From the Department of Molecular Oncology, and the
¶¶ Department of Tumor Biochemistry, The Tokyo Metropolitan
Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo
113-8613, Japan, the § Department of Internal Medicine,
University of Tokyo Branch Hospital, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo
112-8688, Japan, the ** Department of Orthopaedic Surgery, and
Oral and Maxillofacial Surgery, Graduate
School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-8655, Japan, the
Department of Biochemistry, The Cancer
Institute of the Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan, and the ¶ Core
Research for Evolutional Science and Technology, Japan Science
and Technology Corporation, Japan
Received for publication, August 17, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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Cyclin-dependent kinase inhibitory
proteins (CKIs) are negative regulators of the cell cycle. Of all CKIs,
only p57Kip2 plays an essential role(s) that other
CKIs cannot compensate for in embryonic development. Recently, we found
that p57Kip2 is degraded through the ubiquitin-proteasome
pathway in osteoblastic cells stimulated to proliferation by
transforming growth factor (TGF)- Cell division is determined by whether cells meet the requirements
for entry into the S phase. Without these requirements, cells may
progress from the G1 phase toward quiescence or terminal differentiation. In these regulatory processes,
cyclin-dependent kinases
(CDKs)1 positively drive the
progression of the cell cycle, whereas CDK inhibitory proteins (CKIs)
act as negative regulators by binding to cyclin-CDK complexes (reviewed
in Refs. 1 and 2). CKIs have been classified into two families in
mammals, i.e. the INK4 (inhibitor of CDKs) family and the
Cip/Kip (CDK interacting protein/kinase inhibitory protein) family. The
INK4 family (p16Ink4a, p15Ink4b,
p18Ink4c, and p19Ink4d) inhibits only CDK4 and
CDK6, whereas the Cip/Kip family (p21Cip1,
p27Kip1, and p57Kip2) inhibits all CDKs
functioning during the progression of the G1/S phase.
There are convincing lines of evidence addressing the importance of the
Cip/Kip family proteins in the proliferation and/or differentiation of
cells. Compared with the tremendous progress in the p21Cip1
and p27Kip1 studies, much less is known about the role of
p57Kip2 in the cell cycle. Interestingly, only
p57Kip2 is shown to be essential for mouse embryogenesis
(3, 4), whereas the lack of p21Cip1 or p27Kip1
does not show gross developmental defects (2). Thus, it is of interest
to explore the relationship between p57Kip2 expression and
cell proliferation under physiological circumstances. However,
p57Kip2 is hard to study due to its low expression level in
many cells. We recently found that p57Kip2 accumulates in
rat osteoblastic cells when arrested by serum starvation. Furthermore,
TGF- Current studies have advanced our understanding of the intracellular
signaling pathway of TGF- In the present study, we examined whether or not the Smad pathway is
responsible for the accelerated degradation of p57Kip2 in
mouse osteoblastic cells treated with TGF- Materials--
Sources of materials were as follows: recombinant
human TGF- Isolation and Culture of Osteoblastic Cells--
Primary mouse
osteoblastic cells were isolated from calvariae of 1-day-old mice,
strain ddY, by sequential enzymatic digestion as described previously
(12, 13). The cells prepared from calvariae by enzymatic digestion are
composed of heterogeneous cell populations, but they express various
osteoblastic phenotypes including the ability to form bone nodules
in vitro, and are useful for examining sequential changes of
phenotypes occurring during osteoblast differentiation, as reported
before (13). The cells were cultured in
After thawing the cells, 5 × 104 cells in 6-well
plastic plate or 2 × 104 cells in 12-well plate were
cultured with Constructions of Recombinant Adenoviruses--
To obtain a high
transfection efficiency in primary osteoblastic cell cultures, we used
a recombinant adenovirus system. Recombinant adenoviral vectors
carrying hemagglutinin (HA)-tagged human TGF- Proteasome Inhibitor--
Osteoblastic cells were treated with
MG132 (Z-Leu-Leu-Leu-aldehyde) at a final concentration of 2.5 µM for 12 h prior to harvesting of cells to avoid
cell toxicity.
Immunoblot Analysis--
Cells were rinsed with ice-cold
phosphate-buffered saline and lysed directly in SDS sample
buffer. The lysates were sonicated briefly and clarified by
centrifugation at 15,000 × g for 5 min at 4 °C. For
immunoblot analysis, the samples were separated on 12.5% SDS-PAGE.
Proteins were then electrotransferred to polyvinylidene difluoride
membranes (Immobilon-P; Millipore Corp., Bedford, MA), immunoblotted
with respective antibodies, and visualized using an enhanced
chemiluminescence detection system (Renaissance; PerkinElmer Life
Sciences, Boston, MA).
Pulse-Chase analysis--
Serum starved osteoblastic cells in
35-mm dishes were labeled for 16 h with 70 µCi/ml
[35S]Met and [35S]Cys (Amersham Pharmacia
Biotech, Piscataway, NJ) in Northern Blot Analysis--
Total cellular RNAs from cultured
cells were extracted using TRIZOL reagent (Life Technologies, Inc.).
These RNAs were electrophoresed on 1.2% agarose formaldehyde gels and
transferred onto nylon membrane filters (Nytran 0.45; Schleicher & Schuell, Keene, NH). Hybridization and washing the filters were carried
out as described previously (15). The cDNA probe for a 377-base
pair SmaI-HindIII fragment from mouse
p57Kip2 was labeled with [ p57Kip2 Proteolysis Is Induced by TGF-
Furthermore, addition of a proteasome inhibitor MG132 blocked the
reduction of p57Kip2 caused by TGF-
To test whether TGF- Degradation of p57Kip2 Is Induced by Constitutively
Active Form of TGF- The Smad Pathway Accelerates the Degradation of p57Kip2
by TGF-
As mentioned before, Smad7 competes with Smad2 and Smad3 for its
binding to activated ALK-5; thus the inhibitory effect of Smad7
suggests the involvement of Smad2 and/or Smad3 in the destabilization of p57Kip2. Therefore, we examined the effects of Smad2 or
Smad3 with Smad4 (Co-Smad), a partner molecule collaborating with
R-Smad in the presence or absence of Smad7 on the instability of
p57Kip2. In this experiment, a low concentration of
TGF-
To further test whether Smad-mediated transcription is regulated for
p57Kip2 degradation, we tested the effect of several
transcription inhibitors. When osteoblastic cells were treated with
actinomycin D for 24 h in the absence of TGF-
On the other hand, actinomycin D or Previously, we reported that TGF- Second, we found that this effect of TGF- Finally, we found that the TGF- The other possibility is that the degradation machinery itself may be
activated by Smad-mediated transcription. In the ubiquilation pathway,
ubiquitin-protein ligase, E3, plays an important role in the selection
of proteins for degradation, because it specifically binds to the
protein substrate (25-28). Therefore, it is tempting to speculate that
the newly induced protein by TGF- It is noteworthy that the level of p57Kip2 is very low in
proliferating osteoblastic cells, but serum deprivation caused its
dramatic accumulation (Ref. 5 and this study). The exposure of TGF- On the other hand, the level of p27Kip1 was unaffected in
TGF- TGF- Notably, TGF-1 (Urano, T., Yashiroda, H.,
Muraoka, M., Tanaka, K., Hosoi, T., Inoue, S., Ouchi, Y., and
Toyoshima, H. (1999) J. Biol. Chem. 274, 12197-12200). We report here that TGF-
1-induced p57Kip2
proteolysis is mediated through transcription by the Smad pathway. When
the constitutively active form of the TGF-
type I receptor ALK-5(TD)
was ectopically expressed in osteoblastic cells, p57Kip2
that had been accumulated by serum starvation causing the cell-cycle arrest was rapidly degraded in a manner analogous to TGF-
1
stimulation. Moreover, Smad2 or Smad3 with Smad4 enhanced the
proteolytic pathway of p57Kip2. The degradation of
p57Kip2 evoked by TGF-
1 was blocked by forced expression
of an inhibitory Smad called Smad7 or by the addition of actinomycin D
or
-amanitin. These results indicate that accelerated degradation of
p57Kip2 by TGF-
1/Smad signaling is mediated through a
newly synthesized factor(s) that modifies p57Kip2 or the
ubiquitin-proteasome pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 that stimulates proliferation of osteoblastic cells induces
rapid degradation of p57Kip2 through the
proteasome-dependent pathway (5).
(reviewed in Refs. 6-8). TGF-
exerts
its effect via two types of serine/threonine kinase receptors: TGF-
binds first to the type II receptor, which consequently activates the
type I receptor by direct association. Signals from the activated type
I receptor are known to be transmitted into the nucleus through various
mediator molecules. Among these, the best characterized molecules are a
family of proteins termed Smad (reviewed in Refs. 9-11). To date,
eight different Smads have been identified in mammals, and these are
classified into three subgroups, i.e. the receptor-regulated
Smads (R-Smads), the common-partner Smads (Co-Smads), and the
inhibitory Smads (I-Smads). R-Smads are directly phosphorylated by type
I receptors, then complexed with Co-Smads, and ultimately translocate
into the nucleus. The R-Smad/Co-Smad heterooligomers are capable of
binding to DNA directly or indirectly via other DNA-binding proteins
and thus regulate positively or negatively the transcription of a
multitude of target genes. In contrast, I-Smads inhibit the
phosphorylation of R-Smads by activated type I receptors by interfering
with their association, leading to prevention of the assembly of
R-Smads with Co-Smads. In the TGF-
signaling pathway, Smad2 and
Smad3 function as R-Smads, Smad4 as a Co-Smad, and Smad7 as an I-Smad
(9-11).
1. To this end, we used
adenovirus-based vectors able to obtain high efficiencies of
transfection in primary cell culture for expressions of various mediator proteins which are known to locate downstream of TGF-
1 signaling. We report here that proteasomal degradation of
p57Kip2 in osteoblastic cells derived from the calvariae of
newborn mice treated by TGF-
1 is mediated by Smad proteins and that
the TGF-
1-induced p57Kip2 proteolysis is regulated at
the transcriptional level.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (R & D Systems Inc., Minneapolis, MN); MG132
(Z-Leu-Leu-Leu-aldehyde; Peptide Institute, Inc., Osaka, Japan);
actinomycin D and
-amanitin (Sigma);
-minimal essential medium
(
-MEM; Life Technologies, Inc., Grand Island, NY); fetal calf serum
(JRH Biosciences, Lenexa, KS); antibiotics-antimycotic (Life
Technologies, Inc.; 100 units/ml of penicillin G, 100 units/ml of
streptomycin, 0.1 µg/ml of amphotericin B); mouse monoclonal anti-HA
antibody (Berkeley Antibody Co., Richmond, CA); mouse monoclonal
anti-FLAG M2 antibody (Sigma). Polyclonal antibodies against mouse
p27Kip1 and p57Kip2 were raised in rabbits
using synthetic peptides corresponding to the C-terminal amino acids of
each protein (5).
-MEM containing 10% fetal
calf serum (FCS) and antibiotics-antimycotic. Cells at the second
passage were frozen to stock for each experiment.
-MEM containing 10% FCS for 16 h. The culture
medium was then changed into
-MEM containing 0.5% FCS, and the
cells were cultured under serum starvation for 48-72 h before TGF-
1
treatment or viral infection.
type I receptor
(ALK-5), FLAG-tagged Smads, and
-galactosidase (LacZ) cDNAs were
constructed as described previously (14). Infection of the recombinant
adenoviruses was performed at a multiplicity of infection (m.o.i.) of
50-100 plaque forming units/cell. More than 80% of the cells were
infected as determined by staining of the cells for
-galactosidase.
The expression of recombinant proteins with these adenoviruses was
obtained at the peak about 3 days after infection, so we cultured
osteoblastic cells for 3 or 4 days after viral infection prior to use.
-MEM without methionine/cystein (Sigma)
containing 0.5% FCS, then chased with
-MEM containing 0.5% FCS
with 0.4 mg/ml methionine for the time periods indicated in the
presence or absence of TGF-
1 (1 ng/ml). Cells were lysed in Nonidet
P-40 lysis buffer as described previously (5) and the soluble extracts
were subjected to immunoprecipitation with polyclonal
anti-p57Kip2 antibody. The resulting
immunoprecipitates were analyzed by SDS-PAGE and autoradiography, and
the p57Kip2 band was quantified with an image analyzer
(Fujix BAS 2000).
-32P]dCTP using
the multiprime labeling kit (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in Mouse
Osteoblastic Cells--
Previously, we reported that
p57Kip2 was degraded by TGF-
1 stimulation through the
ubiquitin-proteasome pathway using rat osteoblastic cells (5). To
further explore the mechanism of TGF-
1 signaling for
p57Kip2 proteolysis, we used various adenovirus vectors
carrying Smad cDNAs and osteoblastic cells isolated from the
calvariae of newborn mice (14). At first, we tested whether or not
TGF-
1 induces p57Kip2 degradation in the mouse
osteoblastic cells as in rat cells. As shown in Fig.
1, p57Kip2 accumulated in
serum-starved mouse osteoblastic cells. Treatment with TGF-
1
resulted in almost complete loss in the cellular level of
p57Kip2 during 16-24 h. On the other hand,
p27Kip1 remained unchanged irrespective of TGF-
1
stimulation, although its cellular level slightly increased due to
serum deprivation.
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Fig. 1.
TGF- 1 reduces the
level of p57Kip2, but not p27Kip1, in mouse
osteoblastic cells. Mouse osteoblastic cells that had been
cultured in serum-starved media for 72 h were further incubated
for various times as indicated in the presence of TGF-
1 (1 ng/ml).
Whole cell lysates were subjected to SDS-PAGE, followed by immunoblot
analysis using anti-p57Kip2 (upper panel) and
anti-p27Kip1 (lower panel) antibodies. Control
represents the lysates of the cells without serum deprivation.
1 without affecting
the p57Kip2 level in the absence of TGF-
1 (Fig.
2), suggesting that TGF-
1 induces the
instability of p57Kip2. To confirm directly this
assumption, we conducted pulse-chase analysis. As shown in Fig.
3, endogenous
[35S]p57Kip2 metabolically labeled
with [35S]Met and [35S]Cys under
serum-starved conditions was rapidly disappeared with an apparent
half-life of 8-10 h when cells were cultured in the presence of
TGF-
1, whereas [35S]p57Kip2 was fairly
stable without treatment with TGF-
1, indicating that TGF-
1
certainly promotes the degradation of p57Kip2. MG132
treatment of these cells also appreciably increased the p27Kip1 level (Fig. 2), which is consistent with previous
reports that the degradation of p27Kip1 is processed in a
proteasome-dependent manner in a variety of cells (16).
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Fig. 2.
A proteasome inhibitor MG132 prevents the
degradation of p57Kip2 in osteoblastic cells stimulated by
TGF- 1. Serum-starved osteoblastic cells
(see Fig. 1) were cultured for 24 h in the presence (+) or absence
(
) of TGF-
1 (1 ng/ml). A proteasome inhibitor MG132 was added for
12 h at the final concentration of 2.5 µM prior to
harvesting of the cells. The immunoblot analysis was carried out as for
Fig. 1. All experiments were conducted in duplicate.
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Fig. 3.
Pulse-chase analysis of endogenous
p57Kip2 in osteoblastic cells. Cells
were cultured in serum-starved media containing [35S]Met
and [35S]Cys for 16 h, then chased in the presence
or absence of TGF- 1 (1 ng/ml) as a function of time. The
immunoprecipitated [35S]p57Kip2 was subjected
to SDS-PAGE and autoradiography (upper panel). Relative
intensities of p57Kip2 bands determined with an image
analyzer are shown (lower panel). The data show typical
results of three independent experiments which were essentially the
same.
1 affects the synthesis of p57Kip2,
we measured the mRNA level of p57Kip2 after TGF-
1
stimulation by Northern blot analysis. As shown in Fig.
4, the amount of p57Kip2
mRNA slightly decreased after 24 h and thus TGF-
1 may
decrease the p57Kip2 synthesis, affecting partly the level
of p57Kip2 in osteoblastic cells. In addition, the
possibility that TGF-
1 also regulates p57Kip2 at the
translational level cannot be completely ruled out. Nevertheless, considering almost complete loss of cellular p57Kip2 level
upon TGF-
1 stimulation, it is clear that TGF-
1 predominantly regulates p57Kip2 proteolysis by the proteasome
pathway.
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Fig. 4.
The effect of TGF- 1
on the level of p57Kip2 mRNA in osteoblastic
cells. The experimental conditions were the same as for Fig. 1.
Total RNAs were prepared at various times as indicated. The level of
p57Kip2 mRNA (upper panel) was measured by
Northen blot analysis. The amounts of loaded RNAs were checked by
ethidium bromide fluorescent staining of 28 S and 18 S ribosomal RNAs
(lower panel). All experiments were conducted in
triplicate.
Type I Receptor--
It is conceivable that
TGF-
1 binds first to the type II receptor, which consequently
activates the type I receptor (6-8). Of seven different type I
receptors of the TGF-
superfamily (originally termed activin
receptor-like kinase (ALK)-1 to ALK-7), ALK-5 is the type I receptor
for TGF-
(17, 18). Therefore, we next examined whether a
constitutively active form of ALK-5 operates the down-regulation of
p57Kip2 in osteoblastic cells. Replacement of Thr at
position 204 in ALK-5 by acidic amino acids such as Asp (termed
ALK-5(TD)) leads to constitutive activation of the type I receptor
without ligands or type II receptor (19). Thus, we expressed HA-tagged
ALK-5(TD) using an adenoviral vector in serum-starved osteoblastic
cells. As shown in Fig. 5 (upper
panel), p57Kip2 was decreased, depending on the
increment of the ALK-5(TD) protein (lower panel), and
disappeared after 72 h viral infection. However, no obvious change
in the p57Kip2 level was found in the osteoblastic cells
infected with control vector that carries a
-galactosidase (LacZ)
cDNA or nothing. The level of p27Kip1 remained
unchanged, irrespective of the expression of the ALK-5(TD) protein
(Fig. 5, middle panel). Thus, it was concluded that
p57Kip2 is surely degraded through the downstream signaling
pathway of the TGF-
type I receptor.
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Fig. 5.
The degradation of p57Kip2 is
accelerated by forced expression of the constitutively active form of
TGF- type I receptor. Serum-starved
osteoblastic cells (see Fig. 1) were infected for various times as
indicated with adenoviruses carrying the constitutively active form of
human TGF-
type I receptor (ALK-5(TD)-HA, abbreviated simply as
ALK5) cDNA at a m.o.i. of 100. The cells infected with adenoviruses
carrying
-galactosidase (LacZ) or nothing were analyzed similarly as
controls. Serum starvation was continued after viral infection. The
immunoblot analysis was carried out as for Fig. 1, except that the
expression of a HA-tagged ALK-5(TD) protein was measured by reblotting
the same membrane with anti-HA antibody (bottom panel). Some
experiments were conducted in duplicate.
1--
To date, it becomes clear that the Smad family
proteins are involved in the signaling pathway evoked by the TGF-
type I receptor (9-11). Therefore, we examined whether Smad-mediated
transcription accelerates the instability of p57Kip2. The
activated TGF-
type I receptor, ALK-5 phosphorylates R-Smads, such as Smad2 and Smad3, whereas an I-Smad, such as a Smad7,
interferes with the phosphorylation of Smad2 and Smad3 by preventing
their interaction with ALK-5. If the Smad pathway is involved in the down-regulation of p57Kip2, I-Smad would suppress the
reduction of p57Kip2 induced by TGF-
1. When mouse
osteoblastic cells were infected with the adenoviruses carrying the
Smad7 cDNA prior to TGF-
1 treatment, the
signal-dependent breakdown of p57Kip2 was
considerably inhibited (Fig.
6A). In addition, the similar expression of Smad7 also prevented the decrease in p57Kip2
induced by ALK-5(TD) (Fig. 6B). However, the
p27Kip1 level showed no significant change in these
conditions.
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Fig. 6.
An inhibitory Smad7 blocks the
accelerated degradation of p57Kip2 induced by
TGF- 1 or ALK-5(TD). A, the
osteoblastic cells that had been cultured in serum-starved media for
48 h were infected for 72 h with adenoviruses carrying Smad7
cDNA at a m.o.i. of 50 and further incubated in the presence (+) or
absence (
) of TGF-
1 (1 ng/ml) for 24 h. Control experiments
were performed as for Fig. 5. The immunoblot analysis was carried out
as for Fig. 1, except that the expression of FLAG-tagged Smad7 was
measured by reblotting the same membrane with anti-FLAG M2 antibody
(bottom panel). All experiments were done in duplicate.
B, the effect of Smad7 on p57Kip2 proteolysis
induced by ALK-5(TD) was examined as described in A, except
that adenovirus carrying the constitutively active form of human
TGF-
type I receptor (ALK-5(TD)-HA) was infected as described in the
legend to Fig. 5.
1 incapable of promoting p57Kip2 proteolysis (Fig.
7, left lane) was added for
24 h after viral infection, which might be required for
phosphorylation of expressed Smad2 and Smad3. When Smad2 or Smad3 was
coexpressed with Smad4, p57Kip2 disappeared in the
osteoblastic cells infected with adenoviruses carrying these Smads
(Fig. 7). The forced expression of Smad7 prevented the decrease in
the p57Kip2 level induced by coexpressions of Smad2 or
Smad3 with Smad4. No significant change in the p57Kip2
level was observed in the control expressing LacZ. These results strongly indicate that the degradation of p57Kip2 by
TGF-
1 is mediated through the Smad2/4 or Smad3/4 pathway. In
contrast, the p27Kip1 level remained unchanged,
irrespective of coexpression in any given combinations of Smads (Fig.
7).
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Fig. 7.
The effect of forced expressions of various
Smad proteins on the stability of p57Kip2 and
p27Kip1. Serum-starved osteoblastic cells (see Fig. 6)
were infected for 48 h with Smad2/4 or Smad3/4 with or without
Smad7 as indicated (m.o.i. of 100 for each). A low concentration of
TGF- 1 (0.03 ng/ml) was added for 24 h after viral infection
which had no effect of the level of p57Kip2. Adenoviruses
carrying
-galactosidase (LacZ) at the same m.o.i. were used as a
control. The immunoblot analysis was carried out as for Fig. 1, except
that the expression of FLAG-tagged Smad2, Smad3, Smad4, and Smad7 was
measured by reblotting the same membrane with anti-FLAG M2 antibody
(bottom panel).
1, the
p57Kip2 level was not affected, however, the
TGF-
1-induced degradation of p57Kip2 was blocked (Fig.
8A), implying that a
protein(s) newly synthesized by TGF-
1 stimulation is involved in the
proteolytic elimination of p57Kip2. The similar suppressive
effect for the TGF-
1-induced decrease of p57Kip2 was
observed by treatment with another transcription inhibitor,
-amanitin (Fig. 8B). Considering the fact that the
p57Kip2 level remained unchanged for 24 h during
exposure to actinomycin D or
-amatinin (Fig. 8), p57Kip2
is found to be apparently stable without TGF-
1 stimulation (see "Discussion"). This also indicates that the disappearance of
p57Kip2 by TGF-
1 treatment is not attributed to the
down-regulation of the p57Kip2 production. Taken together,
these results suggest that Smad-mediated transcription is involved in
the induction of p57Kip2 proteolysis, which may play an
essential role in the growth control of osteoblastic cells in
vivo.
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Fig. 8.
The effect of actinomycin D
(A) and -amanitin
(B) on the level of p57Kip2 and
p27Kip1 in the presence or absence of
TGF-
1. Serum-starved osteoblastic cells
(see Fig. 1) were cultured for 24 h with (+) or without (
)
TGF-
1 (1 ng/ml) in the presence (+) or absence (
) of actinomycin D
(abbreviated Act. D) (0.1 µg/ml) or
-amanitin (5 µg/ml). The immunoblot analysis was carried out as for Fig. 1. All
experiments were conducted in duplicate.
-amatinin caused disappearance
of p27Kip1, irrespective of TGF-
1 stimulation (Fig. 8).
These results indicate that p27Kip1, unlike
p57Kip2, are rapidly turned over.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 causes rapid degradation of
p57Kip2 in osteoblastic cells isolated from the calvariae
of rat fetuses that had been serum-starved to induce the cellular
p57Kip2 level (5). The process was inhibited by various
proteasome inhibitors, such as lactacystin and MG132, and a
polyubiquitylation of p57Kip2 was detected in an in
vitro assay using TGF-
1-treated cell extracts, strongly
indicating that the ubiquitin-proteasome pathway operates in the
degradation of p57Kip2. In the present work, first, we
observed the proteasomal degradation of p57Kip2 in mouse
osteoblastic cells induced to proliferate by TGF-
1 (Figs. 1 and 2).
Thus, the TGF-
1-dependent selective degradation of
p57Kip2 in mouse osteoblastic cells was essentially the
same as rat cells, except that the degradation of p57Kip2
in the latter cells was considerably faster compared with that in
former cells for unknown reasons.
1 is mediated by the
TGF-
type I receptor, because forced expression of the
constitutively active form of the TGF-
type I receptor ALK-5(TD)
resulted in the accelerated degradation of p57Kip2
analogous to TGF-
1 stimulation (Fig. 5), and because one member of
the I-Smads, Smad7, blocked its degradation induced by TGF-
1 or
ALK-5(TD) (Fig. 6). Third, we observed a stimulatory effect on the
down-regulation of p57Kip2 when Smad2 or Smad3 was
expressed simultaneously with Smad4 under the low concentration of
TGF-
1 (Fig. 7). We also observed that Smad7 greatly suppressed the
down-regulation of p57Kip2 induced by coexpressions of
Smad2 or Smad3 with Smad4. These findings strongly indicate that the
Smad pathway mediates the degradation of p57Kip2 in
osteoblastic cells stimulated to proliferate by TGF-
1. However, the
possibility that the TGF-
type I receptor also activates Smad-independent pathways, i.e. extracellular
signal-regulated kinase/mitogen-activated protein (MAP) kinase, c-Jun
N-terminal kinase/serum-activated protein kinase, and p38 MAP kinase
pathways (20-24) to cause p57Kip2 degradation cannot be
completely excluded, because we have not yet examined the effect of the
compounds capable of inhibiting these pathways. Nonetheless we favor
the idea that the Smad pathway plays a central role in the
destabilization of p57Kip2, because coexpression of
R-Smad2/3 and Co-Smad4 mimicked the effect of TGF-
1 treatment
causing loss of p57Kip2 and because loss of
p57Kip2 induced by these R- and Co-Smads, like TGF-
1
stimulation, was suppressed by I-Smad, Smad7. In the present
experimental conditions, we have added a low concentration of TGF-
1
(0.03 ng/ml) that was insufficient to induce p57Kip2
proteolysis for activation of Smad2 and Smad3. Thus, Smad7 blocks competitively for the binding of Smad2 or Smad3 to the activated TGF-
type I receptor, although the possibility that Smad7 directly suppresses the binding of Smad2 or Smad3 to Smad4 cannot be completely ruled out. Whatever Smad7 acts, it is clear that the Smad pathway is
involved in p57Kip2 proteolysis induced by TGF-
1.
1-induced degradation of
p57Kip2 was abrogated by actinomycin D (Fig. 8A)
or
-amanitin (Fig. 8B), which had no effect on the
p57Kip2 level per se. The effect of these
transcriptional inhibitors is quite interesting in considering the
mechanistic insights of how TGF-
1 accelerates the degradation of
p57Kip2. It is well known that the TGF-
/Smad signaling
system acts as transcriptional factors capable of activating a diverse
spectrum of target genes (9-11). According to this scenario, a newly
synthesized protein(s) may affect the stability of p57Kip2.
For this, two possible mechanisms can be considered. One possibility is
that the presumptive new protein may modify the p57Kip2
protein, which directs p57Kip2 to the ubiquitin-proteasome
machinery. To date, various signals are known to act as a degradation
signal for a multitude of cellular proteins (reviewed in Ref. 25).
Indeed there is accumulating evidence addressing the importance of
phosphorylation of most target molecules involved in the cell-cycle
progression or signal transduction, such as p27Kip1, cyclin
E, I
B, and
-catenin, as a prerequisite for their ubiquilation and
subsequent proteasomal degradation (reviewed in Refs. 26-28). Therefore, it is likely that p57Kip2 is also phosphorylated
prior to its ubiquilation, although phosphorylation of
p57Kip2 has not yet been reported so far. Moreover, to our
knowledge, there is no available information that p57Kip2
is modified post-translationally in other ways, such as acetylation or
oxidation. Further study is required to clarify whether
p57Kip2 is actually modified in response to TGF-
1 stimulation.
1 might belong to E3, which
selectively targets p57Kip2 for ubiquilation. Consistent
with this notion, recently it was reported that an F-box protein acting
as a substrate recognition module of a large multisubunit
ubiquitin-ligase called SCF (skp1-cdc53 or a cullins-F-box protein
complex) is extremely unstable and regulated at transcriptional level
(29-31). In other cases, for example, the anaphase-promoting complex
or cyclosome E3·ligase complex is known to be controlled by
phosphorylation during the M-phase traverse of the cell cycle (32).
Therefore, TGF-
1 may affect the activity of a specific E3 capable of
ubiquilating p57Kip2. To determine the mechanism underlying
this hypothesis, it is essential to search for the E3-ligase
responsible for ubiquilation of p57Kip2, which is in progress.
1 to such nongrowing cells induces down-regulation of
p57Kip2, strongly indicating the importance of
p57Kip2 proteolysis in regulating proliferation and
possible differentiation of osteoblastic cells. This notion is also
supported by the findings that MG132 prevented the TGF-
1-induced
loss of p57Kip2 (Fig. 2) and TGF-
1 had a little effect
on the level of the p57Kip2 mRNA (Fig. 4), indicating
that TGF-
1 affects primarily the degradative process of
p57Kip2. Moreover, actinomycin D or
-amanitin showed no
appreciable alteration of the level of p57Kip2 without
TGF-
1 stimulation, suggesting that p57Kip2 is fairly
stable under nondividing conditions (Fig. 8). This assumption was
ascertained by pulse-chase analysis of metabolically labeled
[35S]p57Kip2. p57Kip2 did not
disappear appreciably in the absence of TGF-
1, while it was rapidly
degraded with an apparent half-life of 8-10 h under the presence of
TGF-
1 in osteoblastic cells (Fig. 3).
1 stimulation and was reduced by treatment with actinomycin D or
-amanitin irrespective of treatment with TGF-
1, indicating that turnover of p27Kip1 is rapid in osteoblastic cells (Fig.
8). Our results also indicate that regulatory mechanisms for these two
CKIs, p27Kip1 and p57Kip2, considerably differ
in osteoblastic cells. However, serum starvation also caused
accumulation of p27Kip1 to a lesser extent (Fig. 1),
indicating that it may somehow be involved in the growth control of
osteoblastic cells.
provides a variety of signals for numerous cells and
interestingly often exerts apparently contradictory effects, depending on the type of cells (6-8). For instance, it mainly suppresses the
growth in many epithelial cells, but promotes it in certain cells, such
as osteoblastic cells. The mechanism of these diverse effects remains
unknown, but it could be due to how the regulatory factors that
positively (e.g. CDKs) and negatively (e.g. CKIs) drive the cell-cycle progression are induced by the TGF-
signaling. However, the details are still unknown at present.
, which is locally produced by osteoblasts and
accumulated abundantly in bone matrix, is thought to have important roles in bone remodeling (33). It is worth noting that, among the gene
knockout studies of the Cip/Kip family proteins, only p57Kip2-deficient mice showed developmental abnormalities
(3, 4). 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. Therefore, it is tempting to speculate that p57Kip2 can be
involved in the regulation of cell growth and differentiation of cells
of a certain cell lineage, including osteoblasts and chondrocytes. In
considering these in vivo roles of p57Kip2, the
new regulatory system of p57Kip2 by TGF-
signaling
linked to proteolysis sheds new light on the mechanisms underlying the
development and/or differentiation of osteoblasts and perhaps certain
other cells in vivo.
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ACKNOWLEDGEMENTS |
---|
We are grateful to all members of the Tanaka laboratory for helpful advice and discussion.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the program grants-in-aid for Scientific Research on Priority Areas (Intracellular Proteolysis) from the Ministry of Education, Science, Sports, and Culture of Japan (to K. T.) and by a Research Grant from the Princess Takamatsu Cancer Research Fund, the Toray Science Foundation, and the Uehara Memorial Foundation (to H. T.).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. Fax: 81-3-3823-2237; E-mail: tanakak@rinshoken.or.jp.
Present address: Institute of Clinical Medicine,
University of Tsukuba, 1-1-1, Tennodai, Tsukuba-city, Ibaraki 305-8575, Japan.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M007499200
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ABBREVIATIONS |
---|
The abbreviations used are:
CDK, cyclin-dependent kinase;
CKI, cyclin-dependent
kinase inhibitory protein;
TGF-1, transforming growth factor-
1;
R-Smad, receptor-regulated Smad;
I-Smad, inhibitory Smad;
Co-Smad, common-partner Smad;
-MEM,
-minimal essential medium;
FCS, fetal
calf serum;
HA, hemagglutinin;
m.o.i., multiplicity of infection;
PAGE, polyacrylamide gel electrophoresis;
E3, ubiquitin-protein ligase;
ALK, activin receptor-like kinase;
Kip, kinase inhibitory protein;
Cip, CDK
interacting protein.
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