arrest with
adipocyte differentiation. A, summary of p18, p21, and
p27 protein expression during the switch between adipocyte growth and
differentiation. Changes in p18 (green), p21
(red), and p27 (blue) protein expression during
3T3-L1 preadipocyte differentiation were summarized from densitometry
measurements of data presented in panels A and
B of Fig. 2. The kinetics of histone H2B mRNA expression
(shaded gray) illustrated in Fig. 2B
was included for reference to S-phase progression during clonal
expansion. Segments representing three unique states of growth arrest
were illustrated above the summarized data. B, adipocyte
differentiation signals, initiated by chemical inducers (MDI), activate
a sequential cascade of transcriptional events that regulate gene
expression responsible for the development of the functional adipocyte.
In addition, chemical inducers activate synchronous cell cycle
progression referred to as clonal expansion and a cascade of CKIs that
define distinct states of growth arrest characterized for adipocyte
differentiation. The expression of these CKIs is shown to be regulated
by adipocyte transcription factors, thereby providing a molecular
mechanism coupling growth arrest with adipocyte differentiation.
Albeit absent in fully differentiated adipocytes in vitro and in vivo, it should be emphasized that the expression of p21 dramatically increases twice during the course of cultured adipocyte differentiation. The initial up-regulation of p21, which coincides with the G1 phase of clonal expansion, is consistent with reported regulation and function of p21 during early phases of cell cycle progression (38). In contrast, the subsequent up-regulation of p21 correlates directly with "exit" from clonal expansion and is dependent on the differentiation program for expression. Thus, it appears that one mixture of mitogen and hormonal agents utilized in the induction of differentiation activates two kinetically independent peaks of p21 protein expression, suggesting the possibility of two independent regulatory mechanisms based on the progression of proliferation versus differentiation. The decay in p21 in the presence of other CKIs during advanced stages of adipogenesis is consistent with other reports indicating a transient expression of p21 during myocyte differentiation in vitro (39) and during cardiac development in vivo (40). The association of p18, p21, and p27 with other proteins mediating both growth and differentiation is currently being investigated to ascribe specific functions of these CKIs during adipogenesis.
This investigation also presents data, generated from three
fibroblastic cell lines regulating the expression of PPAR by two
independent mechanisms and supplement with two pharmacologically different ligands for PPAR
, that provide direct evidence for a role
of adipogenic transcription factors in regulating CKIs at the level of
mRNA and protein expression. As summarized in Fig. 7B,
cell lines that were PPAR
ligand-dependent for
adipogenic gene expression were also ligand-dependent for
regulation of p18 and p21, suggesting a regulatory role for PPAR
at
some point upstream during the course of adipogenesis. The proximity of
PPAR
in the differentiation paradigm to the regulation of p18 and
p21 has yet to be determined. It is predicted, however, that regulation of p18 during adipogenesis occurs primarily at the level of gene expression, as changes in protein expression correlated with equivalent changes in mRNA accumulation. In preliminary experiments,
inhibition of protein synthesis by cycloheximide in cells expressing
PPAR
prevented p18 mRNA accumulation (data not shown),
suggesting that other proteins downstream of PPAR
are likely to be
involved in mediating p18 gene expression. The notion of an
intermediate transcription factor is consistent with the observed delay
between the expression of PPAR
and p18. Preliminary cycloheximide
studies with p21 were not interpretable, inasmuch as inhibition of
protein synthesis, independent of PPAR
expression, led to a dramatic
increase in p21 mRNA accumulation. However, based on a potential
conserved consensus sequence in the promoter of p21 and the coordinate
kinetics of p21 and PPAR
expression, it is possible that this CKI is
directly regulated at the level of gene expression by PPAR
.
Regulation of p21 protein expression during adipogenesis, however, is
likely to be complex with changes in the magnitude and kinetics of
protein expression occurring without coordinate changes in mRNA accumulation.
Although the data presented here demonstrate an upstream regulatory
role for PPAR in the regulation of p21 during adipocyte differentiation, it is important to note that p21 protein expression presented in this investigation correlated closely with the expression of C/EBP
. Interestingly, a role for C/EBP
has recently been shown
in the regulation of p21 in hepatocytes and fibrosarcoma cells at the
level of protein stability (41, 42). Thus, the possibility exists that
post-transcriptional regulation of p21 protein may, in fact, be
regulated by C/EBP
during adipogenesis. The observation that ectopic
expression of PPAR
also results in the expression of C/EBP
shown
here and elsewhere (24) suggests the possibility of a cascade effect of
these transcription factors in the regulation of p21 at the level of
gene expression and protein stability, respectively. As numerous
reports have emerged demonstrating a synergy of these transcription
factors in the regulation of many aspects of the mature adipocyte, it
would not be surprising to find that the complex regulation of CKI
expression during adipogenesis also involves the combined efforts of
both PPAR
and C/EBP
. Experiments addressing the direct and
indirect mechanisms of CKI expression by these and other transcription
factors during adipogenesis are currently under investigation.
Others have reported that both C/EBP (43) and PPAR
(44), when
ectopically expressed, suppress the growth of various subconfluent,
proliferating fibroblastic cell lines. Although the data presented here
suggest that growth arrest is coupled to adipocyte differentiation
through the expression of CKIs, it should be noted that other growth
arrest mechanisms independent of these cell cycle inhibitors may also
be imparted by adipogenic transcription factors under conditions that
may not support adipogenesis. For example, it has been reported that
C/EBP
regulates the growth-arrest-associated gene, gadd45
(45), and that PPAR
can induce cell cycle withdrawal by inhibition
of E2F binding activity via down-regulation of the protein phosphatase,
PP2A (44). Thus, it is likely that various mediators of adipocyte gene
expression may regulate independent and/or synergistic growth arrest
mechanisms as a process to ensure terminal differentiation. Determining
the function of multiple CKIs during adipogenesis and deciphering the
complex interactions of numerous adipogenic transcription factors in
regulating their expression will provide a better understanding of the
physiological control of adipocyte proliferation through coupling of
growth arrest and cell differentiation.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Howard Greene (3T3-L1
preadipocytes), Charles Sherr (cyclin D1, p15, p16, p18 and p19
cDNAs), Bert Volgelstein (p21 and p27 cDNAs), Steve Elledge
(p57 cDNA), Mu-En Lee (A, B and E cyclin cDNAs), Bruce
Spiegelman (retroviral PPAR vector), and Paul Pilch (Glut4 antibody)
for assistance with this investigation. We also thank Jacqueline
Stephens for helpful discussions and advice on culturing 3T3-L1
preadipocytes and Konstantin Kandror for assistance with isolated rat adipocytes.
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FOOTNOTES |
---|
* This work was supported by National Research Service Award 1F32CA69765-01A1 and Boston Obesity Nutrition Research Center Grant DK46200 (both to R. F. M.) and National Institutes of Health Grant DK51586 (to S. R. F.).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: 715 Albany St., Dept.
of Biochemistry, Boston University School of Medicine, Boston, MA
02118. Tel.: 617-638-4186; Fax: 617-638-5339; E-mail:
farmer{at}med-biochem.bu.edu.
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ABBREVIATIONS |
---|
The abbreviations used are:
cdk, cyclin-dependent kinase;
CKI, cyclin-dependent
kinase inhibitor;
C/EBP, CCAAT/enhancer-binding protein;
PPAR, peroxisome proliferator-activated receptor;
TZD, thiazolidinedione;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
MDI, 3-isobutyl-1-methylxanthine, dexamethasone, and insulin;
kb, kilobase pair(s);
TNF, tumor necrosis factor
;
PPA, proliferating
preadipocyte.
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REFERENCES |
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