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INTRODUCTION |
Proliferation of cells is controlled by positive and negative
regulatory pathways of the cell cycle. The activity of
cyclin-dependent kinases
(CDKs)1 positively drives the
progression of the cell cycle. As for negative regulation, a number of
CDK inhibitory proteins (CKIs) are known (reviewed in Ref. 1). On the
basis of homology and specificities of interactions, CKI can be
classified into two distinct families. The INK family (p16, p15, p18,
and p19) specifically inhibits cyclin D-CDK4/6 activity by binding to
CDK4/6 (2-6), whereas the Cip/Kip family (p21Cip1,
p27Kip1, and p57Kip2) inhibits multiple CDKs
including both CDK4/6 and CDK2 by binding to cyclin-CDK complexes
(7-15). The amounts of CKIs expressed in cells may be crucial in
controlling cells regarding whether to start or to stop proliferating.
p21 and p27 have been reported to be induced by various
anti-proliferative stimuli (1). Conversely, the p27 level was shown to
be decreased following mitogenic stimulation of quiescent cells in a
number of systems (16-19). The ubiquitin-proteasome pathway is known
to play an essential role in the control of p27 protein expression
(17). CKIs may have important roles in the withdrawal of the cells from
the cell cycle in the differentiation process. p21 and p27 are both
shown to be induced during the differentiation process of certain cell
types including muscle, neuronal, and hematopoietic cells and in some
cases introduction of p21 or p27 into cognate precursor cells is shown
to induce their differentiation (20-24). Also, the accumulation of
CKIs has been shown in many terminally differentiated cells in mouse
embryo (1). However, among the studies of mice deficient in CKIs, only
p57 is shown to be essential for mouse embryogenesis, suggesting that
p57 may have specific roles in mouse development that cannot be
compensated by other CKIs (25, 26). Developmental defects shown in p57 knockout mice include a cleft palate and abnormal endochondral ossification, attributable to increased apoptosis or proliferation at
the expense of cell differentiation.
In the present study, we sought to investigate the possible involvement
of CKIs in the control of cell growth and differentiation of
osteoblastic cells. Osteoblasts isolated from the calvaria of rat fetus
are useful in an in vitro culture system for studying the
mechanisms of osteoblast differentiation (27). By analyzing this
system, we found that p57 is accumulated in cells starved by low serum.
Growth stimulation of these cells by transforming growth factor
1
(TGF
1) resulted in a dramatic decrease in p57. Further analysis
using a set of proteasome inhibitors demonstrated that p57 was degraded
through the proteasomal degradation pathway. Because TGF
1 inhibits
the differentiation of these cells, our findings suggest a possible
involvement of p57 and its proteasomal degradation in the control of
cell growth and differentiation of osteoblastic cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Primary rat osteoblastic cells were isolated
from calvariae of 21-day-old Sprague-Dawley rats embryos as described
previously (28). The cells were maintained in
-MEM containing 10%
FCS and antibiotics. Cells at the second passage were used for all experiments.
Antibodies and Immunological Methods--
Rabbit antisera
against mouse p27, p57, and CDK2 were raised against synthetic peptides
corresponding to the C terminus of each protein. Anti-mouse p21
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
All antibodies cross-react efficiently with the respective rat
homologs. Cells were plated in 6-well plastic dishes with
-MEM
containing 10% FCS and were cultured in
-MEM containing 0.5% FCS
for 72 h prior to the experiment. The cells were then treated with
TGF
1 (1 ng/ml) for the times indicated. Cells were rinsed twice with
ice-cold phosphate-buffered saline and lysed in 180 µl of Nonidet
P-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM NaF, 5 mM EDTA, 5 mM EGTA, 2 mM sodium vanadate, 0.5% sodium
deoxycholate, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, and 0.1% Nonidet
P-40), and the lysates were cleared by centrifugation at 15,000 × g for 5 min at 4 °C. For immunoblot analysis, the samples
were separated on 9 or 12.5% SDS-PAGE. Western blotting was performed
using an enhanced chemiluminescence detection system (Amersham
Pharmacia Biotech).
CDK2 Binding to CKIs--
To investigate the amount of CDK2
bound to each of the CKIs, total cell lysates were immunoprecipitated
with rabbit polyclonal antibodies against p27 and p57 or monoclonal
antibody against p21. Briefly, 2 µl of antisera or 2 µg of a
monoclonal antibody was added to 200 µg of total cell lysates and
incubated for 2 h at 4 °C. The immunocomplexes were brought
down with protein G-Sepharose beads (Amersham Pharmacia Biotech), and
the pellets were washed four times with ice-cold Nonidet P-40 lysis
buffer. The samples were separated on 12.5% SDS-PAGE and analyzed by
immunoblots for CDK2, as described above.
Histone H1 Kinase Assays--
Immunoprecipitations with
anti-CDK2 antibodies were performed as described above.
Immunoprecipitates were then washed twice with kinase buffer (80 mM sodium
-glycerophosphate, pH 7.4, 20 mM
EGTA, 15 mM Mg(OAc)2, and 1 mM
dithiothreitol) and were mixed with 10 µl of kinase buffer containing
50 µM ATP, 1.25 µCi of [
-32P]ATP, and
1 µg of histone H1. The samples were incubated at 25 °C for 30 min, and the reactions were terminated by addition of 30 µl of
Laemmli sample buffer. The samples were separated on 12.5% SDS-PAGE;
the gel was dried and visualized by autoradiography.
Proteasome Inhibitors--
MG132 (Z-Leu-Leu-Leu-aldehyde) and
Z-Leu-Leu-aldehyde (z-LL-H) were purchased from Peptide Institute, Inc.
(Osaka, Japan). Lactacystin was purchased from Calbiochem (La Jolla,
CA). Cells were treated for 8 h with MG132 at a final
concentration of 2.5 µM and with lactacystin and z-LL-H
at final concentrations of 20 µM.
In Vitro Translation and Ubiquitination Experiments--
Mouse
p57 was transcribed and translated from pCMX-mp57 using TNT rabbit
reticulocyte lysate (Promega) with [35S]methionine.
pCMX-mp57 was constructed by inserting a mouse p57 cDNA, which was
generated by polymerase chain reaction using mouse embryo cDNA as a
template, into a pCMX plasmid vector (29). To 20 µl of each sample
the following was added: 9 µl of 10× buffer (500 mM
Tris-HCl, 100 mM MgCl2, 5 mM
dithiothreitol, 50 µM MG132, and 3 µg/ml
ubiquitin-aldehyde (MBL, Nagoya, Japan), 9 µl of 10× ATP
regenerating system (20 mM ATP, 100 mM creatine
phosphate, 100 IU/ml creatine kinase, and 10 IU/ml inorganic
pyrophosphatase) and 40 µl of cell extract (2.7 mg/ml) of
TGF-
1-treated rat osteoblastic cells. For the GST-ubiquitin
experiment, 40 µg of bacterially purified GST-ubiquitin, which was
generously provided by F. Osaka (Kato Cytoprotein Network Project,
Exploratory Research for Advanced Technology, Japan), was included in
the reaction. The reaction mixtures were incubated at 37 °C, and
20-µl samples were taken at 0 and 3 h. The reaction was
terminated by the addition of Laemmli sample buffer, and the samples
were run on a 9% polyacrylamide gel, dried, and visualized by autoradiography.
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RESULTS |
Down-regulation of p57 by TGF
1--
TGF
1 has been shown to
induce proliferation of osteoblastic cells isolated from the calvaria
of rat embryos (30, 31). We examined the expressions of p21, p27, and
p57 in this system to see if these CKIs are involved in the regulation.
All three CKIs were expressed in osteoblastic cells serum-starved in
-MEM containing 0.5% FCS. Treatment of these cells with TGF
1 for
24 h resulted in a 5-fold increase in [3H]thymidine
incorporation (data not shown), which is consistent with previous
reports (30, 31). The amount of p57 was dramatically decreased within
the first 12 h, whereas some decrease was also seen in p21 and p27
expressions during the growth induction by TGF
1 treatment (Fig.
1). These results suggested that the
decrease in the expressions of CKIs may have important roles in the
growth stimulation of rat osteoblastic cells by TGF
1.

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Fig. 1.
TGF 1 reduces the
expression of p57. Rat osteoblastic cells serum-starved prior to
the experiment were further incubated in the presence (+) or absence
( ) of TGF 1. Whole cell lysates were prepared at the times
indicated in the top row. The levels of p27 (top
panel), p57 (middle panel), and p21 (bottom
panel) were examined by immunoblots employing the respective
antibodies.
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Binding of CKIs with CDK2--
The Cip/Kip family proteins (p21,
p27, and p57) control cell cycle progression by binding and inhibiting
cyclin-CDK complexes, including cyclin E/A-CDK2 complexes (7-15).
Therefore, we examined the possible changes in the amount of CDK2 bound
to each CKI before and after TGF
1 stimulation (Fig.
2A). Before the TGF
1
treatment, a significant amount of CDK2 was shown to be bound to p27
and p57 but not to p21. As expected, after growth stimulation by
TGF
1, a decreased amount of CDK2 bound to p27, and none to p57 nor
p21 were seen in these cells. Also, the activity of CDK2 in these cells, as determined by phosphorylation of histone H1 with anti-CDK2 immunoprecipitates, was induced by TGF
1 treatment (Fig.
2B). These results suggest that TGF
1 stimulates the cell
proliferation of osteoblastic cells by reducing the amounts of p57 and
p27 and releasing CDK2 bound to the CKIs, which in turn results in the induction of CDK2 activities.

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Fig. 2.
TGF 1 increases CDK2
activity. A, the level of CDK2 associated with p27,
p57, and p21 as indicated in the top row, in cells without
( ) or with (+) TGF 1 treatment were examined by immunoprecipitation
(IP) with respective antibodies. B, anti-CDK2
immunoprecipitates from TGF 1 untreated ( ) or treated (+) cells
were assayed for the kinase activity using histone H1 (H1)
as a substrate.
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p57 Is Degraded through the Proteasome--
Our data demonstrated
that the dramatic reduction of p57 expression by the TGF
1 may have
important roles in the control of osteoblastic cell growth stimulation.
Although little is known about the control of p57 expression, the
related CKI, p27, has been shown to be controlled by the
ubiquitin-proteasome degradation pathway (17). Therefore, we tested
whether the proteasomal degradation pathway is involved in the
TGF
1-induced decrease in p57. Addition of MG132, a proteasome and
calpain inhibitor (32-34), to the culture medium completely blocked
the decrease in p57 protein by TGF
1 treatment (Fig.
3A), suggesting the possible
involvement of the proteasomal degradation pathway. To determine
whether the effect of MG132 was through specific inhibition of the
proteasome pathway, we further tested the effects of a calpain
inhibitor, z-LL-H (34), and a proteasome specific inhibitor,
lactacystin (35). p57 was shown to be stabilized by MG132 and
lactacystin, whereas z-LL-H had no effect (Fig. 3B). These
results clearly indicated that p57 is degraded specifically through the
proteasome pathway in the osteoblastic cells treated with TGF
1.

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Fig. 3.
p57 is degraded through the proteasome
pathway. A, serum-starved osteoblastic cells were
treated without ( ) or with (+) TGF 1 and without ( ) or with (+) a
proteasome inhibitor MG132 as indicated. Extracts were prepared at the
times shown in the top row. B, cells stimulated
without ( ) or with (+) TGF 1 were treated for 8 h without ( )
or with (+) a series of proteasome and protease inhibitors as
indicated.
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p57 Is Ubiquitinated in Vitro--
Ubiquitination is known to have
important roles in the control of the porteasomal degradation pathway.
Therefore, we tested whether p57 can also be modified by
ubiquitination. p57 was prepared by in vitro translation in
rabbit reticulocyte lysate and was incubated with an addition of
osteoblast cell extract, with or without bacterially produced
GST-ubiquitin (Fig. 4). After 3 h of
incubation, in addition to slower migrating bands seen in both reactions that may correspond to the ubiquitinated p57, several distinct bands were seen in the sample incubated with GST-ubiquitin (Fig. 4, last lane). Of these, a 92-kDa band nicely
corresponds to p57 covalently modified by one GST-ubiquitin, as
GST-ubiquitin is approximately 35 kDa. The fact that these bands were
only seen in the sample incubated with GST-ubiquitin indicated that the bands indeed represent GST-ubiquitinated p57s, which confirms that
p57 can be ubiquitinated.

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Fig. 4.
p57 is ubiquitinated in
vitro. p57, transcribed and translated in
vitro, was submitted to an ubiquitination assay as described under
"Experimental Procedures." Each reaction was incubated without ( )
or with (+) GST-ubiquitin (GST-Ub) for the time indicated.
p57 denotes the original position of unmodified p57. The
asterisks indicate the additional bands appearing in the
right-hand lane that correspond to p57 covalently bound with
GST-ubiquitin. Positions of molecular size markers are indicated on the
left side.
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DISCUSSION |
TGF
is thought to have important roles in bone remodeling.
TGF
is locally produced by osteoblasts and is accumulated abundantly in bone matrix tissue (31, 36, 37), and several studies have shown that
TGF
may regulate not only cell growth but also differentiation
procedure (36-38). Consistent with the previous report (38), induction
of cellular alkaline phosphatase activity, a bone phenotype marker that
precedes the onset of mineralization, was inhibited during
TGF
1-induced stimulation of primary rat osteoblast proliferation in
our experiments (data not shown). CKIs are shown to play important
roles in the induction of cell differentiation (20-24); therefore, our
results strongly suggest that p57 may also play an important role in
regulating the differentiation procedure of osteoblasts. It is worth
noting that among the gene knockout studies of the Cip/Kip family
proteins, only p57-deficient mice showed developmental abnormalities
(25, 26). Abnormalities shown in these mice include short limbs, a
defect attributable to abnormal endochondral ossification, which may be
caused by delayed cell cycle exit during chondrocyte differentiation
(25, 26). Similar ossification defects have been observed in
double-knockout mice of the retinoblastoma-related proteins, p107 and
p130 (39). Chondrocytes and osteoblasts are thought to be derived from
stem cells of the same origin, and interestingly, primary osteoblastic cells used in this experiment express p107 and p130 but not
retinoblastoma, and p107 became phosphorylated when the cells were
stimulated by TGF
1.2
Therefore, it is tempting to speculate that p57, together with p107,
can be involved in the regulation of cell growth and differentiation of
cells of certain lineage, including osteoblasts and chondrocytes.
Our results also indicated that the expression level of p57 can be
mainly regulated by the ubiquitin-proteasome protein degradation pathway, as is the case with its related CKI, p27. The increasing importance of ubiquitin-mediated proteolysis of the regulators of cell
proliferation such as cyclin D, cyclin E, and p27 has been extensively
examined in a number of recent studies (reviewed in Refs. 40-42), and
our findings have added another CKI in this category. It will be
important to study how signals from TGF
1 can be delivered to the
regulation of this machinery.