Msx2 Exerts Bone Anabolism Via Canonical Wnt Signaling

61 Pages·2008·2.4 MB·English

JBC Papers in Press. Published on May 15, 2008 as Manuscript M800851200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M800851200

Msx2 Exerts Bone Anabolism Via Canonical Wnt Signaling*

Su-Li Cheng, Jian-Su Shao, Jun Cai, Oscar L. Sierra, and Dwight A. Towler

From the Department of Medicine, Division of Bone and Mineral Diseases
Washington University School of Medicine
St. Louis, MO 63110

Running title: Wnt Signals Mediate Msx2 Bone Anabolism

Address correspondence to: Dwight A. Towler MD, PhD, Internal Medicine, Bone and Mineral
Diseases, Washington University School of Medicine, Campus Box 8301, 660 South Euclid Ave.,
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St. Louis, MO 63110. Fax: [phone]; E-mail: [email protected] w
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Msx2 is a homeodomain transcription studied C3H10T1/2 osteoprogenitor cells. ed
factor first identified in craniofacial bone As in bone, Msx2 increased Wnt7 genes and fro
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and human femoral osteoblasts. We down-regulated Dkk1, while inducing the h
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hypothesized that Msx2 might activate osteoblast gene alkaline phosphatase (ALP). ://w
skeletal Wnt signaling. Therefore, we Msx2-directed RNA interference (RNAi) w
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analyzed effects of CMV-Msx2 transgene increased Dkk1 expression and promoter .jb
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(Msx2Tg) expression on skeletal physiology activity, while reducing Wnt7a, Wnt7b, and .o
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and composition. Skeletal Msx2 expression ALP. Moreover, Msx2 inhibited Dkk1 b/
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was increased 2- to 3- fold by Msx2Tg, with promoter activity, and reduced RNA g
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expanded protein accumulation in marrow, polymerase association with Dkk1 st o
secondary ossification centers, and chromatin. RNAi-mediated knockdown of n J
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periosteum. Microcomputed tomography Wnt7a, Wnt7b, and LRP6 significantly nu
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established increased bone volume in reduced Msx2- induced ALP. Msx2 exerts ry
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Msx2Tg mice, with increased numbers of bone anabolism in part by reducing Dkk1 , 2
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plate-like trabeculae. Histomorphometry expression and enhancing Wnt signaling, 1
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revealed increased bone formation in thus promoting osteogenic differentiation of
Msx2Tg mice vs. non-Tg sibs, arising from skeletal progenitors.
increased osteoblast numbers. While
decreasing adipogenesis, Msx2Tg increased Msx21 -- a.k.a. Hox-8 -- is a
osteogenic differentiation via mechanisms homeodomain transcription factor first
inhibited by Dkk1, an antagonist of Wnt characterized by Sharpe as a calcitriol-
receptors LRP5 and LRP6. Bone from regulated transcript in osteoprogenitors
Msx2Tg mice elaborated higher levels of isolated from human femur (1), and
Wnt7 canonical agonists, with diminished subsequently shown to be highly expressed in
Dkk1 -- changes that augment canonical the murine embryonic craniofacial skeleton
signaling. Analysis of non-Tg and Msx2Tg (2). Soon thereafter, Msx2 was identified as a
siblings possessing the TOPGAL reporter transcriptional repressor of the osteoblast –
confirmed this; Msx2Tg upregulated specific osteocalcin (OC) promoter(3,4).
skeletal beta-galactosidase expression (p < Studies of Msx2 –regulated gene expression in
0.01) along with Wnt7a, and Wnt7b, and bone have emphasized its role as a
reduced circulating Dkk1. To better transcriptional repressor of the late osteogenic
understand molecular mechanisms, we phenotype(5-7). For example, in the
Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
developing tooth -- where stage-specific (16); this demonstrates the exquisite
osteogenic gene expression profiles are sensitivity of the developing human
spatially resolved -- Msx2 and OC exhibit craniofacial skeleton to Msx2 gene dosage
reciprocal patterns of mRNA accumulation (10,17). While details have emerged as to
(8). Msx2 suppresses OC gene expression in a how Msx2 inhibits osteoblast terminal
cell-autonomous fashion, mediated via differentiation via cell-autonomous actions (5-
antagonistic protein-protein interactions 7), little is known of the mechanisms whereby
between Msx2 and Runx2-containing Msx2 promotes osteoblast-mediated bone
complexes that support OC promoter activity formation. The mechanistic underpinnings
(6,7). of the low-turnover osteopenia demonstrated
However, elegant genetic studies have in Msx2-/- long bone during post-natal growth
demonstrated that Msx2 also promotes have yet to be determined (9). In seminal
craniofacial bone mineralization, and is studies of calvarial bone formation, Maxson
necessary for robust trabecular and cortical showed that Msx2 promotes the proliferative
bone formation (9,10). Msx2-/- mice exhibit expansion and survival of neural crest-derived
parietal foramina, characterized by reduced osteoblasts (10,11).
mineralization in calvarial fields that give rise Recently, we identified that Msx2
to membranous bone(9). Moreover, Msx2-/- participates in the ectopic medial artery D
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mice exhibit a global, low-turnover calcification characteristic of type II diabetes n
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osteopenia(9). Simultaneous deletion of both (18,19). Murine models of diabetic medial ad
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Msx2 and Msx1 – a homologous Msx gene calcification spatially resolved adventitial fro
family member – results in the complete Msx2-expressing cells and the ALP-positive m
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absence of craniofacial bone (9,11). medial calcifying vascular cells (CVCs) that ttp
Furthermore, a non-synonymous CCC to CAC direct matrix mineralization; therefore, we ://w
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mutation at the 7th codon of the Msx2 deduced that Msx2-expressing cells elaborate w
homeodomain causes Boston-type autosomal paracrine signals that control osteogenic .jbc
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dominant craniosynostosis, characterized by differentiation of neighboring progenitors(19). rg
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precocious mineralization and differentiation Indeed, we showed that conditioned media y
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of osteoblasts in the calvarial suture(12). In from Msx2-expressing C3H10T1/2 cells ue
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vivo and in vitro data indicate that this possessed a pro-osteogenic, adipostatic t o
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Msx2(P148H) variant is most likely a gain-of- activity (19) resembling that of canonical Wnt Ja
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function variant; Msx2(P148H) exhibits ligands such as Wnt3a(20), Wnt7a (21), and a
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increased DNA binding to the Msx Wnt10b(22,23). Molecular profiling by 3
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CTGAATTRG binding cognate(13,14). quantitative RT-qPCR demonstrated , 20
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Intriguingly, however, the frequency of upregulation of canonical Wnt agonists Wnt3a 9
transgene-induced craniosynostotic bone is and Wnt7a in aortic tissues (19). By contrast,
three time more frequent in wild-type Msx2 expression of aortic Dkk1, the prototypic
transgenic mice than Msx2(P148H) transgenic vertebrate inhibitor of canonical Wnt
mice (71% vs. 27%, respectively)(13). This signaling, was concomitantly down-regulated
suggests that in vivo the Pro148H mutation by Msx2 (19). Exogenous recombinant Dkk1
perturbs other important Msx2 functions (13). antagonized Msx2 regulation of osteogenesis
Indeed, the region of the homeodomain N- and adipogenesis(19). Thus, Msx2-producing
terminal arm altered by the P148H substitution vascular adventitial cells enhanced aortic Wnt
participates in regulatory protein-protein signaling and promoted ectopic mineral
interactions with Dlx5(5,15) and other deposition via osteogenic mechanisms (19).
transcription factors(5-7). Consistent with the To better understand the mechanisms
Msx2 gain-of-function model arising from whereby Msx2 augments orthotopic long bone
CMV-Msx2 transgenic mice, distal trisomy of formation, we have evaluated the skeletal
chromosome 5q – the physical region phenotype of adult CMV-Msx2 (Msx2Tg)
encompassing the Msx2 gene – has been transgenic mice(19). We show that
identified in six patients with craniosynostosis augmenting Msx2 expression in long bone
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directs bone mesenchymal progenitors to down-stream of the transcription initiation
osteogenic lineage, enhancing skeletal site, within the early regions encoding the
osteoblast numbers, mineralizing surface, PolII-dependent primary transcripts. A second
trabecular number, and trabecular bone amplimer pair directed ~1.5 kb into the Dkk1
formation via canonical Wnt signals. gene (5’-GAA AGC ATC ATT GAA AAC
CTT GGT-3’ and 5’-GCC TTC CCC GCA
EXPERIMENTAL PROCEDURES GTA ACA-3’) was also used to confirm the
effects of Msx2 on PolII- Dkk1 chromatin
Antibodies, ChIP assays, luciferase assays, interactions. For western blot assays, antigen-
plasmids, and other reagents --- Antibodies antibody complexes were detected by alkaline
for Msx2 (sc-15396, lot #C0404, polyclonal phosphatase-dependent CSPD
antibody), Wnt7a/b (sc-32865, lot #F2006, chemiluminescence (Tropix, Applied
rabbit polyclonal), Wnt1 (sc-5630, lot #E2605, Biosystems, Foster City, CA). Conjugated
goat polyclonal antibody), Wnt3a (sc-28824, secondary antibodies, blocking reagents, and
lot #K0205, rabbit polyclonal antibody), peroxidase-based immunovisualization kits
Dkk1, (sc-25516, lot #J1404, rabbit polyclonal were purchased from Vector Laboratories
antibody), actin (sc-8432), and eIF2alpha (sc- (Burlingame, CA). Custom synthesized
11386) were purchased from Santa Cruz oligodeoxynucleotides were synthesized and D
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Biotechnology (Santa Cruz, CA). The Wnt1 purchased from Invitrogen (Carlsbad, CA). n
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antibody (ab15251-500, lot #315275, rabbit TOPGLOW (cat. # 21-204) and FOPGLOW ad
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polyclonal antibody) purchased from Abcam (cat. # 21-205) luciferase reporter plasmids fro
was also used, and proved to be superior for were purchased from Millipore. PCR was m
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Wnt1 immunohistochemistry. The alkaline used to generate cDNA inserts for expression ttp
phosphatase-conjugated goat anti-rabbit plasmids for pcDNA3-Wnt7a and pcDNA3- ://w
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secondary antibody used for western blots Wnt7b using methods previously described(8). w
with rabbit primary immunoreagents was sc- Dkk1 promoter-luciferase reporter constructs .jbc
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2057 (lot I12503, Santa Cruz). Wnt10b 1141 DKK1LUC (-1141 to -1), 451 rg
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antibody (rat anti-mouse, MAB 2110, clone DKK1LUC (-451 to -1), 170 DKK1LUC (- y
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254206) was purchased from R&D Systems 170 to -1), 120 DKK1LUC (-120 to-1), and 70 ue
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(Minneapolis, MN). Goat polyclonal DKK1LUC (-70 to -1) were generated by t o
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antibody to LRP5 (sc-21390) and the donkey ligating the indicated segments of the mouse Ja
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anti-goat secondary antibody (sc-2022) were Dkk1 promoter into pGL2 Basic (Promega); a
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both purchased from Santa Cruz methods have been previously detailed(25). 3
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Biotechnology. VECTASTAIN Elite ABC The numbering of basepairs is relative to the , 20
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peroxidase immunovisualization kits directed start site of Dkk1 exon 1 (physical basepair 9
towards goat (#PK-6105) or rabbit (#PK- position 30,620,374 of mouse chromosome
6101) primary antibodies were purchased from 19, RefSeq Gene NM_010051). The control
Vector Laboratories (Burlingame, CA). minimal promoter – reporter RSVLUC has
Antibodies to PolII (N-20, sc-899, Santa Cruz been previously described(25). Transient
Biotechnology) and phosphor-PolII (H5, transfections and luciferase assays were
MMS-129R, Covance) were used in ChIP carried out as previously detailed(6). Custom
assays as previously described(6,24). The synthesized siRNA reagents were purchased
amplimer pairs 5’-CGG AGG TCC CGA from Qiagen (Valencia, CA). Commercially
AGT TGA G-3’ and 5’-AAA GGT CAG available siRNAs were purchased from Santa
GAA AAG AGA GGT CAC T-3’ and 5’-ATT Cruz Biotechnology as indicated. SYBR
ATT GGT GAC TTG GTG GTG ATC T-3’ Green was purchased from either Roche
and 5’-ATT TTA TGA GGC ACA GTT GAT Applied Science (Indianapolis, IN) or Applied
GTC TT-3’ were used for quantifying DNA in Biosytems. Total β-catenin ELISA kit (#900-
precipitates by fluorescence qPCR for the 135) was purchased from Assay Designs (Ann
Dkk1 and osteopontin genes, respectively. Arbor, MI). All other reagents were purchased
Primer pairs were directed to regions just either from Invitrogen, Fisher Scientific (St.
3
Louis, MO), or Sigma-Aldrich (St. Louis, growth hormone 3’-UTR and polyadenylation
MO). signal from pcDNA3 (Invitrogen). This 3.2 kb
fragment was used to generate transgenic mice
Assays of osteogenic and adipogenic potential made by male C57Bl/6 pronuclear injection at
of primary mouse bone cell cultures -- the Washington University Mouse Genetics
Calvarial osteoblast were prepared from Core. PCR genotyping for the CMV-Msx2-
neonatal mouse calvaria by timed collagenase bGHpolyA transgene was directed toward
digestion using methods previously uniquely juxtaposed 3’-cDNA Msx2 (5’-TGT
described(26). Bone mesenchymal cells were GCT CCC CAT CCC GCC TGT TGG ACT
isolated from the marrow compartment of long CTA-3’) and vector 3’-UTR sequences (5’-
bones of 4-month-old Msx2Tg and WT mice AAG GAC AGT GGG AGT GGC ACC TTC
using methods previously described (27), then CAG GGT-3’) using the indicated primers,
tested for the capacity to undergo osteogenic following the protocol of Stratman(31).
or adipogenic differentiation. For osteogenic TOPGAL mice(32) (TCF/LEF Optimal
differentiation, cells were seeded in 96-well Promoter / Galactosidase reporter transgenic
tissue culture plates at a density of 1 x 105 mice) were purchased from Jackson Labs
cells/well, left undisturbed for 7 days, then (mouse stock #004623; genotyping
maintained in mineralization medium(α-MEM amplimers: 5’-GAG TGA CGG CAG TTA Do
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medium containing 10% FBS, 50 µg/ml TCT GGA AGA TCA GGA-3’ and 5’-GGA n
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ascorbic acid and 10 mM β-glycerophosphate) AAC CGA CAT CGC AGG CTT CTG CTT ad
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for 3 additional weeks. Cultures were CAA TCA-3’). TOPGAL mice were bred with fro
subsequently stained for calcium deposition Msx2Tg mice to generate Msx2Tg;TOPGAL m
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using Alizarin red. Wells containing red animals on a mixed C57Bl/6:CD1 ttp
nodules with 15 or more cells were considered background. ://w
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positive. The osteogenic potential was w
determined as the percent of positive wells in Immunohistochemistry and western blot .jbc.o
total wells. For adipogenic differentiation analysis -- Multiple commercially - available brg/
assays, bone mesenchymal cells were seeded immunoreagents were tested for sensitivity y g
at 1 x 107 cells per 10 cm culture dish. Seven and specificity, and only those that proved ue
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days later, cells were treated with adipogenic sensitive and specific are reported here. Prior t on
medium containing insulin (5 µg/ml), to immunohistochemistry, the specificity of Jan
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dexamethasone (10-7 M), and indomethacin the antibody reagents was first gauged by ary
Western blot analysis as previously detailed 3
(50 µM) for 9 additional days. Cultures were 1
stained with the lipotrophic dye Oil Red O as (30), using protein extracts prepared from (a) , 201
C3H10T1/2 cells transduced with either 9
previously detailed (28,29). The numbers of
control SFG-LacZ virus or with SFG-Msx2,
adipocyte colonies were enumerated, and data
and then (b) mouse primary calvarial
expressed as the percent of total cell colonies
osteoblast cell extracts from either WT or
that stained positive with Oil Red O.
Msx2Tg mice. Only those reagents that

generated single immunoreactive bands of the
CMV-Msx2 (Msx2Tg) and TOPGAL
appropriate relative mass following SDS-
transgenic mice – The generation and
PAGE and western blot analysis were utilized
maintenance of CMV-Msx2Tg mice (Msx2Tg)
for immunoperoxidase staining. For neonatal
on the C57Bl/6 murine background has been
long bones, following 48 hour fixation in 10%
previously detailed(19). Msx2Tg mice were
neutral buffered formalin, samples were
generated essentially as described by Maxson
imbedded in paraffin without decalcification,
(13). The eukaryotic pcDNA3-Msx2
and 5 micron longitudinal sections prepared
expression construct DT21.14 (30) was
for Msx2, Wnt7a/b, and Wnt1
digested with BglII and NruI to release the 3.2
immunohistochemistry. For adult long bones,
kb fragment encoding the CMV immediate
ossicles were fixed for 48 – 72 hours in 10%
early promoter, mouse Msx2 cDNA (with N-
neutral buffered formalin, then decalcified for
terminal Met-FLAG tag), and the bovine
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3 weeks at 4 ºC in 0.375 M EDTA, pH = 8, in OCT (Sakura Tissue-Tek). Subsequently,
with constant gentle stirring. Decalcified adult 25 micron thick longitudinal frozen sections of
long bones were imbedded in paraffin, and 5 non-decalcified specimens were analyzed by
micron sections cut for subsequent histochemistry for β-galactosidase (LacZ)
immunohistochemistry using methods enzyme activity as previously detailed for
previously described(19). Briefly, after mouse aortas(19), comparing LacZ staining of
deparaffinization of the sections with xylene proximal tibiae in Msx2Tg;TOPGAL vs.
and rehydration via graded aqueous ethanolic TOPGAL animals.

baths, endogenous peroxidases were quenched For dynamic and static
by treatment with 1% H202 in methanol for 20 histomorphometric studies, 6 month old male
minutes, rinsed with water for one minute mice (7 Msx2Tg, 11 WT siblings) were
followed by 10 more minutes of rinsing in sequentially dosed at 8 days and 3 days prior
PBS, then incubated in blocking solution (10% to sacrifice, first with subcutaneous
normal serum cognate to the secondary tetracycline (1 mg/0.1 ml per 30 gram body
antibody and 5% BSA in PBS) for 30 min at weight) and then with subcutaneous calcein (1
25 ºC. Sections were then incubated with 2 mg/0.1 ml per 30 gram body weight),
ug/ml primary antibody in blocking solution respectively. Following euthanasia via
overnight (> 18 hours) at 4 ºC. The next exsanguination under ketamine-xylazine Do
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morning, after washing thrice for 10 minutes anesthesia, right hindlimbs were dissected en n
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with PBS at RT, antigen-antibody complexes bloc, and the combined femur, genu, and tibia ad
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were tagged with VECTASTAIN Elite ABC dissected free of adherent muscle and fascia. fro
peroxidase kits directed towards either goat Carefully liberated right femurs were fixed in m
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(#PK6105) or rabbit (#PK6101) primary 10% neutral buffered formalin at 4 ºC for 2 ttp
antibodies as appropriate (vide supra), by days, washed twice for 15 minutes in PBS, ://w
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incubating sections with 1:200 dilution of the then stored in 70% ethanol until embedded in w
biotinylated secondary antibody for 30 Osteo-Bed Resin for preparation of .jbc
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minutes. After washing and incubation with longitudinal plastic sections. Dynamic and rg
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ABC anti-biotin reagent, specimens again static histomorphometry was performed under y
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washed 2 x 10 min with sterile PBS and a fee-for-service contract with MDS Pharma ue
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complexes visualized by diaminobenzidine Services, formerly SkeleTech, Inc. (Bothell, t o
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staining (#SK-4100), with stain intensity WA), using Osteomeasure Software (33), and Ja
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monitored by microscopy. Reactions were following nomenclature and recommendations ary
stopped by rinsing with tap water. Equal time of the American Society for Bone and Mineral 31
of stain development was utilized for both WT Research Histomorphometry Committee (34). , 20
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and MsxTg tissue sections monitored in For bone formation rate (BFR), the tissue 9
parallel. After dehydration in graded ethanol volume referent (TV) – i.e., BFR/TV (%) –
to xylene, samples were mounted using was chosen to facilitate comparisons with
VectaMount permanent mounting media and contemporary reports of Wnt-regulated(35),
digital images captured using a Leica 4100 Dkk1-regulated(36), and other anabolic
DM microscope coupled with a DFC 420 responses (37-41) in the mouse skeleton.
digital camera, operated with FW 4000
software (Leica Microsystems, Bannockburn, Conebeam microCT imaging -- The left tibiae
IL) as previously detailed(19). obtained from 6 month old mice described
above were used to assess trabecular bones
Histochemistry and histomorphometric using Scanco microCT 40 (Scanco USA Inc,
methods -- Histochemistry for LacZ activity Southeastern, PA) at an energy setting of
in Msx2Tg;TOPGAL and TOPGAL mice was 55kV, current setting of 145 mA, and with an
carried out essentially as previously integration time of 300 ms. A fixed threshold
detailed(19). Long bone from of 270 Hounsfield units was used to separate
Msx2Tg;TOPGAL and TOPGAL siblings were bone from background. A total of 300-350
excised, rinsed twice in PBS, then embedded transverse CT slices were obtained and three-
5
dimensional analysis was performed on Two step RT-qPCR was used quantify gene
trabecular bones in the 50 slices (16 um expression in total RNA extracted from either
thick/slice) starting at about 0.1 mm below the mouse bone (44) or cultured cells (28)using
lowest point of the growth plate. Trabecular the respective methods previously detailed. A
morphometric parameters including the mixture of random hexamers and oligo-dT
percentage of bone volume per total volume was used to prime cDNA synthesis, so that
(%BV/TV), trabecular thickness (Tb Th, um), relative mRNA accumulation could be
trabecular number (per mm3), trabecular normalized to the signal obtained for 18S
spacing (um), structure model index, and ribosomal RNA in parallel. Amplimer pairs
connectivity density (connections per mm3) used for quantitative RT-qPCR were as
were acquired by direct method of follows: Msx2: 5’- CCG CCG CCC AGA
calculation(42). CAT A -3’ and 5’- CTT CCG GTT GGT CTT
GTG TTT C -3’; Wnt1: 5’- AAA TGG CAA
Serum ELISAs for tartrate resistant acid TTC CGA AAC CG -3’ and 5’- CGA AGA
phosphatase (TRAP5c), CTX, and Dkk1 -- TGA ACG CTG TTT CTC G -3’; Wnt3a: 5’-
After overnight fasting, mice were CAT GCA CCT CAA GTG CAA ATG -3’
anesthetized and blood withdrawn from and 5’- TGA GGA AAT CCC CGA TGG T -
descending aorta in heparinized syringes. 3’; Wnt5a: 5’- CCA CGC TAA GGG TTC D
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Blood samples were layered on top of CTA TGA G-3’ and 5’-TGT CCT ACG GCC n
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Microtainer Serum Separator tubes (#365956; TGC TTC A-3’; Wnt7a: 5’- TGG ATG CCC ad
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Bate 4cokCto. n S eDriac wkienrseo no)b taainnde di nacfutebra cteedn troifvuegrnatiigohnt GCTGCG GATGTA ATTCT - 3-3’ ’a; n dW 5n’t-7 bC: C G5’ -ATCTCC CTGGGC from
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at 2,000 x g for 20 min at room temperature. AGG ACC GCA TGA A-3’ and 5’-GGT CCA ttp
Serum concentration of osteoclast-derived GCA AGT TTT GGT GGT A-3’; Wnt10b, 5’- ://w
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tartrate-resistant acid phosphatase form 5b was CGA GAA TGC GGA TCC ACA A-3’ and w
determined by using MouseTRAP Assay kit 5’-CCG CTT CAG GTT TTC CGT TA-3’; .jbc
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(Code TR103; IDS Inc., Fountain Hills, AZ). bacterial beta-galactosidase/LacZ: 5’-GGT rg
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Commercial ELISAs for CTX (RatLaps, TAC GAT GCG CCT ATC TAC AC-3’ and y
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1RTL4000, Nordic Bioscience Diagnostics via 5’-CTC CGC GGG AAC AA CG-3’. ALP: ue
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IDS Inc.) and Dkk1 (DuoSet DY1765; R&D 5’-ACA CCA ATG TAG CCA AGA ATG t o
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Systems) were carried out per the TCA-3’ and 5’-GAT TCG GGC AGC GGT Ja
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manufacturer’s instructions. TAC T-3’; Dkk1 5’- GCT GCA TGA CGC a
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ACG CTA T -3’ and 5’- AGA GGG CAT 3
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Transfection and retroviral transduction -- GCA TAT TCC ATT T -3’; Runx2: 5’- AGG , 20
1
The pseudotyped retrovirus SFG-LacZ AGG GAC TAT GGC GTC AA-3’ and 5’- 9
(43)was the kind gift of Dr. Dan Ory TCG GAT CCC AAA AGA AGC TTT-3’;
(Washington University in St. Louis). SFG Osx: 5’-CCC TTC TCA AGC ACC AAT GG-
retroviruses expressing Dkk1, Msx2, 3’ and 5’-AAG GGT GGG TAG TCA TTT
Msx2(P148H), and Msx2(T147A) were GCA TA-3’; BSP: 5’- CAG AGG AGG CAA
generated using methods previously detailed GCG TCA CT -3’ and 5’- GCT GTC TGG
(28). Transduction of C3H10T1/2 cells and GTG CCA ACA CT -3’; LRP5: 5’-GAG CGA
primary mouse mesenchymal cells was GGA GGC CAT CAA-3’ and 5’- GCC CGA
performed as detailed. (28). Cells transduced GAT GAC AAT GTT CT-3’; LRP6: 5’-TGT
with SFG-LacZ encoding β-galactosidase GGG CCT GAC CGT GTT-3’ and5’- TTC
were used as a negative control. The GAG CCT GGA CCT TGG T-3’; 18S rRNA:
expression of Msx2 and Dkk1 in SFG-Msx2 5’-CGG CTA CCA CAT CCA AGG AA -3’
and SFG-Dkk1 transduced cells was verified and 5’- GCT GGA ATT ACC GCG GCT -3’.
via real-time RT-qPCR analysis of mRNA
(vide infra). Knock-down of Msx2-Wnt signaling
components by siRNA in C3H10T1/2 mouse
Gene expression by fluorescence RT-qPCR – mesenchymal osteoprogenitors --
6
Commercially available siRNAs directed to incubation for 6 h at 37 ºC in a humidified
murine Wnt1 (sc-36840), Wnt5a (sc-41113), 5% CO2 cell culture chamber, an
Wnt7a (sc-41115), Wnt7b (sc-41117), additional 1 ml of 20% FBS in DMEM
Wnt10b(sc-37186), and control siRNA (sc-
was added per well, and lipofection
44230) were purchased from SantaCruz
allowed to continued overnight. The next
Biotechnology. Sequences for these reagents
morning, the lipofection media was
can be obtained upon request from the
aspirated, and transfected monolayers cells
manufacturer (1-800-457-3801 x 125).
re-fed with fresh C3H10T1/2 growth
Custom siRNAs used for knockdown of
media (10% FBS in DMEM). Twenty-
LRP5, LRP6, and Msx2 were ordered from
Qiagen (Valencia, CA) as high- performance four hours later, total cellular RNA
purity (HPP) - grade reagents. The siRNA harvested as previously detailed (19) using
designed for Msx2 knockdown was derived Ambion Turbo DNase treatment and removal
from the viral RNAi strategy previously kit (#AM1907; Applied Biosystems) to
described by Cossu et al. (45) Custom remove all traces of genomic DNA.
sequences for mouse LRP5 and LRP6 Quantitative RT-qPCR was carried out in
knockdown were designed using Easy siRNA triplicate using methods previously
Software (Protein Lounge, San Diego, CA; described(28). Efficiency of target mRNA D
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www.proteinlounge.com). Specific reagents knockdown of > 30% (range 30% to 50%) was n
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used for Msx2, LRP5, and LRP6 knockdown observed for all siRNA reagents presented, ad
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were as follows: Msx2,5’- r(CAG UAC CUG using RT-qPCR for targeted mRNA d fro
UCC AUA GCA G)dTdT-3’ and 5’-r(CUG accumulation as an assay. For RNAi using m
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CUA UGG ACA GGU ACU G)dTdT-3’; primary bone marrow cell cultures from ttp
LRP5, 5’-r(GGA GAU CCU UAG UGC UCU Msx2Tg mice, the above protocol was also ://w
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G)dTdT-3’ and 5’-r(CAG AGC ACU AAG implemented, except that TransIT- TKO w
GAU CUC C)dTdT-3’; LRP6, 5’-r(GAG Transfection Reagent (Mirus, Madison, WI) .jbc
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AAU GCA ACG AUU GUA G)dTdT-3’ and was used to introduce the siRNA reagents. rg
5’-r(CUA CAA UCG UUG CAU UCU by/
g
C)dTdT-3’ and ordered from Qiagen Statistics – For statistical testing of dynamic ue
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(Valencia, CA). histomorphometry, microCT, and gene t o
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Targeted mRNA degradation using expression results, analyses were performed Ja
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siRNA technology was performed essentially using Student’s t-test as previously a
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as described by Brazas and Hagstrom (46), described(44). All data are presented as the 3
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according to the protocol provided by mean ± standard error of measurement , 20
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Santa Cruz Biotechnology. Briefly, (S.E.M.) to facilitate meaningful comparisons 9
C3H10T1/2 cells were seeded at 2 x (95% confidence interval +/- 1.96 S.E.M.)
105/well in 6-well cluster plates (35 mm
RREESSUULLTTSS
diameter wells) in DMEM containing 10%

FBS the day before lipofection. To
CMV-Msx2 transgenic mice exhibit higher
prepare lipid-siRNA complexes, 80 pmol
bone volume with greater numbers of plate-
of the indicate siRNA duplex in 100 ul of
like trabeculae as compared to wild type
Transfection Medium (sc-36868) and 6
littermates -- We previously demonstrated
uL of siRNA Transfection Reagent (sc- that Msx2 stimulated osteogenic
29528) in 100 uL of Transfection Medium differentiation but inhibited adipogenesis in
were combined, incubated for 30 minutes cultured C3H10T1/2 mesenchymal cells
at 25 ºC, and then diluted with 800 uL of (28,29). We wished to better understand the
pre-warmed, Transfection Medium. Cell physiological relevance of osteogenic-
adipogenic lineage allocation by Msx2 on
were rinsed once with serum free DMEM,
post-natal bone homeostasis. Therefore, we
and 1000 uL of lipid-siRNA admixture
extended our studies to the evaluation of bone
described above applied per well. After
7
and body composition in our CMV-Msx2 trabecular bone volume (Tb.BV/TV; 40%
transgenic (Msx2Tg) mice(19) -- a murine increase), trabecular number (Tb.N; 20%), and
model previously demonstrated by Maxson to trabecular connectivity density (80% increase;
efficiently recapitulate key features of Msx2 Figure 2A-2C). The structure model index
actions in craniosynostosis(13). Msx2 mRNA (SMI) – an index that diminishes as trabeculae
was significantly increased in calvarial become more sturdy and plate-like (52) -- was
osteoblasts (2.57±0.07 fold) and bone marrow 40% lower in Msx2Tg mice (Figure 2D; p =
cell cultures (1.81±0.17 fold) in Msx2Tg mice 0.04); thus, trabeculae in Msx2Tg mice were
as compared with WT littermates (p < 0.05 for more plate-like than those of their WT
both). Western blot and digital image analysis sibs(52). While Msx2Tg-dependent increases
of osteoblast extracts isolated from long bone in tibial trabecular thickness did not reach
and calvaria confirmed that Msx2 protein significance by microCT (not shown), static
levels paralleled the transgene-mediated morphometry of femoral trabeculae did
increases in mRNA accumulation (Figure establish a significant 15% increase in femoral
1A). We localized the spatial patterns of trabecular thickness (Tb.Th) -- and confirmed
Msx2 expression in long bone from Msx2Tg Msx2-dependent increases in Tb.N and
mice by immunoperoxidase staining in 3 day Tb.BV/TV (Figure 3, vide infra). Long bone
old mice. Overall signal intensity was cortical thickness was also significantly Do
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increased in Msx2Tg vs. WT siblings, due to increased by 33% in Msx2Tg vs. WT siblings n
lo
expanded Msx2 immunoreactivity in the bone (p < 0.05). Thus, augmenting Msx2 ad
e
d
marrow compartment, the periosteum, and expression in bone significantly increases fro
secondary ossification centers (Figure 1B; trabecular marrow bone mass accrual. m
h
please also see online supplement). Secondary ttp
(epiphyseal) ossification centers begin to form Msx2Tg mice exhibit greater osteoblast ://w
w
in mice in the first post-natal week (47,48); in numbers and increased trabecular bone w
long bone sections where secondary formation -- Detailed static and dynamic .jbc
.o
ossification centers were visualized, Msx2 histomorphometry(34,53) of secondary rg
b/
expression in associated hypertrophic spongiosa in the distal femur revealed the y
g
chondrocytes was also expanded in Msx2Tg cellular mechanisms of Msx2 long bone ue
s
mice (Figure 1B). The recently described anabolism. Significant increases in trabecular t on
accumulation of Msx2 protein in a subset of bone volume / total volume (Tb.BV/TV; Jan
u
early hypertrophic zone chondrocytes was also Figure 3A), Tb.N (Figure 3B), and Tb.Th ary
noted (49), and was enhanced in Msx2Tg vs. (Figure 3C) in Msx2Tg mice (n = 7) vs. non- 31
WT sibs (Figures 1B). No immunoreactivity transgenic siblings (n = 11) were observed at 6 , 20
1
was observed in the absence of primary months of age. Importantly, the trabecular 9
antibody (Figure 1B, right panel). In the bone formation rate / tissue volume (BFR/TV;
epiphyseal ossification center, Msx2 appeared tissue volume referent) was increased 76% (p
to localize primarily to chondrocyte nuclei = 0.03; Figure 3D). A similar change was
(Figure 1C). However, in marrow and noted when bone surface referent was used for
periosteal venues, both nuclear and BFR (WT = 52.5 +/- 9.1 um3/um2/year vs.
cytoplasmic Msx2 immunoreactivity were Msx2Tg = 73.7 +/- 8.4 um3/um2/year, p =
clearly noted (Figure 1D) – presumably 0.065 for the 40% BFR/BS increase). A 2.7-
reflecting stage-specific re-localization of fold increase in osteoid surface / bone surface
Msx2 in bone mesenchymal progenitors as was seen with no change in osteoid thickness
recently described(50). (not shown), indicating normal and unchanged
To examine the effects of Msx2 on matrix mineralization. Since mineralizing
bone content and structure, we performed surface (MS/BS; Figure 3E) was increased
conebeam microCT analysis of the proximal without a change in matrix apposition rate
tibia (51), comparing Msx2Tg vs. WT siblings. (MAR; Figure 3F), this strongly suggested
Consistent with increased BMD (vide infra), Msx2Tg-induced increases in BFR/TV were
Msx2Tg mice exhibited significant increases in due to increased numbers of osteoblasts.
8
Osteoblast numbers were indeed increased; the obese Msx2Tg and non-Tg littermates (p
osteoblast perimeter / bone perimeter =0.43; Figure 4A). Serum leptin, an
(Ob.Pm/B.Pm) was 50% greater in Msx2Tg vs. adipokine marker of body fat content(55), was
non-Tg sibs (p = 0.001; Figure 3G). By also reduced in Msx2Tg vs. non-Tg sibs after
contrast, osteoclast numbers were not dietary challenge challenge (WT = 1773 +/-
increased (Oc.Pm/B.Pm; Figure 3H). 384 pg/ml; Msx2Tg = 452 +/-174; p<0.001;
Increased bone formation without significant Figure 4B). Consistent with the whole body
changes in osteoclast-mediated bone changes in tissue composition, examination of
resorption was confirmed by serum marker proximal tibiae revealed reduced marrow fat
analysis(54); serum TRAP5c (tartrate-resistant accumulation by histology in Msx2Tg mice;
acid phosphatase; WT = 1.09 +/- 0.11 IU/mL Figure 4C is representative of results
vs. Msx2Tg = 1.01+/- 0.17 IU/mL; p = 0.68) observed from 8 animals studied, 4 of each
and CTX (collagen C-terminal telopeptide ; genotype (see online supplement for additional
WT = 29.7 +/- 4.2 ng/ml vs. Msx2Tg = 23.0 data). Quantitative analysis of long bone
+/- 3.6 ng/ml; p = 0.28) did not differ between mesenchymal cells isolated from 4 WT and 7
Msx2Tg and non-Tg sibs. Thus, Msx2Tg Msx2Tg mice fed high fat diets for 4 months
increases long bone mass and trabecular bone demonstrated a reduction in oil red O-positive
formation by increasing the total number of adipocytic colony formation (WT = D
o
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trabecular bone-forming osteoblasts, without 76.4±0.8%, Msx2Tg = 35.7±6.0% oil red-O n
lo
significant changes in osteoclast-mediated positive colonies, Figure 4D). Thus, as ad
e
d
bone resorption. compared to non-Tg WT littermates, Msx2Tg fro
mice exhibit globally enhanced bone mass, m
h
Msx2 transgenic mice exhibit globally higher with reciprocal reductions in body fat and long ttp
bone mineral density, with lower body and bone marrow fat. ://w
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marrow fat as compared to wild type w
littermates -- Global analysis of body Long bone mesenchymal cells derived from .jbc
.o
composition by dual electron X-ray Msx2 transgenic mice exhibit enhanced rg
b/
absorptiometry (DXA) (44)in 1 month old sibs osteogenic differentiation in vitro in addition y
g
confirmed and extended the detailed to reduced adipogenic differentiation -- To ue
s
histomorphorphometry of hind-limb long further study the mechanisms of increased t o
n
bone; whole body DXA indicated that the bone formation and reduced adipose in Ja
n
u
Msx2Tg skeleton globally exhibited a small Msx2Tg mice, we isolated long bone ary
but statistically significant 3.3% greater bone mesenchymal cells and calvarial osteoblasts 31
mineral density (BMD; WT = 30.38±0.39 from WT and Msx2Tg mice, and compared , 20
1
mg/cm2; Msx2Tg = 31.39±0.37, p = 0.05) -- their osteogenic activities in cultures. The 9
with a 10% percent reduction in total body fat osteogenic potential of Msx2Tg bone
(WT = 14.4±0.53%; Msx2Tg = 13.03±0.42%, mesenchymal cell cultures, which contain both
p = 0.04) vs. non-transgenic littermates. No osteoblast and adipocyte precursor cells, was
difference in overall body weight was increased 1.5-fold relative to WT littermate
observed (WT = 14.74 +/- 0.28 gm; Msx2Tg = bone mesenchymal cell cultures (WT =
15.16 +/- 0.52 gm; p = 0.23). The significant 37.5±5.6%, Msx2Tg = 56.3±8.4% mineralized
differences in BMD and fat mass persisted colonies, Figure 5A). Moreover, calvarial
after 4 months of challenge with high fat diets osteoblasts from Msx2Tg mice exhibited
with content typical of westernized societies; significantly increased alkaline phosphatase
whole body BMD remained significantly (ALP) activity (2.08 ± 0.07 fold, Figure 5B)
elevated (WT = 51.84±0.46 mg/cm2 vs. and equivalently elevated mRNA
Msx2Tg = 52.92±0.42 mg/cm2, p = 0.04) and accumulation for ALP, osterix (Osx), and bone
fat mass was reduced (WT = 45.3±0.84% vs. sialoprotein (BSP; Figure 5C). Intriguingly,
no change was observed in the relative
Msx2Tg = 43.1±0.61%; p = 0.02) in Msx2Tg
accumulation of Runx2 message (Figure 5C).
mice (Figure 4A). Again, no difference in
Accumulation of the nuclear transcriptional
overall body weight was detected between
9
co-adapter, β-catenin, has been shown to be the Msx2 transgene (Figure 6B). Thus,
critical to elaboration of osteoblast Msx2Tg induction of osteogenic development
development downstream of osteogenic Wnt in transgenic osteoblasts is dependent upon
signaling(56). Therefore, cellular extracts of endogenous paracrine canonical Wnt signaling
bone mesenchymal cell culture from WT and cascades.
Msx2Tg mice were prepared, and β-catenin
protein accumulation quantified by ELISA. The Msx2 transgene upregulates Wnt7 but
Total cellular β-catenin protein was elevated suppresses Dkk1 protein accumulation in
1.6-fold in Msx2Tg vs. non-Tg long bone cell calvarial osteoblasts and long bone
cultures (Figure 5D, p = 0.02), and 2 fold in mesenchymal cells -- Our studies of arterial
calvarial osteoblasts (data not shown). Thus, calcification have revealed that Msx2 controls
the ex vivo osteogenic potential of osteogenic lineage allocation of aortic vascular
mesenchymal progenitors isolated from progenitors (28) via paracrine Wnt signals
Msx2Tg mice is enhanced compared to non-Tg (19). The body composition changes observed
WT sibs -- consistent with the increased in response to Msx2Tg resembled those of the
osteoblast numbers observed in Msx2Tg mice canonical ligand Wnt10b(20,22,23).
long bone as quantified by histomorphometry. Moreover, since SFG-Dkk1 inhibited Msx2Tg
actions, this strongly suggested that Do
w
Msx2Tg activation of osteogenic modulation of Wnt and Dkk1 protein nlo
differentiation is antagonized by Dkk1, an biogenesis contributes to Msx2 bone ad
e
d
inhibitor of canonical Wnt signaling – The anabolism (vide supra; and ref. (28). To test fro
reciprocal control of bone mesenchymal cell this notion, we analyzed protein accumulation m
h
osteogenesis and adipogenesis by the Msx2 of Wnt1, Wnt3a, Wnt7 (antibody recognizes ttp
transgene was highly reminiscent of effects of both Wnt7a and Wnt7b), Wnt10b, and Dkk1 – ://w
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canonical Wnt signaling(22,23). Wnt10b has prominent genomic targets of Msx2 first w
been shown to promote osteogenesis and identified in aortic adventitial myofibroblasts .jbc.o
suppress adipogenic differentiation from transduced with SFG-Msx2 (19). As brg/
multipotent marrow osteoprogenitors (22,23). compared to non-Tg sibs, Msx2Tg upregulated y g
If paracrine canonical Wnt signals contribute Wnt1 and Wnt7 in calvarial osteoblasts ca. 3- ue
s
to the effects of Msx2Tg on bone marrow fold, but suppressed Dkk1 protein t on
osteogenesis, then Dkk1 – a highly specific accumulation (Figure 7A, expression Jan
u
inhibitor of LRP5/6 Wnt receptor signaling normalized to eIF2α protein signal intensity). ary
complexes(36) – should antagonize Msx2Tg No change was observed in Wnt3a or Wnt10b 31
actions. We thus generated a retroviral protein accumulation. In long bone , 20
1
expression construct, SFG-Dkk1, to augment mesenchymal cells, little if any change 9
Dkk1 production by bone mesenchymal cells. occurred in Wnt1 and Wnt10b protein levels
We first tested effects of SGF-Dkk1 (Figure 7A). However, Wnt7 was once again
transduction on C3H10T1/2 cell cultures increased, and Dkk1 decreased, by the Msx2
previously transduced with SFG-Msx2; in this transgene; accumulation of Wnt7 protein was
validated cell culture model, Msx2 actions are increased ca. 10-fold in Msx2Tg animals vs.
antagonized by exogenous recombinant non-transgenic sibs (Figure 7A). As compared
purified Dkk1(19). As shown in Figure 6A, to calvarial osteoblasts, Wnt3a protein levels
SFG-Dkk1 completely abrogated the effects of were lower in long bone mesenchymal cells,
SFG-Msx2 on ALP induction in C3H10T1/2 but were increased 2-fold by the Msx2
cells. We next evaluated effects of SFG-Dkk1 transgene.
transduction on osteogenic differentiation Since Wnt7 protein accumulation was
using calvarial osteoblasts isolated from consistently and dynamically regulated by the
Msx2Tg vs. non-Tg siblings. As compared to Msx2Tg in primary cell culture,
control virus (SFG-LacZ), transduction with immunohistochemistry was used to spatially
SFG-Dkk1 profoundly inhibited the resolve the expression of Wnt7 in Msx2Tg
mineralized nodule formation upregulated by long bone. In neonatal mice, Wnt7
10