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J. Biol. Chem., Vol. 277, Issue 26, 23794-23799, June 28, 2002
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§,§,
**
From the Cardiovascular Research Institute, the
¶ Howard Hughes Medical Institute, the Cancer Center,
and the ** Department of Pediatrics, University of
California, San Francisco, California 94143-0130
Received for publication, February 27, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Hypertrophy occurs in postmitotic muscle as an
adaptive response to various physiological and pathological stresses.
Studies in vascular smooth muscle cells and primary cardiomyocytes
suggest that angiotensin II-mediated hypertrophy activates signaling
pathways associated with cell proliferation. Regulation of
cyclin-dependent kinase (Cdk)-cyclin activities is
essential to cell size control in lower eukaryotes, yet their role in
the hypertrophic response in muscle is incompletely understood. We
describe an in vitro model of hypertrophy in C2C12 skeletal
myoblasts and demonstrate that induction of hypertrophy involves
transient activation of Cdk4, subsequent phosphorylation of Rb, and
release of HDAC1 from the Rb inhibitory complex. We also demonstrate
that E2F-1 becomes transcriptionally active yet remains associated with
Rb. We propose a model whereby partial inactivation of the Rb complex
leads to derepression of a subset of E2F-1 targets necessary for cell
growth without division during hypertrophy.
Hypertrophy occurs in postmitotic cardiac and skeletal muscle as a
fundamental adaptive process in response to various stresses in both
physiological and pathological situations. A number of studies indicate
that AngII1 acts as a
hypertrophic stimulus in vascular smooth muscle cells (1) and in
primary cultures of cardiomyocytes (2). Recently, AngII has been shown
to be required for optimal overload-induced skeletal muscle hypertrophy
(3). These observations suggest that AngII can act as a hypertrophic
stimulus both in vitro and in vivo across
myogenic cell types.
The signaling events responsible for AngII-mediated hypertrophy have
been extensively studied. AngII has been shown to induce several
immediate-early genes, such as c-fos, c-jun,
egr-1, and c-myc, primarily through the G
protein-coupled angiotensin receptor subtype 1 in both myogenic and
nonmyogenic cells (2, 4), indicating that mitogenic and hypertrophic
stimuli appear to share certain intracellular responses.
Cell cycle entry and G1 progression are controlled
primarily by Cdk-cyclin complexes through their actions on the E2F-1-Rb complex (5). Cdk4 and Cdk6, assembled with their regulatory subunits,
the D-type cyclins, are activated in response to mitogenic stimuli,
heralding cell cycle entry and G1 progression (6). Active
Cdk4/6-cyclin D1 phosphorylates Rb during early G1; this leads to the up-regulation of cyclin E, its assembly with Cdk2, and
activation of the Cdk2-cyclin E complex, which in turn
hyperphosphorylates Rb (7). Hyperphosphorylated Rb releases and thereby
activates the transcription factor E2F-1, allowing the expression of
genes necessary for DNA replication and mitosis (8).
Although regulation of cyclin-Cdk activities is essential to cell size
control in lower eukaryotes (6), their role in the hypertrophic
response in skeletal muscle is incompletely understood. In this study,
we describe an in vitro model of muscle cell hypertrophy using C2C12 cells. We demonstrate for the first time that the hypertrophic response in these cells involves the transient activation of Cdk4, but not Cdk2, with subsequent phosphorylation of Rb, release
of HDAC1 from the Rb inhibitory complex, and activation of the
transcription factor, E2F-1. We propose a model by which partial
inactivation of the Rb complex leads to the derepression of a subset of
E2F targets necessary for cell growth during hypertrophy.
Antibodies--
Monoclonal antibodies to cyclin D1, cyclin D3,
p21Waf1/Cip1, p27Kip1, and E2F-1, and
polyclonal antibodies against cyclin ECdk2, Cdk4,
and HDAC1 were obtained from Santa Cruz Biotechnology. Monoclonal
antibody against Rb, which recognizes hypophosphorylated and
hyperphosphorylated Rb, was obtained from Pharmingen. Polyclonal phospho-Rb (S780) antibody was obtained from Cell Signaling
Technology.
Cell Culture--
Actively growing C2C12 skeletal myoblasts
(ATCC) were maintained in Dulbecco's modified Eagle's medium with
10% heat-inactivated fetal bovine serum (Invitrogen). The cells were
made quiescent by serum withdrawal for 48 h and stimulated to
proliferate by adding 20% fetal bovine serum. Myogenic differentiation
was induced in subconfluent cultures by the addition of 2% horse
serum. Cellular hypertrophy was stimulated with AngII (100 nM; Sigma).
DNA and Protein Synthesis Assays--
Quiescent cells were
stimulated with AngII or 20% dialyzed fetal bovine serum for 24 h. The cells were pulse-labeled for 2 h with 2 µCi/ml
[3H]thymidine to measure DNA synthesis or 1 µCi/ml
[3H]leucine (PerkinElmer Life Sciences) to measure
protein synthesis and then suspended in cold 10% trichloroacetic acid
for 30 min at 4 °C. The trichloroacetic acid-precipitable material
was washed with cold 5% trichloroacetic acid, cold 70% ethanol, and
deionized water and then solubilized in 0.1 M NaOH.
[3H]Thymidine and [3H]leucine incorporation
was measured by liquid scintillography.
Flow Cytometry--
All of the cultures were analyzed after
24 h of treatment, as described previously (9). Briefly,
106 cells were stained with propidium iodide to exclude
dead cells from evaluation and Hoechst 33342 (Molecular Probes) to
measure DNA content in living cells. Flow cytometry was performed using a Becton Dickinson VANTAGE SE with dual argon ion lasers at 488- and
363-nm light output. Propidium iodide and Hoechst signals were acquired
using 630/22- and 457/10-mm band pass filters, respectively. All of the
analyses of DNA content were performed on 100,000 collected events
using CellQuest software (Becton Dickinson).
Immunoblot Analysis and Immunoprecipitation--
Immunoblot
analysis was performed as described previously (9).
Immunoprecipitations were performed using standard methods (10) with
modifications. The cells were lysed by sonication in cold lysis buffer
(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% Nonidet P-40, 10%
glycerol, 1 mM dithiothreitol, 1 mM NaF, 100 mM Na3VO4, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µM leupeptin, 10 µg/ml
aprotinin, 0.01 mM 4-(2
aminoethyl)-benzenesulfonyl-fluoride) and centrifuged at
16,000 × g for 10 min at 4 °C. The lysate
supernatants were precleared with protein G-Sepharose (Amersham
Pharmacia Biotech) for 60 min at 4 °C. The protein was quantitated
in precleared lysate supernatants by BCA assay (Pierce). 500 µg of
protein was incubated with primary antibodies for 3 h at 4 °C,
followed by the addition of protein G-Sepharose for an additional 60 min. The beads were washed four times with lysis buffer, resuspended in
40 µl of lysis buffer, and boiled in sample buffer (100 mM Tris, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol,
200 mM dithiothreitol, 0.2% bromphenol blue) for 5 min
before separation by gradient SDS-PAGE.
Cyclin-dependent Kinase Assays--
Cdk2 and Cdk4
activities were measured by kinase assay using histone H1 (Roche) or
GST-Rb (amino acids 769-921; Santa Cruz Biotechnology) as substrates
using procedures modified from standard methods (11). Cdk2 or Cdk4 was
immunoprecipitated as described above. The immune complexes were washed
three times with lysis buffer and three times with kinase buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol). Cdk2 activity was assayed by resuspending immune complexes in 40 µl of kinase buffer containing 30 µM ATP, 5 µCi of [ Transcriptional Reporter Assay--
E2F4B-Luc (12) containing
four consensus E2F-binding sites and E1B TATA upstream of
Photinus pyralis luciferase was provided by F. Dick and N. Dyson (Massachusetts General Hospital Cancer Center). E2F1-Luc (13)
containing the human E2F-1 promoter fused to P. pyralis
luciferase was provided by W. Kaelin (Dana-Farber Cancer Institute).
C2C12 cells were transiently transfected using LipofectAMINE PLUS
(Invitrogen) according to the manufacturer's instructions, and
luciferase activity was assayed in whole cell lysates using the dual
luciferase reporter assay system (Promega) as described previously
(14). After recovering from transfection, the cells were quiesced by
serum withdrawal and then stimulated with 100 nM AngII or
10% fetal bovine serum. pRL-TK (Promega), encoding Renilla
reniformis luciferase downstream of a herpes thymidine
kinase promoter, was included in each transfection for normalization of
transfection efficiency.
AngII Stimulates Hypertrophy and G1 Arrest in C2C12
Myoblasts--
AngII has been shown to induce hypertrophy in cardiac
myocytes and vascular smooth muscle cells (1, 2); however, its effect
on skeletal muscle growth has not been demonstrated. To determine
whether AngII stimulates hypertrophy in C2C12 myoblasts, we treated
quiescent cells with AngII (100 nM) in serum-free medium. Protein synthesis, as measured by [3H]leucine
incorporation, increased 2.6-fold with AngII stimulation compared with
quiescent cells, and 1.4-fold compared with proliferating cells (Fig.
1A). AngII had no significant
effect, however, on [3H]thymidine incorporation, an
indicator of DNA synthesis (Fig. 1A). As a result, the
[3H]leucine/[3H]thymidine ratio, an
indicator of cellular hypertrophy, increased from 1.6 in serum-induced
proliferating cells or 2.2 in quiescent myocytes to 5.0 in
AngII-stimulated cells undergoing hypertrophy.
We also measured relative cell size by flow cytometry using forward
angle light scatter. This analysis demonstrated a 12% increase in cell
size in AngII-stimulated cells compared with quiescent cells (Fig.
1B), similar to the increase seen in AngII-stimulated vascular smooth muscle cells (15). Flow cytometry of Hoechst-stained myocytes also showed that AngII-stimulated cells were arrested in the
G0/G1 phase of the cell cycle (Fig.
1C). This was similar to results observed for both quiescent
myoblasts and differentiating myocytes. Taken together, these findings
suggest that AngII stimulates hypertrophy by increasing protein
synthesis in the absence of cell division.
Cdk4, but Not Cdk2, Is Active in C2C12 Myoblasts Undergoing
Hypertrophy--
Because cell cycle re-entry and G1/S
transit in proliferative cells commences with assembly and activation
of Cdk4/6-cyclin D and subsequent activation of Cdk2-cyclin E, we
studied the effect of AngII stimulation on G1 cyclin
expression and Cdk activity in C2C12 cells. AngII had an up-regulatory
effect on cyclin D1, whereas cyclin D3 was down-regulated in
AngII-stimulated cells, compared with quiescent cells (Fig.
2A). In differentiating C2C12 cells, cyclin D1 expression was repressed, whereas cyclin D3 levels were elevated, compared with proliferating cells (Fig. 2A).
These findings are consistent with previous observations that cyclin D1
is a mitogen sensor and the limiting factor in the assembly of active
Cdk4 complexes necessary for G1/S transit (16), whereas cyclin D3 contributes to the irreversible withdrawal of postmitotic cells from the cell cycle and is critical in the maintenance of a
differentiated phenotype (17).
Although D-type cyclins control Cdk4/6 activity in early
G1, cyclin E regulates Cdk2 activity near the
G1/S transition (18). Cyclin E was repressed in
differentiating cells compared with proliferating cells;
however, we observed up-regulation of cyclin E expression in
AngII-stimulated cells (Fig. 2A). Because Cdk activities are
controlled by association with regulatory cyclins and not at the level
of expression, we were not surprised to find equivalent levels of Cdk2
or Cdk4 protein in AngII-stimulated cells compared with quiescent,
proliferating, and differentiating cells (Fig. 2A).
G1 cyclin expression was induced in AngII-stimulated cells,
suggesting cell cycle re-entry. Because these cells arrested in G1, however, we hypothesized that only Cdk4-cyclin D1 would
be active during the hypertrophic response, because Cdk2 activity is
associated with the G1/S transition. To test this, we
measured Cdk activities in C2C12 cells following AngII stimulation.
Cdk4 and Cdk2 were immunoprecipitated from cell lysate using anti-Cdk4 or anti-CDk2 antibody, and kinase activity was determined using GST-Rb
and histone H1 as substrate, respectively. Cdk4 and Cdk2 activities
were increased in proliferating myocytes and not in AngII-stimulated
cells after 24 h (Fig. 2B). To determine whether kinase
activities might be transient, we assayed Cdk4 and Cdk2 activities at
8, 12, 16, 20, and 24 h after stimulation with AngII. Although
Cdk2 activity was not detected at any of these earlier time points
(data not shown), a burst of Cdk4 activity was observed at 12 h
(Fig. 2C). These results demonstrate an early increase in
Cdk4 activity following AngII stimulation. The lack of Cdk2 activity in
AngII-treated cells is consistent with their failure to progress
through the G1-S transition.
Levels of Cdk4-bound Cdk Inhibitors, p21Waf1/Cip1 and
p27Kip1, Are Unchanged in Hypertrophic C2C12
Myoblasts--
G1 progression is controlled through Cdk
activity, and this in turn is regulated in part by CKIs such as
p21Waf1/Cip1 and p27Kip1 (19). To further
examine the mechanism by which C2C12 cells recruit the cell cycle
machinery but remain arrested in G1 during the hypertrophic
response, we determined the levels of p21Waf1/Cip1 and
p27Kip1 expression following AngII treatment (Fig.
3A). As expected, levels of
both proteins were down-regulated in proliferating myoblasts and
up-regulated in differentiating myotubes, compared with quiescent cells. Expression of both proteins was elevated in AngII-treated myoblasts compared with quiescent cells, at levels similar to those
observed in differentiating myotubes. Other investigators have observed
elevated p27Kip1 levels in AngII-stimulated, vascular
smooth muscle cells, resulting in diminished Cdk2 activity and
hypertrophy (15). In addition, up-regulation of
p21Waf1/Cip1 and p27Kip1 has been observed in
the mesangial cell hypertrophic response to high glucose (20, 21). Our
results are consistent with those reported for other cell types and
extend a general role for these G1-active CKIs in the
response to hypertrophic stimuli.
Although increased expression of CKIs may suggest a role for these
proteins in AngII-stimulated hypertrophy in C2C12 cells, their effects
as cell cycle regulators are delivered through interaction with Cdk
complexes. To correlate the increased p21Waf1/Cip1 and
p27Kip1 levels that we had observed in response to AngII
with a downstream effect on Cdk4 activity and G1
progression, we next measured their association with the Cdk4-cyclin D
complex. We observed co-immunoprecipitation of p21Waf1/Cip1
with Cdk4 complexes in differentiating cells, and this association appeared to be decreased in proliferating cells, as expected. Surprisingly, the p21Waf1/Cip1-Cdk4 interaction was not
significantly disrupted in AngII-stimulated cells, compared with
proliferating cells (Fig. 3B).
Our observation that Cdk4 activity is low or absent in differentiating
cells and elevated in proliferating cells and at 12 h following
AngII stimulation suggests that p21Waf1/Cip1 may function
through several mechanisms in the same cell line. Although
p21Waf1/Cip1 appears to associate with inactive Cdk4
complexes in differentiating and quiescent C2C12 cells, it also
interacts with active Cdk4 in C2C12 cells responding to hypertrophic
stimuli. It has been shown in other systems that despite its initial
description as a Cdk inhibitor, p21Waf1/Cip1 may be found
in active Cdk complexes and may facilitate the assembly of active
Cdk4-cyclin D1 complexes in particular (22). Our data suggest the
possibility that both activities of p21Waf1/Cip1 are
present in skeletal muscle myoblasts responding to different stimuli.
Although the interaction between p21Waf1/Cip1 and Cdk4 in
differentiating cells may be inhibitory, p21Waf1/Cip1 also
may stabilize Cdk4-cyclin D1 activity in cells undergoing hypertrophy,
allowing for the brief but nonsustained Cdk4 activity observed.
In contrast, p27Kip1 co-immunoprecipitated with inactive
Cdk2 in differentiating and AngII-treated cells compared with
proliferating C2C12 myoblasts (data not shown). This suggests that
p27Kip1 functions primarily as a Cdk2 inhibitor at the
G1/S transition in hypertrophic C2C12 myocytes.
Rb Is Hyperphosphorylated in Hypertrophic C2C12 Myoblasts--
Rb
is an important in vivo substrate of Cdk4-cyclin D1. As a
critical regulator of the G1/S transition of the cell
cycle, Rb can exist in hypophosphorylated and hyperphosphorylated forms (23). Although the active, hypophosphorylated form binds to and
inhibits E2F transcription factors required for expression of genes
involved in DNA synthesis, the inactive, hyperphosphorylated form
releases E2F, allowing for its activation (24). To determine whether
the transient Cdk4 activity observed in cells undergoing hypertrophy
inactivates Rb, we examined Rb phosphorylation in AngII-stimulated
cells. Whereas hypophosphorylated (active) Rb persisted in quiescent
myoblasts and differentiated myotubes, Rb phosphorylated on serine 780, which has been shown to be preferentially phosphorylated by Cdk4-cyclin
D1 (25), was detected in both proliferating cells and in hypertrophic
cells stimulated with AngII (Fig.
4A). Hyperphosphorylated Rb
similarly was detected in C2C12 myoblasts treated with other known
hypertrophic stimuli, including prostaglandin F2 Rb Hyperphosphorylation by Cdk4-Cyclin D Is Associated with the
Release of HDAC1--
Rb plays a fundamental role in cell cycle
progression through its association with the E2F family of
transcription factors (5). These in turn regulate expression of genes
important for G1/S transition and DNA synthesis (26). Rb
represses transcription from promoters containing E2F binding sites by
complexing with HDAC enzymes (27-29) and SWI/SNF nucleosome
remodeling factors (30). The first molecular event for cell cycle
re-entry caused by mitogenic stimuli in nonmyogenic, proliferating
cells is activation of Cdk4/6-cyclin D1 followed by initial
phosphorylation of Rb by activated Cdk complexes. Phosphorylation of Rb
displaces HDAC1 from the inhibitory HDAC1-Rb-SWI/SNF complex (31). This
partial disruption of the Rb inhibitory complex relieves repression of the cyclin E gene (32). Cyclin E expression and assembly with Cdk2 lead
to formation of active Cdk2-cyclin E complexes, which hyperphosphorylate Rb, further disrupting the inhibitory complex (24).
This leads to the complete release of E2F and transcriptional activation of a wide variety of E2F target genes necessary for DNA
replication and mitosis (8). The association between Rb and HDAC1
recently has been shown to coincide with myogenic differentiation and
myocyte cell cycle withdrawal (33). Because of this and our observation
of Cdk4 activity (Fig. 2C) without subsequent Cdk2 activity
and Rb hyperphosphorylation (Fig. 4A) in C2C12 cells undergoing hypertrophy, we wanted to determine the downstream effect of
this transient Cdk4 activity on HDAC1 association with the Rb
inhibitory complex. To accomplish this, we performed
co-immunoprecipitation experiments of Rb with HDAC1 in AngII-treated
C2C12 cells (Fig. 4B). Although Rb co-immunoprecipitated
with HDAC1 in quiescent and differentiating C2C12 cells, indicating
active repression by the Rb inhibitory complex, the amount of HDAC1
associated with the Rb complex in AngII-treated cells was
markedly reduced. These data show for the first time that HDAC1 is
released upon phosphorylation of Rb by Cdk4-cyclin D complexes in
myogenic cells during the hypertrophic response.
E2F-1 Remains Associated with Rb but Is Transcriptionally Active in
Hypertrophic C2C12 Cells--
Although AngII-stimulated cells arrested
in G1 (Fig. 1C) and did not demonstrate Cdk2
activity, Rb phosphorylation coincident with Cdk4 activity was observed
(Figs. 4A and 2C). In addition, HDAC1 was
partially released from the Rb inhibitory complex (Fig. 4B),
and cyclin E expression was induced (Fig. 2A). This
suggested that E2F-1, which regulates cyclin E expression (32) and is regulated by the Rb complex, may be active at a subset of target promoters during the hypertrophic response. To test this, we first examined whether E2F-1 was released from Rb upon AngII stimulation. Surprisingly, co-immunoprecipitation of E2F-1 with Rb in proliferating and AngII-stimulated cells demonstrated that E2F-1 remained associated with Rb after AngII stimulation, although perhaps to a lesser extent
than in quiescent, unstimulated cells (Fig.
5A).
To determine whether E2F-1 in complex with Rb but after HDAC1 release
was transcriptionally active, we performed reporter assays in
AngII-stimulated C2C12 cells. We used one reporter containing four
tandem repeats of the E2F-1 binding site (E2F4B-luc) to examine E2F-1
DNA binding (12) and another containing the entire E2F-1 promoter
(E2F1-luc) to evaluate transcriptional activation (13), as E2F-1
positively regulates its own expression. With both reporters, we
observed a modest but significant increase (~3-fold) in activity in
C2C12 cells stimulated with AngII for 12 h (Fig. 5B),
compared with unstimulated, quiescent cells. Although this represents
only one-third of the E2F-1 activity seen in proliferating cells, it coincided with the increase in cyclin E expression (Fig.
5B), as well as the peak of Cdk4 activity (Fig.
2C), and Rb phosphorylation (Fig. 4A) also
observed. These data demonstrate for the first time that during the
hypertrophic response, E2F-1 is transcriptionally active without Rb
hyperphosphorylation by Cdk2 or complete release from the Rb inhibitory complex.
The G1 Cell Cycle Machinery Plays an Important Role in
Skeletal Muscle Cell Hypertrophy--
We have shown that AngII
stimulation of C2C12 cells simulates the hypertrophic cell growth and
G1 arrest observed in primary cardiomyocytes and vascular
smooth muscle cells. We have used this model system to elucidate the
mechanisms by which the G1 cell cycle regulatory apparatus
controls the hypertrophic response in myoblasts. The early
G1 Cdk4-cyclin D1 complex is transiently activated in
response to AngII in C2C12 cells, whereas the later G1
Cdk2-cyclin E complex is not. Although p27Kip1 is
associated with inactive Cdk2 during this process,
p21Waf1/Cip1 may have two functions in these cells:
inhibiting Cdk4 activity in differentiating cells and permitting a
transient burst of Cdk4 activity during hypertrophy, perhaps through
stability effects. Coincident with peak Cdk4 activity, Rb is
hyperphosphorylated, resulting in the release of HDAC1 from the Rb
inhibitory complex. Although E2F-1 remains associated with Rb, it
becomes transcriptionally active, inducing the expression of at least
one target gene, cyclin E.
Based on these observations, we propose a model in which hypertrophy
results from the derepression of a subset of E2F-1 targets necessary
for cell growth during the hypertrophic response (Fig. 6). The identification of this subset of
E2F-1-regulated genes and other components of the Rb complex that also
may mediate partial E2F-1 activity will further validate such a model
and provide potential targets for manipulating the hypertrophy response
in muscle tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP, 5 µg of
histone H1 (Roche) and incubating for 30 min at 30 °C. The reactions
were stopped with sample buffer and heating at 85 °C for 5 min. Cdk4
activity was assayed using the same procedure with 1 µg of GST-Rb as
substrate. Phosphorylated histone H1 or Rb was resolved by gradient
SDS-PAGE and analyzed by autoradiography. Cdk4 activity was quantitated
by liquid scintillography of [-32P]Rb excised from the gel.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
AngII elicits a hypertrophic response in
C2C12 myoblasts. Quiescent (Q) C2C12 cells were
stimulated to proliferate with 20% fetal bovine serum (P),
differentiate with 2% horse serum (D), or hypertrophy with
AngII for 24 h. A, protein and DNA synthesis in
AngII-stimulated cells. Protein synthesis was measured as
[3H]leucine incorporation (counts/min/cell number). DNA
synthesis was measured as [3H]thymidine incorporation
(counts/min/cell number). The data shown are the means ± S.E.
(n = 3). The protein/DNA synthesis ratio increased in
response to AngII. B, cell growth with AngII stimulation.
The cells were analyzed by forward angle light scatter
(FALS). A representative experiment is shown
(n = 3). A rightward shift (*) indicated an increase in
cell size with AngII treatment. C, cell cycle progression in
AngII-stimulated cells. The cells were stained for DNA content with
Hoechst 33342 and analyzed by flow cytometry. A representative
experiment is shown (n = 3). The majority of cells
treated with AngII demonstrated 2 N DNA content, compared
with the normal distribution between 2 and 4 N DNA content
seen in proliferating (P) cells.
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Fig. 2.
A transient increase in Cdk4 activity
accompanies the hypertrophic response in AngII-stimulated C2C12
cells. The cells were treated as described in the legend to Fig.
1. A, cyclin and Cdk expression. The cell lysates were
separated by SDS-PAGE and analyzed with indicated antibodies by
immunoblot. A representative experiment is shown (n = 3). Cyclins D1 and E were up-regulated, cyclin D3 was down-regulated,
and G1 Cdk levels were unchanged in AngII-stimulated cells,
compared with differentiating or quiescent cells. B, Cdk
activities with AngII stimulation. Cdk2 and Cdk4 were
immunoprecipitated from cell lysates, and kinase activities were
measured using histone H1 and GST-Rb as Cdk2 and Cdk4 substrates,
respectively. 32P-Labeled substrates were separated by
SDS-PAGE and analyzed by autoradiography. A representative experiment
is shown (n = 3). Cdk2 and Cdk4 are active only in
proliferating cells at this time point. C, Cdk4 activity
over 24 h of AngII stimulation. Cdk4 activity was assayed at
indicated time points (top panel). Kinase activity was
quantitated as picomoles of 32P incorporated/min/mg
substrate (bottom panel). The data shown are the means ± S.E. (n = 3). A transient, ~3-fold increase in
Cdk4 activity was measured following AngII stimulation for 12 h.
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Fig. 3.
The p21Waf1/Cip1-Cdk4 association
is maintained in AngII-stimulated C2C12 myoblasts. The cells
were treated as described in the legend to Fig. 1. A,
CKI expression with AngII stimulation. Cell lysates were separated by
SDS-PAGE and analyzed with the indicated antibodies by immunoblot. A
representative experiment is shown (n = 3). The levels
of p21Waf1/Cip1 and p27Kip1 proteins were
elevated in AngII-treated cells. B, CKI-Cdk4 association
with AngII stimulation. Cdk4 was immunoprecipitated (IP)
from cell lysates. The complexes were separated by SDS-PAGE and
analyzed with antibodies against p27Kip1 or
p21Waf1/Cip1 by immunoblot. Although the association
between p21Waf1/Cip1 and Cdk4 was increased in
differentiating and quiescent (0h) cells, compared with
proliferating cells, there was no significant change in the
p21Waf1/Cip1-Cdk4 association from 0 h (inactive Cdk4)
to 12 h (active Cdk4) of AngII treatment.
and
1,25-dihydroxyvitamin D3 (data not shown). These results suggest that
Rb is inactivated by Cdk4 phosphorylation during the hypertrophic
response in C2C12 cells and that this occurs regardless of the initial
hypertrophic stimulus.
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Fig. 4.
Rb is phosphorylated and HDAC1 is released
from the Rb complex in AngII-treated C2C12 myoblasts. The cells
were treated as described in the legend to Fig. 1. A, Rb is
phosphorylated at S780. Rb was immunoprecipitated (IP) from
cell lysates, separated by SDS-PAGE, and detected by immunoblot using
an anti-Rb antibody that recognizes both hypophosphorylated and
hyperphosphorylated Rb (top panel), or a monoclonal antibody
directed against Rb phosphorylated at Ser780 (bottom
panel). A representative experiment is shown (n = 3). Hyperphosphorylated Rb (ppRb) was readily detected in
proliferating cells, as well as in cells treated with AngII by 12 h, compared with quiescent (0h) and differentiated cells, in
which mostly hypophosphorylated Rb (pRb) was observed
(top panel). This pattern of phosphorylation specifically
was detected at Ser780, a preferred site for Cdk4-cyclin D1
(bottom panel). B, HDAC1 is released from the Rb
complex. HDAC1 was immunoprecipitated from cell lysates after 12 h. The complexes were separated by SDS-PAGE and analyzed by immunoblot
with anti-Rb antibody. The HDAC1-Rb interaction was markedly reduced in
AngII-treated cells.
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Fig. 5.
E2F-1 becomes transcriptionally active in
AngII-stimulated C2C12 cells. The cells were treated as described
in the legend to Fig. 1. A, E2F-1 remains associated with
Rb. Rb was immunoprecipitated (IP) from cell lysates. The
complexes were separated by SDS-PAGE and analyzed by immunoblot with
anti-E2F-1 or anti-Rb antibodies. There was a modest decrease in
Rb-associated E2F-1 after 12 h of AngII stimulation, although the
association persisted at levels higher than in proliferating cells.
B, E2F-1 becomes transcriptionally active. The cells were
transfected with luciferase reporter plasmids containing four tandem
repeats of the E2F-1 binding site (E2F4B-luc) or the E2F-1 promoter
(E2F1-luc). Cyclin E expression was measured as described in the legend
to Fig. 2. A ~3-fold increase in E2F-1 reporter activity was observed
in AngII-stimulated cells at 12 h (top panel),
coincident with an increase in cyclin E expression (bottom
panels).
View larger version (27K):
[in a new window]
Fig. 6.
Model for the role of G1
regulatory elements in the hypertrophic response in C2C12 skeletal
myoblasts. See text for discussion.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Shaun Coughlin, David Stokoe, James Bristow, and Kevin Shannon for helpful discussion and critical reading of this manuscript.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grant HL62174 and American Heart Association Grant-in-Aid 0150062N.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.
§ Supported by National Institutes of Health Grant HL07544.
To whom correspondence should be addressed: University
of California, San Francisco, 505 Parnassus Ave., Box 0130, San
Francisco, CA 94143-0130. E-mail:
hsbernstein@pedcard.ucsf.edu.
Published, JBC Papers in Press, April 19, 2002, DOI 10.1074/jbc.M201980200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: AngII, angiotensin II; Cdk, cyclindependent kinase; CKI, cyclin-dependent kinase inhibitor; HDAC, histone deacetylase; Rb, retinoblastoma protein; GST, glutathione S-transferase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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