From the Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas 78229-3900
Received for publication, April 21, 2003
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ABSTRACT |
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INTRODUCTION |
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Cbfa12 is a transcription factor that belongs to the runt-domain gene family and plays an essential role in osteoblast differentiation, bone development, and postnatal bone formation (25). Its expression and activity are regulated by many bone-derived growth factors, including BMPs (2, 6). BMPs may stimulate osteoblast differentiation by activating Cbfa1 through Smad1, as evidenced by reports that Smad1, the downstream effector of BMP signaling, directly interacts with Cbfa1 (7), but precise signaling mechanisms remain unclear.
The activities of signaling proteins and transcription factors are regulated at both the transcriptional and post-translational levels. Recent reports (810) demonstrate that both Smad1 and Cbfa1 undergo ubiquitin-proteasome-mediated degradation. Protein ubiquitination involves a cascade of enzymatic reactions catalyzed by the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin ligases (11), which play a crucial role in defining substrate specificity and subsequent protein degradation by the 26 S proteasomes. Smurf1 is a member of the Hect family of E3 ubiquitin ligases and has been found to interact with the BMP-activated Smad1 and -5, thereby triggering their ubiquitination and degradation (8).
Hect domain proteins represent a major subclass of E3 ligases and contain a
conserved cysteine located at the carboxylterminal of the Hect domain that is
capable of forming a thioester bond with ubiquitin
(8,
12). Another motif often found
in the Hect family of E3 ligase is the WW domain, which contains two highly
conserved tryptophans and a conserved proline in an 30-amino acid region
(8,
13). The WW domains have a
preference for binding to small proline-rich sequences, PPXY motifs,
and different WW domains possess different substrate specificity. Although
Cbfa1 has been reported to be degraded through the ubiquitin-proteasome
pathway (10), the specific E3
ubiquitin ligase for Cbfa1 has not been identified.
In the present studies, we examined the relationship between Smad1, Cbfa1, and Smurf1 in osteoblast precursor cells. Smad1 interacts directly with Cbfa1 in these cells, and both Smad1 and Cbfa1 are required for BMP-2 signaling. The intracellular concentrations of both Smad1 and Cbfa1 are dependent on osteoblast proteasomal function. Smurf1 mediates Cbfa1 degradation in a ubiquitin proteasome-dependent manner and therefore is a powerful regulator of osteoblast differentiation.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and TransfectionsC2C12 cells were cultured in
Dulbecco's modified Eagle's medium, and 2T3 osteoblast precursor cells were
cultured in -minimal essential medium supplemented with 8% fetal calf
serum. The cDNA expression plasmids were transiently transfected into these
cells using LipofectAMINE Plus reagents (Invitrogen) in a 10-cm-diameter
culture dish for immunoprecipitation assay, 6-well culture plates for Western
blotting analysis, and 24-well plates for the luciferase assay. 5, 1, and 0.2
µg of expression plasmid were used for these transfections. After
transfection (48 h), cells were lysed with lysis buffer. In proteasome
inhibitor experiments, transfected cells were incubated with the proteasome
inhibitors for 2, 6, or 24 h, the proteasome inhibitor was removed, and the
cells were then cultured an additional 24 h.
Immunoprecipitation and ImmunoblottingExpression plasmids
were transfected into C2C12 cells separately or together for
immunoprecipitation. In Western analysis, the total amounts of transfected
plasmids in each group were equalized by addition of empty vector cDNA. 48 h
after transfection, cells were washed three times with phosphate-buffered
saline and solubilized in lysis buffer (150 mM NaCl, 1% Triton
X-100, 0.5% doc, and 50 mM Tris buffer, pH 7.5). For Western
blotting, 0.1% SDS was included in the lysis buffer. The protease inhibitors
aprotinin (10 µg/ml), leupeptin (10 µg/ml), and phenylmethylsulfonyl
fluoride (1 mM) were added to the lysis buffer. Cell lysates were
centrifuged for 10 min at 4 °C at 10,000 x g and incubated
with anti-Myc antibody for 4 h at 4 °C, followed by immunoprecipitation
with protein G-agarose (Roche Applied Science) at 4 °C overnight.
Immunoprecipitates were washed with lysis buffer five times, added to 1x
reducing buffer containing 0.5 M -mercaptoethanol, and boiled
for 3 min. The immunoprecipitation and Western blotting samples were separated
by SDS-PAGE, transferred to nitrocellulose membrane, immunoblotted with
anti-FLAG or anti-Myc antibody, and visualized with horseradish
peroxidase-coupled anti-mouse IgG antibody (Amersham Biosciences) with an
enhancement by ECL detection kits (Amersham Biosciences). A monoclonal
antibody against human Smad1 and a polyclonal antibody against human Smurf1
were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. A polyclonal
antibody against human Cbfa1 was purchased from Oncogene Research Product,
Cambridge, MA.
Luciferase AssayOligonucleotides containing 6 copies of
Cbfa1 response element, OSE2, (6xOSE2) and 12 copies of Smad1 binding
element (12xSBE) were synthesized in the DNA laboratory at The
University of Texas Health Science Center at San Antonio. The oligonucleotides
were cloned in front of the osteocalcin basal promoter (155/+1), which
was amplified by RT-PCR, and cloned in pGL3 vector. C2C12 and 2T3 cells were
co-transfected with Cbfa1 expression plasmid and 6xOSE2-OC-pGL3 reporter
plasmid in the presence or absence of Smurf1 or mutant Smurf1 expression
plasmid. caBMPR-IB and Smad1 expression plasmids were co-transfected with
12xSBE-OC-luc or 12xSBE-SV40-luc in the presence or absence of
mutant Cbfa1. Cell lysates were extracted 48 h later, and luciferase activity
was measured and normalized by -galactosidase activity.
Alkaline Phosphatase (ALP) Activity and Osteocalcin Production C2C12 and 2T3 cells were plated into 6-well culture plates grown to 60% confluency, transfected transiently with Smurf1 or mutant Smurf1 expression plasmids using LipofectAMINE Plus reagents (Invitrogen), or treated with proteasome inhibitors. 48 h after transfection, the medium was collected; the cells were washed twice with ice-cold phosphate-buffered saline, and cell lysates were extracted with 0.05% Triton X-100. ALP activity in cell lysates was measured using a Sigma ALP assay kit (Sigma), and osteocalcin in the medium was measured with a mouse osteocalcin immunoradiometric assay (IRMA) kit (Immutopics, Inc., San Clemente, CA).
Calvarial Periosteal Bone Formation AssayProteasome inhibitor 1 (PS1) was injected subcutaneously over the parietal bone of the calvariae of 2-month-old ICR Swiss mice (14). Mice received either vehicle (20 µl of phosphate-buffered saline) alone or PS1 (0.2, 1, and 5 mg/kg/day, daily for 5 days). Mice were sacrificed 14 days after commencing injections, and calvarial bones were removed. The bones were decalcified in 14% EDTA, bisected coronally midway between the coronal and lambdoid sutures, dehydrated through graded alcohols, and embedded in paraffin. Transverse 3-µm-thick sections were cut and stained with Hematoxylin and Eosin. Newly formed bone was identified histologically using an E400 microscope (Nikon Inc., Melville, NY) linked to a color video monitor (PVM-14M2MDU Trinitron, Sony Corp., Japan) and image capture techniques.
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RESULTS AND DISCUSSION |
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To determine whether Smurf1-mediated degradation of Cbfa1 is ubiquitin-dependent, we co-transfected Smurf1, Cbfa1, and ubiquitin expression plasmids in C2C12 cells. After immunoprecipitating the Cbfa1 protein, a typical ubiquitinated Cbfa1 protein ladder was observed (Fig. 1c), indicating that Smurf1-mediated Cbfa1 degradation is ubiquitin-dependent. To examine whether Smurf1-mediated degradation of Cbfa1 is proteasome-dependent, we treated transfected C2C12 cells with 1 µM PS1 for 2 h. Expression of Smurf1 resulted in total degradation of Cbfa1, an effect completely abolished by treatment with PS1 (Fig. 1d). Similar results were obtained when another structurally different proteasome inhibitor, epoxomicin, was used (data not shown). These results demonstrate that Smurf1-mediated Cbfa1 degradation is proteasome-dependent.
To determine whether Smurf1 physically interacts with Cbfa1, Cbfa1, and
mutant Smurf1 (Smurf1, C710A), expression plasmids were co-transfected
into C2C12 cells. Because it is known that Smurf1 mediates the degradation of
proteins with which it interacts
(8), we attempted to stabilize
the protein complex with Smurf1 using an expression plasmid of the catalytic
point mutant of Smurf1 (
Smurf1) as described by Zhu et al.
(8). Using epitope-tagged
proteins in co-immunoprecipitation and Western blot analyses, we found that
Cbfa1 co-precipitates with
Smurf1
(Fig. 1e). A putative
PY motif, presumably bound with the WW domain of Smurf1 protein, was found in
the carboxyl-terminal of Cbfa1, suggesting that Smurf1 directly binds Cbfa1
and mediates its degradation.
To determine whether Smurf1-mediated Cbfa1 degradation causes functional
changes in Cbfa1, we examined the ability of Cbfa1 to activate a specific
reporter gene, 6xOSE2-OC-Luc
(15), in C2C12 cells. A Cbfa1
expression plasmid and 6xOSE2-OC-Luc reporter construct were
co-transfected into C2C12 cells in the presence and absence of Smurf1 or
Smurf1. Expression of Cbfa1 significantly increased activity of
6xOSE2-OC-Luc reporter, whereas co-transfection of Smurf1 with Cbfa1
significantly reduced Cbfa1-induced luciferase activity of this reporter gene.
In contrast, co-transfection of
Smurf1, lacking in catalytic activity,
had no significant effect on Cbfa1-induced luciferase activity of the reporter
gene (Fig. 1f). These
results demonstrate that Smurf1 mediates Cbfa1 degradation and leads to a
decrease in Cbfa1 activity. That there was no significant effect of
Smurf1 on Cbfa1-induced luciferase activity of the reporter gene may be
because activation of the reporter gene by expressed Cbfa1 had already
plateaued.
Because it has been reported that Cbfa1 degradation is cAMP-dependent in
osteoblasts (10), in the
present studies we also treated C2C12 cells with forskolin, an agent that
stimulates adenylyl cyclase and examined expression of endogenous Smurf1,
Cbfa1 degradation, and the effect of Smurf1 on forskolin-induced Cbfa1
degradation. Although forskolin treatment increased endogenous Smurf1 level
only slightly (Fig.
2a), Cbfa1 protein level was significantly reduced by
forskolin; this reduction was blocked in cells transfected with
Smurf1
(Fig. 2b). These
results suggest that Smurf1 may be involved in the cAMP-induced Cbfa1
degradation.
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Smurf1 Mediates Smad1 Degradation in OsteoblastsAlthough Smurf1 has been shown to mediate Smad1 degradation in COS cells transfected with a Smad1 expression plasmid, the effects of Smurf1 on Smad1 degradation in osteoblasts, and in particular degradation of endogenous Smad1 protein by proteasomal-dependent mechanism in osteoblasts, have not been examined. In the present studies, we confirmed the effect of Smurf1 on Smad1 degradation in osteoblast precursor cells and examined the regulatory roles of proteasome inhibitors on Smad1 protein. We first examined the effect of Smurf1 on steady-state protein levels of Smad1 in C2C12 and 2T3 cells and found that Smurf1 mediates Smad1 degradation in a dose-dependent manner (Fig. 3a). The effect of Smurf1 on Smad1 degradation is ubiquitin-proteasome-dependent because the catalytic mutant of Smurf1 (C710A) had only minor effects on Smad1 degradation (Fig. 3b) and the proteasome inhibitor epoxomicin reversed the effect of Smurf1 on Smad1 degradation in a dose-dependent manner (Fig. 3c). Similar effects were observed when other structurally different proteasome inhibitors such as PS1, lactacystin, and MG-132 were utilized (data not shown). To further determine the effects of proteasome inhibitors on the steady-state protein levels of endogenous Smad1, the C2C12 cells were treated with different proteasome inhibitors for 6 h. The treatment with proteasome inhibitors increased Smad1 levels 2 6-fold (Fig. 3d), demonstrating that Smad1 is under Smurf1-mediated ubiquitin-proteasome regulation in osteoblasts.
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Cbfa1 Is Required for Activation of BMP-2 SignalingCbfa1
has been reported to interact with Smad1, but details of the transactivation
mechanism of these two signaling molecules have not been clearly defined.
Therefore, we examined whether Cbfa1 is specifically required for BMP
signaling by first confirming the interaction of Cbfa1 with Smad1 in
osteoblast precursor cells. Epitope-tagged Cbfa1 and Smad1 expression plasmids
were co-transfected into C2C12 and 2T3 cells. Co-precipitation of Cbfa1 and
Smad1 was detected in both cells (Fig.
4a). To determine the role of Cbfa1 in BMP signaling, we
generated multiple copies of Smad1 response element (12xSBE) and cloned
those upstream of an osteocalcin and a SV40 basal promoter. Treatment of BMP-2
or expression of caBMPR-IB stimulated luciferase activity of the
12xSBE-OC-Luc reporter but not the 12xSBE-SV40-Luc reporter
(Fig. 4b). Because the
osteocalcin basal promoter contains one copy of the Cbfa1 response element
(OSE2), we next examined whether binding of Cbfa1 to this response element in
the osteocalcin basal promoter is required for BMP-2 signaling. A mutant Cbfa1
(Cbfa1) expression plasmid was generated as described by Ducy et
al. (5), and C2C12 cells
were transfected with caBMPR-IB expression plasmid and the 12xSBE-OC-Luc
reporter construct with or without
Cbfa1. Expression of
Cbfa1
completely blocked the effect of caBMPR-IB on the 12xSBE-OC-Luc reporter
(Fig. 4c). caBMPR-IB
or BMP-2 had only minor effects on the osteocalcin basal promoter (data not
shown). To further define the role of Cbfa1 in BMP signaling, the OSE2 site in
the osteocalcin basal promoter was mutated. The 12xSBE-OC-Luc reporter
with OSE2 mutation (12xSBE-
OC) completely lost its response to
BMP-2 or caBMPR-IB (Fig.
4d). These results suggest that Cbfa1 is required for
BMP-2 and Smad1 to activate downstream target genes in osteoblasts.
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Function of Smurf1 in Osteoblast DifferentiationTo examine
the function of Smurf1 in osteoblast differentiation, a Smurf1 or
Smurf1 expression plasmid was transfected into C2C12 cells, and ALP
activity and osteocalcin production were measured. Expression of Smurf1
significantly reduced ALP activity as well as osteocalcin production in C2C12
cells (data not shown). In contrast, transfection of
Smurf1 increased
basal as well as BMP-2-induced ALP activity and osteocalcin production in
these cells (Fig. 5, a and
b). Similar results were obtained when the same
expression plasmids were transfected into 2T3 cells (data not shown). These
results suggest that Smurf1 mediates Cbfa1 and Smad1 degradation and inhibits
function of Cbfa1 and Smad1.
Smurf1 acts in a dominant-negative fashion
to inhibit the degradation of Cbfa1 and Smad1.
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To establish a correlation between Smurf1-mediated Smad1 degradation and
osteoblast differentiation, we analyzed changes in endogenous Smad1 protein
after C2C12 and 2T3 cells were transfected with mutant Smurf1 or treated with
proteasome inhibitors. We found that proteasome inhibitors increase endogenous
Smad1 levels 2 6-fold (Fig.
3d). Transfection of Smurf1 causes a 5-fold
increase in protein levels of endogenous Smad1. Overexpression of Smad1
(6-fold higher than endogenous Smad1) significantly enhanced ALP activity and
osteocalcin production in C2C12 cells (Fig.
5, a and b). These results suggest that enhanced
Smad1 levels by
Smurf1 or proteasome inhibitors promote osteoblast
differentiation.
Proteasome Inhibitors Induce Osteoblast Differentiation and Bone FormationTo further determine the role of proteasome inhibitors in osteoblast function and bone formation, we examined the effects of PS1, a synthetic proteasome inhibitor, on ALP activity and the BMP signaling reporter in C2C12 cells and on periosteal bone formation in rodents in vivo. We found that in C2C12 cells, treatment with PS1 increased ALP activity (Fig. 6a) and luciferase activity of the 12xSBE-OC-Luc reporter (Fig. 6b) in a dose-dependent manner. Increasing concentrations of PS1 (0.2, 1, and 5 mg/kg/day) were injected daily for 5 days into subcutaneous tissues over the calvariae of mice. The mice were sacrificed 2 weeks after the injections, and calvariae were processed for histology. PS1 induced significant new bone formation (Fig. 6, c and d). Similar effects were also obtained when epoxomicin, a naturally occurring proteasome inhibitor, was administered in vivo (data not shown). Moreover, these proteasome inhibitors administered systemically stimulated bone formation in intact and ovariectomized mice (16). These results demonstrate that inhibition of Smurf1 and proteasome degradation can lead to increased osteoblast function and suggest that part of the mechanism of proteasome inhibitor on new bone formation may be due to regulation of intracellular protein levels of Cbfa1 and Smad1.
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We have demonstrated for the first time that 1) E3 ubiquitin ligase Smurf1 mediates Cbfa1 degradation through the ubiquitin-proteasome pathway, 2) Cbfa1 is required for BMP-2 signaling, and 3) expression of mutant Smurf1 stimulates osteoblast differentiation. These findings suggest that Smurf1 plays an important role in osteoblast differentiation by regulating Cbfa1 and Smad1 function.
Cbfa1 is a critical transcription factor in osteoblast differentiation and function (2). BMPs have been shown to stimulate osteoblast differentiation in vitro and in vivo (1), in part by up-regulating Cbfa1 expression (2, 6) and/or through direct interaction with Smad1 and Cbfa1 (7). Smad1 is a downstream mediator of BMP receptors and plays a central role in BMP receptor signaling (17). In the present studies, we found that Cbfa1 is required to activate BMP-2 signaling. It has been reported (18, 19) that Smad1 and Cbfa1 binding sites co-localize in the promoters of several bone-related genes. Taken together with our results, this suggests that Smad1 and Cbfa1 co-activate downstream target genes in osteoblasts.
Expression of mutant Smurf1 inhibits forskolin-induced Cbfa1 degradation in C2C12 cells, suggesting that Smurf1 may be involved in Cbfa1 degradation induced by a cAMP-dependent protein kinase pathway. Parathyroid hormone (PTH) induces proteasomal degradation of protein substrates in osteoblasts (20), and it has recently been reported that the anabolic effect of PTH requires Cbfa1-dependent signaling (21). We speculate that PTH may induce Cbfa1 degradation via cAMP-dependent signaling and that Smurf1 is involved in this process. However, this remains to be determined.
To maintain efficient signal transduction and gene activation, cells must precisely regulate levels of signaling proteins and transcription factors during cellular processes. Under physiological conditions, Smurf1 may mediate degradation of the Cbfa1-Smad1 protein complex, but further investigation is required for unequivocal proof. Because Smurf1 mediates degradation of two critical proteins in the BMP signaling pathway and overexpression of Smurf1 modulates osteoblast differentiation and function, we propose that Smurf1 plays an important role in bone formation in vivo. It has been shown that Smurf mutations in Drosophila lead to enhanced decapentaplegic (a homologue of BMP-2/4) signaling and downstream target gene expression (22). Our findings suggest that Smurf1 may be a critical protein regulating Cbfa1 and Smad1 functions and, ultimately, osteoblast differentiation and bone formation.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 210-614-0770 (ext. 239);
Fax: 210-614-0797; E-mail:
chend1{at}uthscsa.edu.
1 The abbreviations used are: BMP, bone morphogenetic protein; Cbfa1,
Core-binding factor 1; Smad, Sma/Mother against decapentaplegic;
Smurf1, Smad ubiquitin regulatory factor 1; PS1, proteasome inhibitor 1; SBE,
Smad1 binding element; caBMPR-IB, constitutively active type IB BMP receptor;
OC, osteocalcin; ALP, alkaline phosphatase; E1, ubiquitin-activating enzyme;
E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; HA,
hemagglutinin.
2 Other names for Cbfa1/Runx2 are PEBP2A, AML-3, and Osf-2.
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ACKNOWLEDGMENTS |
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REFERENCES |
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