Msx2 Exerts Bone Anabolism via Canonical Wnt Signaling*□S
Received for publication, February 1, 2008, and in revised form, May 7, 2008 Published, JBC Papers in Press,May 15, 2008, DOI 10.1074/jbc.M800851200
Su-Li Cheng1, Jian-Su Shao1, Jun Cai, Oscar L. Sierra, and Dwight A. Towler2
From the Department of Medicine, Division of Bone andMineral Diseases, Washington University School of Medicine,
St. Louis, Missouri 63110
Msx2 is a homeodomain transcription factor first identified in
craniofacial bone and human femoral osteoblasts. We hypothe-
sized that Msx2 might activate skeletal Wnt signaling. There-
fore, we analyzed the effects of CMV-Msx2 transgene (Msx2Tg)
expression on skeletal physiology and composition. Skeletal
Msx2 expression was increased 2–3-fold by Msx2Tg, with
expanded protein accumulation in marrow, secondary ossifica-
tion centers, and periosteum. Microcomputed tomography
established increased bone volume in Msx2Tg mice, with
increased numbers of plate-like trabeculae. Histomorphometry
revealed increased bone formation in Msx2Tg mice versus
non-Tg siblings, arising from increased osteoblast numbers.
While decreasing adipogenesis, Msx2Tg increased osteogenic
differentiation via mechanisms inhibited by Dkk1, an antago-
nist of Wnt receptors LRP5 and LRP6. Bone from Msx2Tg mice
elaborated higher levels of Wnt7 canonical agonists, with
diminished Dkk1, changes that augment canonical signaling.
Analysis of non-Tg and Msx2Tg siblings possessing the TOP-
GAL reporter confirmed this; Msx2Tg up-regulated skeletal

-galactosidase expression (p < 0.01), along with Wnt7a and
Wnt7b, and reduced circulating Dkk1. To better understand
molecular mechanisms, we studied C3H10T1/2 osteoprogeni-
tor cells. As in bone, Msx2 increased Wnt7 genes and down-
regulated Dkk1, while inducing the osteoblast gene alkaline
phosphatase. Msx2-directed RNA interference increased Dkk1
expression and promoter activity, while reducing Wnt7a, Wnt7b,
and alkaline phosphatase. Moreover, Msx2 inhibited Dkk1 pro-
moteractivityandreducedRNApolymeraseassociationwithDkk1
chromatin. RNA interference-mediated knockdown of Wnt7a,
Wnt7b, and LRP6 significantly reduced Msx2-induced alkaline
phosphatase. Msx2 exerts bone anabolism in part by reducing
Dkk1 expression and enhancing Wnt signaling, thus promoting
osteogenic differentiation of skeletal progenitors.
Msx2, also known as Hox-8, is a homeodomain transcription
factor first characterized by Sharpe and co-workers (1) as a
calcitriol-regulated transcript in osteoprogenitors isolated
from human femur, and subsequently shown to be highly
expressed in the murine embryonic craniofacial skeleton (2).
Soon thereafter, Msx2 was identified as a transcriptional
repressor of the osteoblast-specific osteocalcin (OC)3 promoter
(3, 4). Studies of Msx2-regulated gene expression in bone have
emphasized its role as a transcriptional repressor of the late
osteogenic phenotype (5–7). For example, in the developing
tooth, where stage-specific osteogenic gene expression profiles
are spatially resolved,Msx2 and OC exhibit reciprocal patterns
of mRNA accumulation (8). Msx2 suppresses OC gene expres-
sion in a cell-autonomous fashion, mediated via antagonistic
protein-protein interactions between Msx2 and Runx2-con-
taining complexes that support OC promoter activity (6, 7).
However, elegant genetic studies have demonstrated that
Msx2 also promotes craniofacial bone mineralization and is
necessary for robust trabecular and cortical bone formation (9,
10). Msx2
/
mice exhibit parietal foramina, characterized by
reducedmineralization in calvarial fields that give rise tomem-
branous bone (9). Moreover, Msx2
/
mice exhibit a global,
low turnover osteopenia (9). Simultaneous deletion of both
Msx2 and Msx1, a homologous Msx gene family member,
results in the complete absence of craniofacial bone (9, 11).
Furthermore, a nonsynonymous CCC to CAC mutation at the
7th codon of theMsx2 homeodomain causes Boston-type auto-
somal dominant craniosynostosis, characterized by precocious
mineralization and differentiation of osteoblasts in the calvarial
suture (12). In vivo and in vitro data indicate that this
Msx2(P148H) variant is most likely a gain-of-function variant;
Msx2(P148H) exhibits increased DNA binding to the Msx
CTGAATTRG binding cognate (13, 14). Intriguingly, however,
the frequency of transgene-induced craniosynostotic bone is
three time more prevalent in wild-type Msx2 transgenic mice
than Msx2(P148H) transgenic mice (71 versus 27%, respec-
* Thisworkwas supported, inwholeor inpart, byNational Institutes ofHealth
Grants AR43731 and HL81138 (to D. A. T.) and the Barnes-Jewish Hospital
Foundation. The costs of publicationof this articlewere defrayed inpart by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordancewith 18U.S.C. Section 1734 solely to
indicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S6.
1 Both authors contributed equally to this work.
2 To whom correspondence should be addressed: Internal Medicine, Bone
and Mineral Diseases, Washington University School of Medicine, Campus
Box 8301, 660 South Euclid Ave., St. Louis, MO 63110. Fax: 314-454-8434;
E-mail: dtowler@dom.wustl.edu.
3 The abbreviations used are: OC, osteocalcin; ALP, alkaline phosphatase;
BMD, bone mineral density; BSP, bone sialoprotein; C3H10T1/2, murine
multipotentmesenchymal cell line; ChIP, chromatin immunoprecipitation;
CMV, cytomegalovirus immediately early enhancer/promoter; CTX, type I
collagen C-terminal telopeptide; FBS, fetal bovine serum; Fzd, frizzled Wnt
co-receptor; micro-CT, microcomputed tomography; Msx, muscle seg-
ment homeobox gene; Msx2Tg, transgenic mouse expressing Msx2 cDNA
from the CMVpromoter; Osx, osterix; PBS, phosphate-buffered saline; PKC,
protein kinase C; pol II, RNA polymerase II; RT-qPCR, reverse transcription-
quantitative PCR; siRNA, small interfering RNA; Tg, transgenic mouse; TCF,
T-cell factor; LEF, lymphoid enhancer factor; TOPGAL, TCF/LEFoptimal pro-
moter-galactosidase reporter transgenic mouse; TRAP5c, osteoclast tar-
trate-resistant acid phosphatase; Wnt, Wingless/int gene family; WT, wild-
type sibling mouse; sibs, siblings; DMEM, Dulbecco’s modified Eagle’s
medium; E3, ubiquitin-protein isopeptide ligase; ELISA, enzyme-linked
immunosorbent assay; BFR, bone formation rate; RNAi, RNA interference;
TV, tissue volume.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 29, pp. 20505–20522, July 18, 2008
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
JULY 18, 2008•VOLUME 283•NUMBER 29 JOURNAL OF BIOLOGICAL CHEMISTRY 20505

tively) (13). This suggests that in vivo the P148H mutation per-
turbs other important Msx2 functions (13). Indeed, the region
of the homeodomain N-terminal arm altered by the P148H
substitution participates in regulatory protein-protein interac-
tions with Dlx5 (5, 15) and other transcription factors (5–7).
Consistent with the Msx2 gain-of-function model arising from
CMV-Msx2 transgenic mice, distal trisomy of chromosome 5q,
the physical region encompassing the Msx2 gene, has been
identified in six patients with craniosynostosis (16); this dem-
onstrates the exquisite sensitivity of the developing human
craniofacial skeleton to Msx2 gene dosage (10, 17). Although
details have emerged as to how Msx2 inhibits osteoblast termi-
nal differentiation via cell-autonomous actions (5–7), little is
known of themechanismswherebyMsx2 promotes osteoblast-
mediated bone formation. The mechanistic underpinnings of
the low turnover osteopenia demonstrated in Msx2
/
long
bone during postnatal growth have yet to be determined (9). In
seminal studies of calvarial bone formation, Maxson and co-
workers (10, 11) showed that Msx2 promotes the proliferative
expansion and survival of neural crest-derived osteoblasts.
Recently, we identified that Msx2 participates in the ectopic
medial artery calcification characteristic of type II diabetes (18,
19). Murine models of diabetic medial calcification spatially
resolved adventitial Msx2-expressing cells and the ALP-posi-
tive medial calcifying vascular cells that direct matrix mineral-
ization; therefore, we deduced thatMsx2-expressing cells elab-
orate paracrine signals that control osteogenic differentiation
of neighboring progenitors (19). Indeed, we showed that con-
ditioned media from Msx2-expressing C3H10T1/2 cells pos-
sessed a pro-osteogenic, adipostatic activity (19) resembling
that of canonical Wnt ligands such as Wnt3a (20), Wnt7a (21),
and Wnt10b (22, 23). Molecular profiling by quantitative RT-
qPCR demonstrated up-regulation of canonical Wnt agonists
Wnt3a andWnt7a in aortic tissues (19). By contrast, expression
of aortic Dkk1, the prototypic vertebrate inhibitor of canonical
Wnt signaling, was concomitantly down-regulated by Msx2
(19). Exogenous recombinant Dkk1 antagonized Msx2 regula-
tion of osteogenesis and adipogenesis (19). Thus, Msx2-pro-
ducing vascular adventitial cells enhanced aorticWnt signaling
and promoted ectopicmineral deposition via osteogenicmech-
anisms (19).
To better understand the mechanisms whereby Msx2 aug-
ments orthotopic long bone formation, we have evaluated the
skeletal phenotype of adult CMV-Msx2 (Msx2Tg) transgenic
mice (19). We show that augmenting Msx2 expression in long
bone directs bone mesenchymal progenitors to osteogenic lin-
eage, enhancing skeletal osteoblast numbers, mineralizing sur-
face, trabecular number, and trabecular bone formation via
canonical Wnt signals.
EXPERIMENTAL PROCEDURES
Antibodies, ChIP Assays, Luciferase Assays, Plasmids, and
Other Reagents—Antibodies for Msx2 (sc-15396, lot C0404,
polyclonal antibody), Wnt7a/b (sc-32865, lot F2006, rabbit
polyclonal), Wnt1 (sc-5630, lot E2605, goat polyclonal anti-
body),Wnt3a (sc-28824, lotK0205, rabbit polyclonal antibody),
Dkk1, (sc-25516, lot J1404, rabbit polyclonal antibody), actin
(sc-8432), and eIF2
(sc-11386) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). The Wnt1 antibody
(ab15251-500, lot 315275, rabbit polyclonal antibody) pur-
chased fromAbcamwas also used and proved to be superior for
Wnt1 immunohistochemistry. The alkaline phosphatase-con-
jugated goat anti-rabbit secondary antibody used for Western
blots with rabbit primary immunoreagents was sc-2057 (lot
I12503, Santa Cruz Biotechnology).Wnt10b antibody (rat anti-
mouse, monoclonal antibody 2110, clone 254206) was pur-
chased from R & D Systems (Minneapolis, MN). Goat poly-
clonal antibody to LRP5 (sc-21390) and the donkey anti-goat
secondary antibody (sc-2022) were both purchased from Santa
Cruz Biotechnology. VECTASTAIN Elite ABC peroxidase sec-
ondary antibody immunovisualization kits directed toward
goat (PK-6105) or rabbit (PK-6101) primary antibodies were
purchased from Vector Laboratories (Burlingame, CA). Anti-
bodies to pol II (N-20, sc-899, Santa Cruz Biotechnology) and
phospho-pol II (H5, MMS-129R, Covance) were used in ChIP
assays as describedpreviously (6, 24). The amplimer pairs 5-CGG
AGG TCC CGA AGT TGA G-3 and 5-AAA GGT CAG GAA
AAG AGA GGT CAC T-3 and 5-ATT ATT GGT GAC TTG
GTG GTG ATC T-3 and 5-ATT TTA TGA GGC ACA GTT
GAT GTC TT-3 were used for quantifying DNA in precipitates
by fluorescenceqPCR for theDkk1 andosteopontin genes, respec-
tively. Primer pairs were directed to regions just downstream of
the transcription initiation site, within the early regions encoding
the pol II-dependent primary transcripts. A second amplimer pair
directed 1.5 kb into the Dkk1 gene (5-GAA AGC ATC ATT
GAA AAC CTT GGT-3 and 5-GCC TTC CCC GCA GTA
ACA-3) was also used to confirm the effects of Msx2 on pol
II-Dkk1 chromatin interactions. For Western blot assays,
antigen-antibody complexes were detected by alkaline phos-
phatase-dependent disodium 3-(4-methoxyspiro[1,2-diox-
etane-3,2-(5-chloro)-tricyclo[3.3.13,7]decan]-4-yl)phenyl
phosphate chemiluminescence (Tropix, Applied Biosystems,
Foster City, CA). Conjugated secondary antibodies, blocking
reagents, and peroxidase-based immunovisualization kits were
purchased from Tropix or Vector Laboratories (Burlingame,
CA). Custom-synthesized oligodeoxynucleotides were synthe-
sized and purchased from Invitrogen. TOPGLOW (catalog
number 21-204) and FOPGLOW (catalog number 21-205)
luciferase reporter plasmids were purchased from Millipore.
PCR was used to generate cDNA inserts for expression plas-
mids for pcDNA3-Wnt7a and pcDNA3-Wnt7b using meth-
ods described previously (8). Dkk1 promoter-luciferase
reporter constructs 1141 DKK1LUC (
1141 to
1), 451
DKK1LUC (
451 to
1), 170 DKK1LUC (
170 to
1), 120
DKK1LUC (
120 to
1), and 70 DKK1LUC (
70 to
1)
were generated by ligating the indicated segments of the
mouse Dkk1 promoter into pGL2 Basic (Promega); methods
have been detailed previously (25). The numbering of base
pairs is relative to the start site of Dkk1 exon 1 (physical base
pair position 30,620,374 of mouse chromosome 19, RefSeq
Gene NM_010051). The control minimal promoter-reporter
RSVLUC has been described previously (25). Transient
transfections and luciferase assays were carried out as
detailed previously (6). Custom-synthesized siRNA reagents
were purchased from Qiagen (Valencia, CA). Commercially
available siRNAs were purchased from Santa Cruz Biotech-
Wnt Signals MediateMsx2 Bone Anabolism
20506 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 29•JULY 18, 2008