CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation.

Abstract

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

Full text

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T H E J O U R N A L O F C E L L B I O L O G Y
JCB: ARTICLE
© 2008 Li et al.
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 181 No. 6 959–972
www.jcb.org/cgi/doi/10.1083/jcb.200711044
JCB
959
X. Li, M. Huang, and H. Zheng contributed equally to this paper.
Correspondence to Zhijie Chang: zhijiec@tsinghua.edu.cn
Abbreviations used in this paper: AA, ascorbic acid; ? -GP, ? -glycerophosphate;
BMP, bone morphogenetic protein; BSP, bone sialoprotein; OCN, osteocalcin;
TPR, tetratricopeptide repeat.
The online version of this article contains supplemental material.
Introduction
Runx2, a runt domain family protein, is an essential transac-
tivator for osteoblast differentiation and bone formation. In
Runx2-null mutant mice, both intramembraneous and endo-
chondral ossifi cation are absent ( Komori et al., 1997 ; Otto
et al., 1997 ). The expression of Runx2 is a milestone for
mesenchymal cells ’ commitment to osteoblasts ( Ducy et al.,
1997 ; Komori et al., 1997 ; Otto et al., 1997 ). During the dif-
ferentiation of precursor cells into osteoblasts, Runx2 is ac-
tivated and induces osteoblast marker gene expression by
binding to a cis-acting element, OSE2 ( Ducy et al., 1997 ).
Several studies indicate that Runx2 is involved in the com-
mitment and differentiation of cells in the osteoblast and
adipocyte lineages ( Chen et al., 1998 ; Gori et al., 1999 ;
Lecka-Czernik et al., 1999; Enomoto et al., 2004 ; Hong et al.,
2005 ). Multiple biological functions of Runx2 have been
demonstrated recently ( Nam et al., 2002 ; Taniuchi et al.,
2002 ; Ito and Miyazono, 2003 ; Woolf et al., 2003 ; Aberg et al.,
2004 ; Yoshida et al., 2004 ; Yoshida and Komori, 2005 ; Hinoi
et al., 2006 ; Pratap et al., 2006 ; Whiteman and Farrell, 2006 ;
Young et al., 2007 ).
Runx2 is tightly regulated at both the transcriptional and
posttranslational levels. In particular, Runx2 activity can be
regulated through a ubiquitin – proteasome-mediated protein
degradation mechanism. Smurf1 was the fi rst factor identi-
fi ed as an E3 ligase for Runx2 ubiquitination and degradation
( Zhao et al., 2003; Zhao et al., 2004 ). Smurf1-induced Runx2
degradation can be enhanced by Smad6 ( Shen et al., 2006 ) and
TNF ( Kaneki et al., 2006 ), as Smad6 interacts with Runx2 and
TNF promotes the expression of Smurf1 expression in osteo-
blasts. Recently, Schnurr-3 (Shn3) was reported to recruit the
E3 ligase WWP1 to mediate Runx2 degradation ( Jones et al.,
2006 ). The osteoblast activity is augmented in the Shn3 defi -
ciency mice that generate adult onset osteosclerosis with in-
creased bone mass.
CHIP (C terminus of Hsc70-interacting protein) is a cochap-
erone protein identifi ed through its interaction with Hsc/Hsp70
R
paper, we report that CHIP (C terminus of Hsc70-interact-
ing protein)/STUB1 regulates Runx2 protein stability via a
ubiquitination-degradation mechanism. CHIP interacts
with Runx2 in vitro and in vivo. In the presence of in-
creased Runx2 protein levels, CHIP expression decreases,
whereas the expression of other E3 ligases involved in
Runx2 degradation, such as Smurf1 or WWP1, remains
unx2, an essential transactivator for osteoblast dif-
ferentiation, is tightly regulated at both the tran-
scriptional and posttranslational levels. In this
constant or increases during osteoblast differentiation.
Depletion of CHIP results in the stabilization of Runx2,
enhances Runx2-mediated transcriptional activation, and
promotes osteoblast differentiation in primary calvarial
cells. In contrast, CHIP overexpression in preosteoblasts
causes Runx2 degradation, inhibits osteoblast differentia-
tion, and instead enhances adipogenesis. Our data sug-
gest that negative regulation of the Runx2 protein by CHIP
is critical in the commitment of precursor cells to differenti-
ate into the osteoblast lineage.
CHIP promotes Runx2 degradation and negatively
regulates osteoblast differentiation
Xueni Li , 1 Mei Huang , 1 Huiling Zheng , 2 Yinyin Wang , 1 Fangli Ren , 1 Yu Shang , 1 Yonggong Zhai , 3 David M. Irwin , 4
Yuguang Shi , 5 Di Chen , 6 and Zhijie Chang 1
1 Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Medicine,
Tsinghua University, Beijing 100084, China
2 Northwest Agriculture and Forestry University, Shaanxi, Yangling 712100, China
3 Beijing Key Laboratory, College of Life Sciences, Beijing Normal University, Beijing 100875, China
4 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5G 1L5, Canada
5 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033
6 Department of Orthopaedics, University of Rochester, Rochester, NY 14642

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JCB • VOLUME 181 • NUMBER 6 • 2008 960
Figure 1. Runx2 interacts with CHIP. (A) Runx2 physically interacts with CHIP in vitro. A GST pull-down assay was performed with the purifi ed GST or GST-
CHIP protein and Myc-Runx2 protein expressed by 293T cells. (B) Interaction of Runx2 with CHIP occurs in the nucleus of mammalian cells. Cytoplasmic
(Cy) and nuclear (Nu) extracts were used in coimmunoprecipitation assays with anti-HA antibody, and the precipitated complexes were analyzed with an

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961 CHIP REGULATES R UNX 2 ACTIVITY • Li et al.
anti-Myc antibody against Myc-Runx2. (C) Endogenous Runx2 and CHIP proteins interact. MC3T3-E1 cells were treated with 10 μ M MG132 for 4 h before
lysis. (top) Whole cell lysates were immunoprecipitated with an anti-CHIP antiserum or a preimmune serum (Pre). The immunoprecipitates were visualized
with an anti-Runx2 antibody (65 kD*; Santa Cruz Biotechnology, Inc.). (bottom) Reversed coimmunoprecipitation assays were performed using anti-Runx2
antibodies (MBL International) and IgG. (D) The C terminus of Runx2 interacts with CHIP. Schematic illustration of Runx2 and its truncated constructs is
shown on top. QA, QA domain; RHD, runt homology domain; NMTS, nuclear matrix targeting signal. A GST pull-down assay was performed using the
purifi ed GST-CHIP protein and different deletions of the hRunx2 protein expressed in mammalian cells. (E) TPR domain of CHIP is critical for the interaction
with Runx2. Three main functional domains of CHIP (TPR, tetratricopeptide repeats; U-box, U-box domain; Charged, charged domain) are schematically
illustrated on top. A GST pull-down assay was performed using GST-fused CHIP, and the deletion proteins to pull down the mammalian cell expressed Myc-
Runx2 protein. (F) Mutation CHIP(K31A) impairs the ability of CHIP to interact with Runx2. Lysates were prepared from 293T cells expressing HA-tagged
CHIP, CHIP(K31A), or CHIP(H261Q) together with the indicated Myc-Runx2. Coimmunoprecipitation and immunoblotting were performed as described in A.
The result from a longer exposure (5 × ) for the immunoblot is also presented (top).

( Ballinger et al., 1999 ). CHIP promotes the ubiquitination
and degradation of chaperone-bound proteins, such as receptor
tyrosine kinase ErbB2, glucocorticoid receptor, and the mis-
folded cystic fi brosis transmembrane conductance regulator
protein ( Wiederkehr et al., 2002 ). In a recent study using a
CHIP ? / ? cell model, CHIP has been reported to elicit the tran-
scriptional activation of HSF1 during stress recovery ( Qian et al.,
2006 ). We have recently reported that CHIP regulates bone
morphogenetic protein (BMP) and TGF- ? signals by enhancing
Smad protein ubiquitination and degradation ( Li et al., 2004 ;
Xin et al., 2005; Li et al., 2007 ). Here, we show that CHIP pro-
motes Runx2 ubiquitination and degradation and thereby nega-
tively regulates osteoblast differentiation.
Results
Runx2 interacts with CHIP as identifi ed
by yeast two-hybrid and
coimmunoprecipitation analyses in
culture mammalian cells
To search for additional potential regulators of Runx2, we per-
formed a yeast two-hybrid screening experiment using the
full-length mouse type II isoform of Runx2. CHIP was identi-
fi ed as a Runx2-interacting protein. The interaction was con-
fi rmed by a GST pull-down experiment ( Fig. 1 A ). To show
the interaction in mammalian cells, constructs expressing Myc-
Runx2 and HA-CHIP were transfected into 293T cells. Because
CHIP is reported to mostly localize to the cytoplasm ( Ballinger
et al., 1999 ; Hatakeyama et al., 2001 ) and Runx2 is in the
nucleus ( Zaidi et al., 2001 ), we determined whether the two
proteins have any opportunity to interact in intact cells. Sepa-
ration of nuclear and cytoplasm proteins indicates that al-
though the majority of Myc-Runx2 is present in the nucleus,
HA-CHIP is present in both the cytoplasm and the nucleus
(Fig. S1, available at http://www.jcb.org/cgi/content/full/
jcb.200711044/DC1), which is consistent with a previous study
( Huang et al., 2004 ). Coimmunoprecipitation experiments us-
ing cytoplasmic and nuclear fractions show that Myc-Runx2 is
precipitated by HA-CHIP protein in the nuclear extracts ( Fig. 1 B ,
left) but not from the cytoplasmic fraction ( Fig. 1 B , right).
These results suggest that CHIP is capable of interacting with
Runx2 in the nucleus of mammalian cells. Endogenous Runx2
and CHIP protein interaction was observed in MC3T3-E1
cells, a preosteoblast cell line ( Fig. 1 C ). This result fi rmly
confi rmed that Runx2 specifi cally interacts with CHIP under
physiological conditions.
The C terminus of Runx2 and the TPR
domain of CHIP are critical for interaction
Runx2 has two protein isoforms with the glutamine – arginine-
rich domain (QA), DNA-binding runt homology domain, NLS,
nuclear matrix targeting signal, and the transducin-like enhancer
of split/groucho-interacting C-terminal pentapeptide VWRPY
(for review see Stein et al., 2004 ). To map the interaction do-
mains between Runx2 and CHIP proteins, we expressed the hu-
man type I isoform of Runx2 and its deletion mutants ( Zhang et al.,
2000 ) in 293T cells. The GST pull-down experiment results
show that among the different deletions of Runx2 ( Fig. 1 D ,
top), the full-length (hRunx2 1 – 507) and the deletion mutant
hRunx2(88 – 507) show strong interactions with GST-CHIP
( Fig. 1 D , bottom; third and fourth lanes), whereas the deletion
mutant hRunx2(1 – 340) has completely lost, and hRunx2(1 – 424)
only has a weak interaction with GST-CHIP. These experiments
suggest that the C-terminal end of Runx2, which is around the
nuclear matrix – targeting signal motif, contributes to the inter-
action with CHIP. As CHIP contains a tetratricopeptide repeat
(TPR) domain responsible for chaperone binding, a U-box
domain important for E3 ubiquitin ligase activity, and a central
domain rich in charged residues ( Ballinger et al., 1999 ; Murata
et al., 2001 ), interaction with Runx2 was further mapped using
a series of GST-CHIP deletion mutants ( Fig. 1 E , top). The data
show that both GST-TPR and GST – ? U-box fusion proteins in-
teract with Myc-Runx2, but GST – U-box and GST- ? TPR do not
( Fig. 1 E , bottom). This suggests that the TPR domain is neces-
sary and suffi cient for the interaction with Runx2.
The chaperone activity of CHIP is required
for its interaction with Runx2
Next, we investigated whether the Runx2 – CHIP interaction is as-
sociated with the activity of CHIP. For this, we compared the inter-
action of Runx2 with the mouse CHIP(K31A) and CHIP(H261Q)
mutants, as the K31A mutant (in the TPR domain) binds neither
Hsp90 nor Hsp/Hsc70 chaperone ( Xu et al., 2002 ), whereas
H261Q mutant (in the U box) fails to bind the cognate E2 and
abolishes its E3 ligase activity ( Hatakeyama et al., 2001 ). The co-
immunoprecipitation assay results show that the interaction of
HA-CHIP(K31A) with Myc-Runx2 is barely detectable on a short
exposure and is still only weak with a long exposure, and HA-
CHIP(H261Q) demonstrates a strong interaction with Myc-Runx2
( Fig. 1 F ). These results suggest that the interaction of CHIP with
Runx2 is dependent on the K residue in the TPR domain, which is
critical for the chaperone activity, as the CHIP mutation that abol-
ishes interactions with chaperones fails to interact with Runx2.

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JCB • VOLUME 181 • NUMBER 6 • 2008 962
fi fth lanes]). We speculated that CHIP might enhance Runx2
turnover. To examine this hypothesis, Myc-Runx2 (mouse type II)
was coexpressed with increasing amounts of HA-CHIP pro-
tein in 293T cells in the presence or absence of MG132, a pro-
teasome inhibitor. Immunoblotting results demonstrate that
with increasing amounts of HA-CHIP, the Myc-Runx2 protein
levels decrease in the absence of MG132 but remain constant in
the presence of MG132 ( Fig. 2 A ). In a parallel experiment, we
CHIP enhances Runx2 degradation
and ubiquitination
CHIP has been reported to be an E3 ubiquitin ligase mediating
the ubiquitination and proteasome-dependent degradation of
several substrates ( Wiederkehr et al., 2002 ). In our experiments,
we have observed that expression of CHIP signifi cantly reduced
Runx2 protein levels (Fig. S1, top; and Fig. 1, B [third blot] and
F [fourth blot, compare the third lane with the fi rst, fourth, and
Figure 2. CHIP prompts Runx2 degradation and ubiquitination. (A) CHIP mediates the degradation of Runx2 in a dose-dependent manner. Myc-Runx2 was
coexpressed with increasing amounts of HA-CHIP in 293T cells treated with (right) or without (left) 10 μ M MG132 for 4 h. Protein levels of Myc-Runx2 were
measured by immunoblotting with an anti-Myc antibody. EGFP was used as a control. (B) CHIP does not induce degradation of the C-terminal truncated
Runx2. Myc-tagged Runx2 (full length), Runx2(1 – 424), and Runx2(1 – 340) were expressed with increasing amounts of HA-CHIP in 293T cells. Levels of the
Myc-tagged proteins were measured by immunoblotting with an anti-Myc antibody. (C and D) CHIP accelerates turnover of the Runx2 protein. A pulse-chase
assay (C) was performed in COS7 cells transfected with Runx2 together with empty vector, CHIP, or Smurf1. Quantitative presentation of the results with
SEM (error bars; three repeats) is shown in D. (E and F) Knocking down CHIP by siRNA increases Runx2 protein levels. pBS/U6 vector, vector-based siRNA
targeting CHIP (CHIP RNAi), or EGFP (EGFP RNAi) was transfected with or without Myc-Runx2 vector into UMR106 cells. Myc-Runx2 (E) or endogenous
Runx2 protein (F) levels were measured by immunoblotting with an anti-Myc antibody or an anti-Runx2 antibody (65 kD*; Santa Cruz Biotechnology, Inc.).
(G) CHIP prompts Runx2 ubiquitination. Myc-Runx2 and His-ubiquitin (His-Ubi) were coexpressed with or without HA-CHIP in 293T cells treated with 25 μ M
MG132 for 4 h before lysis. Total ubiquitinated proteins were precipitated from whole cell lysates by Ni – nitrilotriacetic acid resin and analyzed by immuno-
blotting with an anti-Runx2 antibody. (H) Runx2 ubiquitination by CHIP occurs in the nucleus. Myc-Runx2 and His-ubiquitin were coexpressed with HA-tagged
CHIP, CHIP(K31A), or CHIP(H261Q) in 293T cells. The nuclear exacts were separated and used for the ubiquitination experiment.

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963 CHIP REGULATES R UNX 2 ACTIVITY • Li et al.
gesting that CHIP is involved in Runx2 ubiquitination. In an ex-
periment with the CHIP mutants, we observed that CHIP(K31A)
did not alter the level of Runx2 ubiquitination ( Fig. 2 H , fourth
lane), presumably because it fails to interact with Runx2.
However, the expression of CHIP(H261Q) caused a decrease in
Runx2 ubiquitination compared with wild-type CHIP ( Fig. 2 H ,
compare the fi fth and third lanes) but a slight increase in Runx2
ubiquitination compared with the control vector ( Fig. 2 H , com-
pare the fi fth and second lanes). Because the later experiments
were performed using nuclear proteins, the results suggest that
CHIP enhances Runx2 ubiquitination in the nucleus.
CHIP decreases Runx2-induced
transcriptional activation
We analyzed how CHIP infl uences Runx2 biological activity
using a luciferase reporter assay in which the fi refl y luciferase
gene is under control of a Runx2-response element ( Zhang et al.,
2000 ). Our data demonstrate that in both NIH3T3 and COS7
cells, the overexpression of Runx2 alone activates reporter ex-
pression four- to sixfold and that overexpression of CHIP sig-
nifi cantly blunts the enhancing effect of Runx2 ( Fig. 3 A ). When
the inhibitory effects of wild-type CHIP, CHIP mutants, and
Smurf1 were compared, we observed that both of the CHIP mu-
tants could not inhibit Runx2-induced transcriptional activation
( Fig. 3 B , third and fourth bar groups) but that Smurf1 had a
similar scale of inhibitory effect as wild-type CHIP ( Fig. 3 B ,
last bar group). CHIP(H261Q) may have a slightly enhanced
effect on the luciferase activity ( Fig. 3 B , fourth bar group).
CHIP-induced attenuation of Runx2 transcriptional activity was
also observed in the UMR106 cells ( Fig. 3 C , second bar group).
Furthermore, when the endogenous CHIP protein was depleted
in the UMR106 cells by CHIP siRNA ( Li et al., 2004 ; Xin et al.,
observed that the protein levels of the two C terminus – truncated
forms of Runx2, which show little or no interaction with CHIP,
remained unchanged ( Fig. 2 B ). These results suggest that CHIP
participates in Runx2 degradation.
Because Smurf1 has been reported to enhance Runx2 deg-
radation ( Zhao et al., 2003, 2004 ), we compared whether CHIP
and Smurf1 have a similar effect on turnover rates of the Runx2
protein. A pulse-chase analysis using COS7 cells expressing
Myc-Runx2 alone or together with CHIP or Smurf1 show that
overexpression of CHIP increases the Runx2 degradation rate
( Fig. 2, C and D [line with open squares]) compared with Smurf1
( Fig. 2, C and D [line with closed triangles]). When the two E3
ligases were coexpressed, we observed an additive effect on
Runx2 degradation (unpublished data). These results indicate
that CHIP is a potent factor enhancing Runx2 degradation.
To determine the role of CHIP on Runx2 degradation un-
der physiological conditions, we knocked down CHIP expres-
sion using siRNA in a rat osteosarcoma cell line, UMR106
( Fig. 2, E [third blot, last lane] and F [bottom, right lane]), in
which endogenous CHIP is moderately expressed (see Fig. 4 G).
This results in a signifi cant increase in the amount of either over-
expressed Myc-Runx2 protein ( Fig. 2 E ) or endogenous Runx2
protein ( Fig. 2 F ). These results suggest that the endogenous
CHIP protein degrades Runx2 in osteoblast-like cells.
As CHIP affects protein degradation through the ubiqui-
tination process, we examined the effect of CHIP on Runx2
ubiquitination. We coexpressed Runx2 and polyhistidine-tagged
ubiquitin in 293T cells with HA-CHIP. Ubiquitinated proteins
were precipitated from cell lysates under denatured conditions
and analyzed by immunoblotting with the anti-Runx2 antibody.
Our results show that coexpression of CHIP is associated with a
marked increase in the ubiquitination of Runx2 ( Fig. 2 G ), sug-
Figure 3. CHIP inhibits Runx2-induced tran-
scriptional activity. (A) Overexpression of CHIP
inhibits Runx2-induced transcriptional activity.
Luciferase assays were performed using
NIH3T3 or COS7 cells transfected with the
indicated expression vectors along with a
Runx2-responsive reporter construct, 6xOSE2-
OC/pGL3 and pRL-TK (internal control).
Values were normalized using the internal
control and presented relative to basal activ-
ity (NIH3T3, fi rst bar; COS7, fi fth bar) with
the mean from three independent repeats.
(B) Mutants of CHIP fail to inhibit Runx2-
induced transcriptional activity. CHIP, CHIP mu-
tants, and Flag-Smurf1 were compared for the
inhibitory effect on Runx2 activity in NIH3T3
cells. (C) Knocking down CHIP by siRNA facili-
tates Runx2-mediated transcription in UMR106
cells. Luciferase assays were performed in
UMR106 cells transfected with Runx2 in the
presence of CHIP, EGFP RNAi, or CHIP RNAi.
(D) CHIP did not affect the transcriptional ac-
tivity of STAT3. NIH3T3 cells transfected with
STAT3 in the presence or absence of CHIP
along with pGL3 – acute phase response ele-
ment and treated with 10 ng/ml interleukin-6.
All of the data are presented as mean with SEM
(error bars) from three independent experi-
ments. The same letter represents a signifi cant
difference within this group in a meaningful
comparison (P < 0.05).

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JCB • VOLUME 181 • NUMBER 6 • 2008 964
Figure 4. Up-regulation of Runx2 protein level parallels reduced expression of CHIP during osteoblast differentiation. (A) von Kossa staining for MC3T3-E1
cells cultured for 3 wk in the presence of AA/ ? -GP was performed to demonstrate the cellular phenotype of osteoblasts. (B) mRNA and protein levels of Runx2
and CHIP during the osteoblast differentiation were compared. RT-PCR and Western blot (see quantitative presentations in Fig. S2, available at http://www.jcb
.org/cgi/content/full/jcb.200711044/DC1) analyses were performed on MC3T3-E1 cells induced by AA/ ? -GP for the indicated number of days. (C) Smurf1
expression is unchanged, and WWP1/Shn3 is increased during osteoblast differentiation. Real-time RT-PCR analysis was performed for the expression of the
indicated genes during osteoblast differentiation of MC3T3-E1 cells. Values are shown relative to cells on day 0. (D) Alizarin red staining for mouse calvarial cells

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965 CHIP REGULATES R UNX 2 ACTIVITY • Li et al.
cultured for 2 wk in the presence of AA/ ? -GP. (E and F) The expression of Runx2, CHIP, WWP1, Shn3, and Smurf1 in mouse calvarial cells was analyzed as
in B and C. (G) Real-time RT-PCR was performed to show expression of the indicated genes in different lineages of mesenchyma-originated cells. Fold induction
was calculated based on the mRNA level of each gene in NIH3T3. (H) Expression of the CHIP (green) and Runx2 (red) proteins in osteoblasts in vivo. Double
immunofl uorescence staining was performed using sections of trabecular (femur; a and b) and calvarial (c and d) bone from newborn mice. Enlarged osteoblasts
(boxed) are shown on the bottom (b and d). Ob, osteoblast; Chon, chondrocyte; Fi, fi broblast-like or other types of cells. Error bars represent SEM.

2005 ), we observed an increase in the luciferase activity either
at basal (without overexpression of Runx2) or under Runx2
overexpression ( Fig. 3 C , last bar group). As CHIP did not af-
fect the activity of the nonrelated reporter pGL3 – acute phase
response element (this reporter responds to interleukin 6 through
the activation of STAT3; Fig. 3 D ), these data suggest that CHIP
decreases Runx2-induced transcriptional activation.
CHIP is down-regulated during
osteoblast differentiation
To investigate the physiological role of CHIP on Runx2 protein
turnover, we examined CHIP expression during osteoblast dif-
ferentiation. Treatment with ascorbic acid (AA) and ? -glycero-
phosphate ( ? -GP) induced the differentiation of MC3T3-E1 cells
into osteoblast-like cells, as indicated by von Kossa – positive
staining ( Fig. 4 A ) and increased AP activity examination (not
depicted). We next analyzed CHIP and Runx2 mRNA and pro-
tein levels at different time points during early osteoblast differ-
entiation. The results show that Runx2 mRNA levels remained
constant during 2 wk of induction ( Fig. 4 B , second blot), whereas
both CHIP mRNA and protein levels signifi cantly decreased over
time ( Fig. 4 B ). In contrast, the levels of Runx2 protein increased
during this culture period ( Fig. 4 B , fi fth blot; and Fig. S2, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200711044/DC1)
concurrent with decreased CHIP protein expression. These re-
sults indicate that decreased CHIP protein levels correlate with
increased Runx2 protein levels despite the maintenance of con-
stant Runx2 mRNA levels (obviously increased in the later stage)
during early osteoblast differentiation. To determine the potential
involvement of other E3 ligases, we also examined the expres-
sion of Smurf1 and WWP1 (and its adaptor Shn3), two known E3
ligases that regulate Runx2 protein turnover, and found that the
Smurf1 mRNA levels were unchanged and that WWP1 (also
Shn3) mRNA levels became elevated ( Fig. 4 C ).
To study the expression of the aforementioned genes in
the later osteoblast differentiation, we isolated mouse calvarial
cells. When these cells were cultured for further differentiation
into mature osteoblasts (indicated by Alizarin red staining in
Fig. 4 D ), we observed that CHIP expression decreased, whereas
Runx2 protein levels increased together with its mRNA levels
( Fig. 4, E and F ). Changes in WWP1, Shn3, and Smurf1 mRNA
levels remained the same as patterns observed in MC3T3-E1
cells, the early osteoblasts.
Further analysis of the expression levels of Runx2, CHIP,
WWP1, Shn3, and Smurf1 in different cell lines derived from
mesenchymal cells was performed. We observed that Runx2 is
not expressed in NIH3T3 (a fi broblast cell line), C3H10T1/2
(a pluripotent mesenchymal cell line), and C2C12 (a progenitor
cell line for myoblasts), is moderately expressed in MC3T3-E1
(a progenitor cell line for osteoblasts), and is abundantly
expressed in UMR106 (an osteosarcoma cell line; Fig. 4 G ).
In contrast, CHIP is expressed at high levels in C2C12, NIH3T3,
and C3H10T1/2 cells but moderately expressed in MC3T3-E1
and UMR106 cells ( Fig. 4 G ). Furthermore, Smurf1 is expressed
ubiquitously in these cell lines, except it is higher in C2C12 cells.
WWP1, similar to Shn3, is barely expressed in C2C12 cells,
moderately expressed in NIH3T3 and C3H10T1/2 cells, and most
highly expressed in MC3T3-E1 and UMR106 cells ( Fig. 4 G ).
As these cell lines represent different differentiation stages of
mesenchymal cells, the expression patterns of these genes are
potentially related to cell differentiation. We were particularly
intrigued by the reversed pattern of Runx2 and CHIP expression
in nonosteoblast (NIH3T3, C3H10T1/2, and C2C12) and osteo-
blast (MC3T3-E1 and UMR106) lineages. Collectively, these
data suggest that CHIP is down-regulated during osteoblast dif-
ferentiation, and this down-regulation maintains Runx2 protein
stability. The coexpression of CHIP and Runx2 in osteoblasts
in vivo was fi nally confi rmed by double immunofl uorescence
staining using sections of calverial and trabecular bone (femur)
from newborn mice ( Fig. 4 H and Fig. S3, available at http://
www.jcb.org/cgi/content/full/jcb.200711044/DC1).
CHIP affects osteoblast differentiation
and mineralization of MC3T3-E1 cells
To further investigate the potential functions of CHIP on osteo-
blasts, we established stable MC3T3-E1 cell lines with the over-
expression of HA-CHIP ( Fig. 5 A ) or depletion of endogenous
CHIP ( Fig. 5 C , left). The stable cell lines were treated with
AA/ ? -GP for different periods of time and analyzed for Runx2
expression. AA/ ? -GP induced an elevation of Runx2 protein lev-
els but not mRNA levels in the mock cells ( Fig. 5 B , left lanes),
whereas Runx2 protein levels decreased in HA-CHIP – expressing
clones ( Fig. 5 B ). These results indicate that overexpression of
CHIP decreases Runx2 protein levels but has no effect on its
mRNA levels in the MC3T3-E1 cells. On the other hand, we ob-
served that CHIP depletion by siRNA in creased Runx2 protein
levels but did not affect its mRNA ( Fig. 5 C ). These data indicated
the role of endogenous CHIP in the regulation of Runx2 protein.
To investigate whether the overexpression of CHIP affects
the differentiation of cells into osteoblasts, we characterized the
cells by induction with AA/ ? -GP for different periods of time.
First, we observed that AP activity was dramatically decreased
in the two HA-CHIP – expressing clones but signifi cantly in-
creased in the two CHIP knockdown clones compared with the
two mock cells ( Fig. 5 D ). Alizarin red staining demonstrated
similar results ( Fig. 5 E ). Furthermore, real-time RT-PCR was
performed to examine expression of the osteoblast differentia-
tion marker genes (bone sialoprotein [BSP] and osteocalcin
[OCN]). Expression of these genes was delayed in the over-
expression clones or enhanced in the depletion clones ( Fig. 5 F ),
whereas the other E3 ligase genes known to participate in Runx2
degradation were not affected (not depicted). These results indicate

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JCB • VOLUME 181 • NUMBER 6 • 2008 966
observation that the overexpression of CHIP decreased Runx2
protein levels, we hypothesized that CHIP may prevent precur-
sor cells from differentiating into osteoblasts but may promote
cells to differentiate into adipocytes. When the two MC3T3-E1 –
CHIP clones were cultured under confl uence for long times,
cells appeared with features of adipocytes, including the accumu-
lation of triglyceride lipid droplets (Oil Red O staining positive;
Fig. 6 A , left; #2 and #8). When these cells were induced using
the adipogenesis inducer DIM (dexamethasone, 3-isobutyl-1-
methylxanthine, and insulin) mixture, the percentages of the
that increased CHIP overexpression prevents or delays cell dif-
ferentiation into mature osteoblasts, and the depletion of CHIP
promotes or enhances this process.
Overexpression of CHIP drives MC3T3-E1
cells to differentiate into adipocytes
but has no effect on the adipocytic
differentiation of C3H10T1/2 cells
MC3T3-E1 cells are committed to differentiate into osteoblasts
by the expression of Runx2 ( Prince et al., 2001 ). Based on the
Figure 5. Stable expression of CHIP inhibits osteoblast differentiation and mineralization. (A) Establishment of cell lines stably expressing HA-CHIP in
MC3T3-E1 cells (#2 and #8). Endogenous CHIP and HA-CHIP expression was measured by immunoblotting with an anti-CHIP antiserum. (B) Stable ex-
pression of HA-CHIP in MC3T3-E1 cells reduces the endogenous Runx2 protein levels. The cell lines were treated with AA/ ? -GP for the indicated days.
The mRNA (top) and protein (bottom) levels of Runx2 were measured by RT-PCR and Western blotting, respectively. (C) Stable depletion of CHIP using
siRNA in MC3T3-E1 cells increased Runx2 protein levels. The cell lines with depletion of CHIP (#9 and #20) were treated with AA/ ? -GP for the indicated
days. mRNA (left) and protein (right) levels of Runx2 were measured by RT-PCR and Western blotting, respectively. (D – F) CHIP inhibits the differentiation of
MC3T3-E1 cells into osteoblast-like cells. CHIP- or CHIP siRNA – expressing cell lines were induced to differentiate into osteoblast-like cells in the presence of
AA/ ? -GP for 4 – 21 d followed by the determination of AP activity (D), calcium accumulation by Alizarin red staining (E), and expression of the osteoblast
marker genes (BSP and OCN) by real-time RT-PCR (F). Error bars represent SEM.

Page 9

967 CHIP REGULATES R UNX 2 ACTIVITY • Li et al.
mediated Runx2 protein turnover, we used C3H10T1/2 cells, a
pluripotent cell line with features of mesenchymal stem cells.
Consistent with previous studies (for review see Rosen and
Spiegelman, 2000; Saito et al., 2002 ), we found that this cell line
does not express Runx2 ( Fig. 4 G ) and can be induced by DIM
into adipocytes ( Fig. 6 E , mock). We then established cell lines
that stably overexpress HA-CHIP in C3H10T1/2 cells ( Fig. 6 D ).
Our data show that DIM treatment results in similar percentages
of differentiated adipocytes in both the mock and stable cell
lines ( Fig. 6, E and F ), suggesting that the overexpression of
CHIP has no effect on the commitment of C3H10T1/2 cells to
the adipocyte lineage. The mRNA levels of PPAR ? and CEBP ?
also showed no signifi cant change between the mock and
C3H10T1/2-CHIP cell lines after DIM treatment for 7 d ( Fig. 6 G
and Fig. S4, c and d). These results suggest that CHIP does not
affect adipogenesis in uncommitted mesenchymal cells (without
Runx2 expression) and only functions in the osteoblastic cell
lineage (with Runx2 expression). Indeed, we also observed that
triglyceride-containing cells increased to 37.8 ± 6.7% (#2) and
28.6 ± 4.3% (#8) compared with the noninduced 6.0 ± 1.9% (#2)
and 3.4 ± 1.4% (#8; Fig. 6, A and B ). No triglyceride-containing
cells could be detected in mock cells cultured in the absence or
presence of DIM mixture.
In addition to cell morphology changes, we also examined
the expression of the adipocyte marker genes PPAR ? and
CEBP ? . Our data show that expression of these genes is dra-
matically increased after 7 d of DIM treatment in the MC3T3-E1 –
CHIP clones ( Fig. 6 C and Fig. S4, a and b; available at
http://www.jcb.org/cgi/content/full/jcb.200711044/DC1). Sig-
nifi cantly elevated PPAR ? and CEBP ? mRNA levels were even
observed in the MC3T3-E1 – CHIP cell clones without treatment
( Fig. 6 C and Fig. S4, a and b). These data suggest that exoge-
nous expression of CHIP directs MC3T3-E1 cells to differenti-
ate into adipocytes.
To determine whether the CHIP-induced reversed differ-
entiation of preosteoblast to adipocyte is dependent on CHIP-
Figure 6. Overexpression of CHIP drives
MC3T3-E1 cells to differentiate into adipocytes
but has no effect on adipocyte differentiation
in C3H10T1/2 cells. (A – C) Overexpression of
CHIP results in the facilitated differentiation of
MC3T3-E1 cells into adipocytes. MC3T3-E1-
CHIP cell lines (#2 and #8) and the mock cells
were cultured in the presence or absence of
DIM mixture for 7 or 14 d. Accumulation of
cytoplasmic triglyceride was detected by Oil
Red O staining (A). A quantitative representa-
tion of positive Oil Red O staining adipocytes.
Cell numbers were counted on three random-
ized fi elds with a phase-contrast microscope.
The percentages of triglyceride-containing
cells are shown with mean and SD (error
bars; B). The overexpression of CHIP induces
the expression of adipocyte marker genes
PPAR ? and CEBP ? . RT-PCR was performed on
day 7 (C). (D) Establishment of cell lines stably
expressing HA-CHIP in C3H10T1/2 cells (#8
and #11). Protein levels of the endogenous
CHIP and HA-CHIP are shown. (E – G) CHIP
does not affect the ability of C3H10T1/2 cells
to differentiate into adipocytes. C3H10T1/
2-CHIP cell lines (#8 and #11) were used in
adipocyte differentiation experiments. Oil Red
O staining shows accumulation of cytoplasmic
triglyceride on day 14 (E) with a quantitative
representation of positive Oil Red O staining
adipocytes (F) and expression of PPAR ? and
CEBP ? on day 7 (G).

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JCB • VOLUME 181 • NUMBER 6 • 2008 968
in wild-type MC3T3-E1 cells induced by AA/ ? -GP, we ob-
served that CHIP expression decreases signifi cantly with cell
differentiation but that Smurf1 expression remains unchanged
( Fig. 4, C and F ). Moreover, in comparison with Smurf1, which
is ubiquitously expressed in different cell lines, CHIP expres-
sion is more restricted to preosteoblast cells ( Fig. 4 ). These ob-
servations indicate that CHIP-facilitated Runx2 degradation is
closely related to its role in osteoblast differentiation.
Recently, WWP1, another Nedd4 family E3 ligase, has
been reported to be responsible for Runx2 ubiquitination and
degradation. WWP1 is recruited to Runx2 by an adaptor protein,
Shn3, and mice lacking Shn3 indeed display adult onset osteo-
sclerosis with increased bone mass as a result of augmented os-
teoblast activity ( Jones et al., 2006 ). In this study, we show that
the expression level of WWP1 is up-regulated during osteoblast
(from both cell lines and primary cultured calvarial cells) differ-
entiation in vitro, which is consistent with a previous study
( Jones et al., 2006 ). Also, WWP1 is expressed at higher levels in
the osteoblast lineages ( Fig. 4 G , MC3T3-E1 and UMR106).
The increased levels of WWP1/Shn3 together with the increased
Runx2 proteins during the osteoblast differentiation are not con-
cordant with the role of WWP1/Shn3 on Runx2 protein degra-
dation. One possible explanation is that WWP1 may not play a
role in Runx2 protein degradation at this stage of osteoblast dif-
ferentiation. Indeed, knocking out Shn3, a WWP1 adaptor, leads
to enhanced mineralization in calvarial osteoblasts but has no
effect on AP induction ( Jones et al., 2006 ). Because elevation of
AP activity occurs before the formation of mineralized matrix
nodules, it could be concluded that Shn3 and WWP1 function at
the mineralization stage, as Runx2 has to be turned off in fully
matured osteoblasts. In our study, we observed impaired AP in-
duction in CHIP-overexpressing MC3T3-E1 cells from an early
stage of differentiation. Therefore, we suggest that both CHIP
and WWP1 are responsible for the degradation of Runx2 but
function at different stages of osteoblast differentiation. We pro-
pose that CHIP maintains the low levels of Runx2 protein in
preosteoblasts and is turned down when differentiation starts to
ensure that Runx2 protein can be quickly elevated to the level
high required to initiate further osteoblast differentiation. When
cells have fully differentiated to mature osteoblasts, WWP1 (or
Shn3) expression is elevated to maintain the common features
of osteoblasts. Our data depict a complex relationship among
the different E3 ligases in regulating Runx2 protein levels dur-
ing preosteoblast differentiation.
It still remains to be determined whether there is an adap-
tor involved in the regulation of CHIP – Runx2 interaction, as in
the case of WWP1 and Shn3. In our study, we observed that the
H261Q mutant of CHIP causes an increase in Runx2 protein
level ( Fig. 1 F ). It is conceivable that CHIP(H261Q) loses its E3
activity because it lacks the capability to bind its cognate E2 en-
zyme. In addition, the H261Q mutant was found to retain, rather
than lose, the ability to ubiquitinate Runx2 ( Fig. 2 H , last lane).
Similar results were also observed on CHIP-mediated E47 deg-
radation ( Huang et al., 2004 ). On the other hand, CHN-1, the
Caenorhabditis elegans orthologue of CHIP, was reported to
form a complex with UFD-2, an enzyme known to have ubiquitin-
conjugating E4 activity in yeast, and this complex is necessary
the overexpression of CHIP by adenovirus starts to enhance
PPAR ? and CEBP ? gene expression in primary calvarial cells
(Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb
.200711044/DC1). Collectively, we propose that by degrading
Runx2 protein, CHIP is an inhibitor for osteoblast differentiation.
The down-regulation of CHIP expression is an important event
to initiate osteoblast differentiation, which releases Runx2 pro-
tein to allow further progress in osteoblast differentiation.
CHIP functions on the primary osteoblasts
Finally, we address whether CHIP functions in primary osteo-
blasts. For this purpose, we generated adenovirus to overexpress
(Ad-CHIP) or deplete (Ad-CHIPi) CHIP in isolated calvarial
cells. After we tested the effi cacy of the virus infection (not de-
picted), we infected calvarial cells with the adenovirus ( Fig. 7 A ).
Western blots demonstrated that the Runx2 protein levels were
largely depleted in the presence of the Ad-CHIP virus but in-
creased in the presence of the Ad-CHIPi virus ( Fig. 7 B ). The Aliza-
rin red staining experiments indicated that the over expression of
CHIP blocked differentiation of the cells into mature osteoblasts
but that depletion of CHIP signifi cantly enhanced osteoblast
maturation compared with the control virus (Ad-EGFP; Fig. 7 C ).
The inhibited osteoblast differentiation by overexpression of
CHIP with the virus was further confi rmed by examining the ex-
pression of osteoblast-specifi c marker genes BSP and OCN ( Fig.
7 D , middle bar groups). Reversely, depletion of CHIP with the
Ad-CHIPi virus resulted in a higher level of the marker gene ex-
pression ( Fig. 7 D , right bar groups). These data strongly indicate
that CHIP has an inhibitory role during osteoblast differentiation.
Discussion
Runx2 is a critical transactivator in osteoblast differentiation.
The activity of Runx2 is tightly regulated at different levels,
including transcription ( Sudhakar et al., 2001a ), translation
( Sudhakar et al., 2001b ), phosphorylation ( Kim et al., 2006 ;
Rajgopal et al., 2006 ), acetylation ( Jeon et al., 2006 ), protein –
protein interactions ( Hong et al., 2005; Dobreva et al., 2006 ),
and ubiquitin-mediated degradation ( Zhao et al., 2003; Jones
et al., 2006 ). The important role of the posttranslational regula-
tion of Runx2 has been demonstrated by experiments showing
that dexamethasone treatment yields a signifi cant increase in
Runx2 protein levels without affecting its mRNA expression
( Prince et al., 2001 ). In this study, we show that CHIP is a novel
posttranslational regulator of Runx2 through several lines of
evidence. Our fi ndings suggest that CHIP has an important role
in the regulation of Runx2 protein levels and cell commitment
in osteoblast precursor cells.
Previously, several other E3 ligases have been reported to
regulate Runx2 protein turnover, including Smurf1, a HECT
domain E3 ubiquitin ligase. Smurf1 has been reported to di-
rectly interact with Runx2 and to induce ubiquitin-dependent
degradation of Runx2 protein ( Zhao et al., 2003 ). In this study,
we compared the infl uences of Smurf1 and CHIP in mediating
Runx2 degradation. We found that both overexpressed CHIP
and Smurf1 mediate Runx2 degradation ( Fig. 2, C and D ) and
thus inhibit Runx2-mediated transcription ( Fig. 3 B ). However,

Page 11

969 CHIP REGULATES R UNX 2 ACTIVITY • Li et al.
C2C12 cells. These observations implicate that the regulation of
CHIP gene expression together with that of WWP1 and Smurf1
yields a specifi c pattern for different fates of the cells and may
be controlled by common factors during cell differentiation.
We have reported that CHIP facilitates the degradation of
Smad proteins in several cell lines ( Li et al., 2004 ; Xin et al.,
2005 ). Because Smad proteins are critical mediators of BMP/
TGF- ? signals, which play important roles in osteoblast differ-
entiation, and activation of BMP signaling has been reported to
activate Runx2 mRNA expression ( Ito and Miyazono, 2003 ),
we questioned whether CHIP overexpression would also block
these signals in osteoblasts. In our experiments, we adopted
and sufficient to multiubiquitylate UNC-45 in vitro ( Hoppe
et al., 2004 ). We presume that other factors such as the Hsc70 –
Skp2 complex might also be involved in the Runx2 ubiquitina-
tion process mediated by CHIP.
It is not clear how CHIP expression is regulated at the
transcriptional level during osteoblast differentiation. Our data
indicate that CHIP is down-regulated when cells differentiate
into osteoblasts and is maintained at high levels in nonosteoblast
lineages. Conversely, WWP1 (also Shn3) is up-regulated when
cells differentiate into osteoblasts but is maintained at a basal
level at the early stage of differentiation (in uninduced MC3T3-E1,
NIH3T3, or C3H10T1/2 cells) or is completely turned off in
Figure 7. CHIP inhibits osteoblast differentiation. (A) Microscopic analysis of GFP expression 4 d after adenoviral infection shows equally effi cient trans-
duction (up to 70%) of primary mouse calvarial cells. GFP is expressed from the same viral vector as CHIP or CHIP siRNA. (B) Examination of the adenovirus
expressing CHIP (Ad-CHIP) or CHIP siRNA (Ad-CHIPi) on endogenous Runx2 protein levels in calvarial cells. GFP levels indicate amounts of virus infection.
(C) Alizarin red staining of mouse calvarial osteoblasts transfected with the adenovirus expressing GFP, CHIP, or CHIP siRNA and cultured in the presence
of AA/ ? -GP for 2 wk. (D) Real-time RT-PCR demonstrates that the mRNA of bone marker genes (BSP and OCN) in the mouse primary calvarial cells infected
the virus in the condition of osteoblast differentiation. Error bars represent SEM. (E) A schematic presentation of the functions of CHIP on osteoblast dif-
ferentiation. Although Runx2 mRNA is expressed in preosteoblast cells, the Runx2 protein level remains at a low level because high CHIP expression leads
to its degradation. When cells commit to differentiate into osteoblasts, CHIP expression decreases, leading to increased Runx2 protein levels, which drives
the cells to become mature osteoblasts. WWP1 seems to enhance Runx2 degradation in the late stage of osteoblast differentiation ( Jones et al., 2006 ).
The question mark indicates an undefi ned stage of the function. This diagram has been modifi ed from Komori, 2005 .

Page 12

JCB • VOLUME 181 • NUMBER 6 • 2008 970
HEK293T, COS7, and C2C12 cells were cultured in DME supple-
mented with 10% FBS. NIH3T3 cells were maintained in DME supple-
mented with 10% newborn calf serum, which was replaced with 10% FBS
the day before transfection. MC3T3-E1 and UMR106 cells were cultured in
? MEM supplemented with 10% FBS. C3H10T1/2 cells were cultured in
basal medium Eagle supplemented with 10% FBS. All of the cells were kept
at 37 ° C in a 5% CO 2 -containing atmosphere. The stable cell lines were se-
lected by 1 mg/ml G418 (Sigma-Aldrich) after transfection of the related
plasmids, and the obtained clones were maintained in media containing
400 μ g/ml G418.
In the induction of osteoblast differentiation, cells were treated with
AA/ ? -GP (50 μ g/ml AA and 10 mM ? -GP for primary mouse calvarial
osteoblastic cells using 5 mM ? -GP) for the indicated days. In the adipo-
genesis experiments, cells were cultured in the presence of DIM (1 μ M
dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μ g/ml insulin)
for up to 14 d. Media were changed every 3 d.
Protein assays and luciferase assay
Protein extraction, immunoblotting, coimmunoprecipitation, ubiquitination,
GST pull down, pulse-chase assays, and luciferase assay were performed
according to previous studies ( Li et al., 2004 ; Xin et al., 2005 ).
Reverse transcription/real-time PCR
DNase-treated RNA was reverse transcribed with SuperScript II reverse
transcription (Invitrogen) using random hexamer primer (Promega). The re-
sulting cDNA was subsequently used for PCR or quantitative real-time PCR
analyses. Primer sequences are listed in Table S1 (available at http://
www.jcb.org/cgi/content/full/jcb.200711044/DC1).
AP activity assay
Cells were plated at a density of 10 4 cells/cm 2 for osteoblast differentiation.
Cultures from indicated days were lysed with 250 μ l of 0.1% Triton X-100/
well. 500 μ l of freshly prepared 20-mM p -nitrophenyl-phosphate in AMP buf-
fer (1.5 M AMP and 0.1 M MgCl 2 , pH 10.3) was mixed with 100 μ l of cell
lysate and incubated for 30 min at 37 ° C until the reaction was terminated
with 2 ml of 0.25-M NaOH solution. The product p -nitrophenol had a colori-
metric determination at 405 nm ( Coelho and Fernandes, 2000 ). Protein con-
centration of the lysate was determined using the BCA protein assay kit
(Thermo Fisher Scientifi c). AP activity was standardized as OD405/OD562.
The experiment was repeated three times, and SEM was calculated.
Histochemical assays
Cultures were fi xed with 95% ethanol for 10 min and rinsed with distilled
water. Phosphate deposits were assessed by von Kossa staining. The fi xed
cultures were covered with a 1.0% AgNO 3 solution and kept for 1 h under
UV light. After rinsing, a 5.0% NaS 2 O 3 solution was added for 2 min, and
cultures were washed again. For calcium staining, the fi xed cultures were
covered with a 1.0% Alizarin red solution (0.028% NH 4 OH, pH 6.4) for
2 min and rinsed with distilled water and acid ethanol (ethanol and 0.01%
HCl). Lipid droplets were examined with Oil Red O staining, and the posi-
tive cells were counted with a microscope (BX-51; Olympus) and CCD
camera (DP70; Olympus).
Immunofl uorescence staining
The bones were fi xed in 10% formalin and embedded with paraffi n. Sec-
tions were incubated with anti-CHIP and anti-Runx2 antibodies overnight at
4 ° C followed by FITC- and TRITC-labeled second antibodies for 60 min at RT.
Nuclei were labeled with DAPI nuclear dye (blue). Sections were mounted
with glycerol and photographed with a microscope (Eclipse C1si; Nikon).
Preparation of primary mouse calvarial osteoblastic cells
Calvariae from newborn mice were dissected aseptically and treated with
0.2% collagenase (Sigma-Aldrich) for 20 min with shaking. Cell suspen-
sion in collagenase was collected and transferred into a 50-ml tube by
adding FBS to 10% (to inactive collagenase). Then, fresh collagenase was
added with shaking for second digestion. Digestion was repeated six times
to obtain as many cells as possible (the cells from the fi rst digestion were
discarded). Cells were plated at a density of 1.5 × 10 4 cells/cm 2 and cul-
tured in ? MEM supplemented with 10% FBS to confl uent.
Adenoviral infection of calvarial osteoblasts
Calvarial cells were transduced with adenoviruses expressing GFP, CHIP,
or CHIP siRNA or the empty virus (pAdTrack-cytomegalovirus). Virus parti-
cles were administered at 50 plaque-forming units/cell in ? -MEM with
1% FBS and incubated for 1 h at 37 ° C. After 1 h, medium was aspirated,
AA/ ? -GP (rather than BMP2) to induce MC3T3-E1 and calvar-
ial cell differentiation. Our observations on the cell differentiation
affected by CHIP suggest that AA/ ? -GP induces cell differenti-
ation through a pathway not directly related to BMP signaling.
Whether CHIP plays a role in BMP signaling to regulate osteo-
blast differentiation in vivo remains to be studied.
The biological signifi cance of CHIP-mediated Runx2 reg-
ulation on preosteoblast differentiation is underscored by its de-
pendence on preosteoblast-specifi c cell types. In contrast to the
MC3T3-E1 cells, which express Runx2 and are committed to
the osteoblast lineage, overexpression of CHIP in C3H10T1/2
cells that are defi cient in Runx2 expression did not change the
differentiation potential (to become adipocytes) of these cells.
Based on these and previous work ( Komori, 2005 ) on Runx2,
we propose a model by which CHIP interacts with Runx2 and
regulates preosteoblast differentiation ( Fig. 7 E ). In this model,
CHIP regulates Runx2 protein levels and thereafter regulates the
commitment of preosteoblast differentiation to osteoblasts and
adipocytes. CHIP levels decrease steadily during preosteoblast
differentiation accompanied by a steady increase in Runx2 pro-
tein levels. This model can explain our observations that the
forced expression of CHIP prevented the committed differen-
tiation of preosteoblasts to osteoblasts, resulting in adipocyte
differentiation. This is consistent with previous studies on the
effect of Runx2 on adipocyte differentiation, as Runx2 has been
shown to be an inhibitor that prevents mesenchymal cells from
entering into the adipocyte lineage ( Lecka-Czernik et al., 1999;
Kobayashi et al., 2000 ; Jeon et al., 2003 ), and Runx2 defi ciency
in chondrocytes stimulates their differentiation to adipocytes
( Enomoto et al., 2004 ). Collectively, our data identifi es for the
fi rst time an important role of CHIP in regulating preosteoblast
differentiation by modulating the protein levels of Runx2
through ubiquitination and protein degradation. This enriches
our knowledge of the regulation of Runx2 protein during the os-
teoblast differentiation in addition to the known negative regu-
lators WWP1/Shn3 and Smurf1/2.
Materials and methods
Antibodies
The antiserum against CHIP was generated by immunizing rabbits with
purifi ed GST-CHIP(1 – 161) in this laboratory. Anti – ? -actin and anti-Runx2
(8G5) were purchased from Sigma-Aldrich and MBL International, respec-
tively. Other antibodies (including anti-PEBP ? A and M70) were purchased
from Santa Cruz Biotechnology, Inc.
Plasmids, cell lines, and cultures
Type II mouse Runx2 cDNA was cloned from pBSA3 (provided by G.
Karsenty, Columbia University, New York, NY; Ducy et al., 1997 ) and in-
serted into the pGBKT7 and pCMV/Myc vectors (Clontech Laboratories,
Inc.). Constructs expressing deletions of type I human Runx2 (PEBP2 ? A) were
provided by Y. Ito (Institute of Molecular and Cell Biology, Singapore; Zhang
et al., 2000 ). pGL3/6xOSE2-OC-luc was described previously ( Zhao et al.,
2003 ). pGEX-5X-3/CHIP and its deletions, pcDNA6/HA-CHIP, pcDNA6/
HA-CHIP(K30A), pcDNA6/HA-CHIP(H260Q), and pACT-2/CHIP, together
with CHIPi constructs were preserved in our laboratory ( Li et al., 2004 ; Xin
et al., 2005 ). The siRNA target sequences for mouse CHIP is 5 ? -AACAG-
GCACTTGCTGACTG-3 ? and for human CHIP is 5 ? -AGCAGGCCCTGGC-
CGACTG-3 ? . His-ubiquitin and Flag-Smurf1 were provided by Y. Chen
(Tsinghua University, Beijing, China). pEFNue/HA-CHIP was generated from
pcDNA6/HA-CHIP. The recombinant adenoviruses expressing GFP, CHIP-
GFP, and CHIPi-GFP were constructed using the pAd Easy system.

Page 13

971 CHIP REGULATES R UNX 2 ACTIVITY • Li et al.
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cultures were rinsed twice with serum-free medium, and fresh medium sup-
plemented with 10% FBS was added to the dishes.
Online supplemental material
Fig. S1 shows the nuclear and cytoplasmic localization of Runx2 and
CHIP proteins. Fig. S2 shows a quantitative analysis of Runx2 and CHIP
protein levels in the induced MC3T3-E1 cells and mouse calvarial cells.
Fig. S3 shows the coexpression of CHIP and Runx in mouse osteoblasts
in femur and calvaria. Fig. S4 quantitatively presents adipocyte-specifi c
marker gene expression in MC3T3-E1 and C3H10T1/2 cells. Fig. S5
shows the expression of adipocyte markers in the primary calvarial cells
infected with Ad-CHIP or Ad-EGFP in the presence or absence of DIM.
Table S1 shows the primer sequences used in real-time PCR analyses.
Online supplemental material is available at http://www.jcb.org/cgi/
content/full/jcb.200711044/DC1.
We are grateful to Xin-Yuan Fu (Indiana University, Bloomington, IN) and Xiaofan
Wang (Duke University, Durham, NC) for their support and suggestions. We
thank Gerard Karsenty, Yeguang Chen, and Yoshiaki Ito for providing constructs.
This work was supported by the Tsinghua Yu-Yuan Medical Sciences
Fund and grants from the National Nature Science Foundation of China
(30530420, 30470888, and 30518002), Chinese National Support Proj-
ect (2006CB910102), and 973 Project (2002CB513007).
Submitted: 9 November 2007
Accepted: 16 May 2008
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