Wnt proteins promote bone regeneration

DOI: 10.1126/scitranslmed.3000231, 29ra30 (2010);2 Sci Transl Med

, et al.Steven MinearWnt Proteins Promote Bone Regeneration

http://stm.sciencemag.org/content/2/29/29ra30.full.htmlfigures, can be found at:

and other services, including high-resolutionA complete electronic version of this article

http://stm.sciencemag.org/content/suppl/2010/04/26/2.29.29ra30.DC1.html "Supplementary Material"

can be found at: Supporting Online Material

http://stm.sciencemag.org/content/2/29/29ra30.full.html#ref-list-1, 18 of which can be accessed free:cites 60 articlesThis article

http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: reproduce this article

permission to of this article or about obtaining reprintsInformation about obtaining

is a registered trademark of AAAS. Science Translational Medicinerights reserved. The title NW, Washington, DC 20005. Copyright 2010 by the American Association for the Advancement of Science; alllast week in December, by the American Association for the Advancement of Science, 1200 New York Avenue

(print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except theScience Translational Medicine

on

May

2, 2

010

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

http://stm.sciencemag.org/content/2/29/29ra30.full.html

http://stm.sciencemag.org/content/suppl/2010/04/26/2.29.29ra30.DC1.html

http://stm.sciencemag.org/content/2/29/29ra30.full.html#ref-list-1

http://www.sciencemag.org/about/permissions.dtl

http://stm.sciencemag.org/

R E S EARCH ART I C L E

BONE

Wnt Proteins Promote Bone RegenerationSteven Minear,1* Philipp Leucht,1,2* Jie Jiang,1* Bo Liu,1 Arial Zeng,3 Christophe Fuerer,3

Roel Nusse,3† Jill A. Helms1†

(Published 28 April 2010; Volume 2 Issue 29 29ra30)

010

The Wnt signaling pathway plays a central role in bone development and homeostasis. In most cases, Wntligands promote bone growth, which has led to speculation that Wnt factors could be used to stimulate bonehealing. We gained insights into the mechanism by which Wnt signaling regulates adult bone repair throughthe use of the mouse strain Axin2LacZ/LacZ in which the cellular response to Wnt is increased. We found that bonehealing after injury is accelerated in Axin2LacZ/LacZ mice, a consequence of more robust proliferation and earlierdifferentiation of skeletal stem and progenitor cells. In parallel, we devised a biochemical strategy to increasethe duration and strength of Wnt signaling at the sites of skeletal injury. Purified Wnt3a was packaged in li-posomal vesicles and delivered to skeletal defects, where it stimulated the proliferation of skeletal progenitorcells and accelerated their differentiation into osteoblasts, cells responsible for bone growth. The end resultwas faster bone regeneration. Because Wnt signaling is conserved in mammalian tissue repair, this protein-based approach may have widespread applications in regenerative medicine.

2, 2

on

May

st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

INTRODUCTION

In the skeleton, adult skeletal stem cells serve two essential functions.First, they are responsible for generating osteocytes that maintainbone mass and bone volume during physiologic bone turnover (1).Second, they are essential for replacing missing bone after skeletal dis-ease or injury (2). In both scenarios, Wnt signals regulate skeletalstem and progenitor cell behavior and control bone formation. Wntproteins are lipid-modified growth factors that are secreted from onecell and transported to adjacent cells, perhaps in association withmembranes (3, 4). Upon binding to a cell surface receptor complex,Wnt signals initiate an intracellular cascade that results in tissue-specific activation of target gene transcription.

Reductions in Wnt signaling, by overexpression of Wnt antagonists,deficiency of Wnt ligands, or mutations in genes encoding Wnt re-ceptors, cause bone loss (5–7). Conversely, enhancement of Wntsignaling results in increased bone volume, abnormal bone density(that is, hyperostosis), and pathological thickening of bone, a condi-tion known as sclerosing bone dysplasia (8–11). Wnt signals achievethese effects by promoting bone-forming osteoblast activity (12, 13),by inhibiting bone-resorbing osteoclast function (14, 15), and by driv-ing the differentiation of many types of multipotent stem cells towardan osteoblast cell fate (16–18). These data suggest that Wnt proteinscould be used as therapeutic agents to stimulate bone formation afterinjury or disease. The use of Wnts for bone regeneration, however,has not been directly tested because of practical difficulties inadministering the highly hydrophobic and insoluble Wnt proteins.Soluble agonists of the Wnt pathway have been identified, but noneact in the absence of Wnt proteins (19–22).

1Department of Surgery, Division of Plastic and Reconstructive Surgery, StanfordSchool of Medicine, Stanford, CA 94305, USA. 2Department of Orthopaedic Surgery,Stanford School of Medicine, Stanford, CA 94305, USA. 3Howard Hughes MedicalInstitute, Department of Developmental Biology, Stanford School of Medicine,Stanford, CA 94305, USA.*These authors contributed equally to this work.†To whom correspondence should be addressed. E-mail: [email protected] (R.N.);[email protected] (J.A.H.)

www.S

There is another difficulty that needs to be resolved before Wntscan be used as therapeutic agents: Wnt signals regulate multiplephases of the skeletogenic program ranging from proliferation andcommitment of skeletal progenitor cells to the maturation of chon-drocytes and osteoblasts (2). Accordingly, the optimal time for Wntpathway stimulation requires a detailed understanding of the func-tion of endogenous Wnt signaling during the entire reparative process.For example, we demonstrated that skeletal injury triggers a rapid in-crease in Wnt signaling at the site of damage (5) and that Wnt signalsare necessary for bone healing (5, 23), but whether exogenous Wntsignals are sufficient to promote bone healing is not known.

RESULTS

Mutating a negative feedback regulator potentiates Wntsignaling in a ligand-dependent mannerTo determine how Wnt signals influence bone homeostasis, we useda strain of mice in which LacZ was inserted into the Axin2 gene (24).Axin2, a target of Wnt signaling (24), encodes a ligand-dependent,negative feedback regulator that dampens Wnt signaling in a cell-autonomous manner. The LacZ insert inactivates Axin2 (24), andthus, homozygous mutant mice (Axin2LacZ/LacZ) provide a geneticmodel in which to assess how loss of a negative Wnt regulator affectsbone homeostasis and bone repair.

We first evaluated how the Axin2 mutation influenced Wnt sig-naling in osteoprogenitor cells in culture. To measure Wnt pathwayactivity, we used a T cell factor (Tcf)–dependent luciferase reporterconstruct, which was infected (through a lentiviral vector) into primaryosteoprogenitor cells isolated from Axin2LacZ/LacZ, Axin2LacZ/+, andwild-type mice. In the unstimulated state, Axin2LacZ/LacZ cells had thesame baseline luciferase reporter activity as wild-type and Axin2LacZ/+

cells (Fig. 1A). After brief exposure to Wnt protein, Axin2LacZ/LacZ cellsshowed higher luciferase reporter activity than either wild-type orAxin2LacZ/+ cells (Fig. 1A). The duration of Wnt signaling was alsoextended in Axin2LacZ/LacZ cells: 70 hours after withdrawing Wnt pro-tein, wild-type and Axin2LacZ/+ cells had returned to near-baseline

cienceTranslationalMedicine.org 28 April 2010 Vol 2 Issue 29 29ra30 1



responsiveness, whereas Axin2LacZ/LacZ cells maintained ~40% oftheir maximum Wnt responsiveness [Fig. 1A; see also (25)]. Thus,Axin2 repressed Wnt signaling when cells had been exposed to

www.S

on

May

2, 2

010

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

Wnt ligands, and removing the Axin2 gene resulted in an amplifiedWnt response in these cells.

Axin2 deletion does not cause pathologic bone accrualDespite the increased response of Axin2LacZ/LacZ osteoprogenitorcells to an acute Wnt exposure, Axin2LacZ/LacZ mice had bones witha normal cortical thickness. Three-dimensional reconstruction withmicro–computed tomography (mCT) demonstrated that wild-typeand Axin2LacZ/LacZ mice had equivalent bone area and bone volumeand equivalent bone mineral density (n = 5 for each condition; Fig. 1,B and C, and fig. S2). Histological analyses verified that bones fromwild-type and Axin2LacZ/LacZ mice had similar cortical anatomy (Fig.1, D and E, n ≥ 12 for each genotype). Notwithstanding this equiv-alence, Axin2LacZ/LacZ mice exhibited an increase in alkaline phos-phatase (ALP) activity. Within the osteogenic cell lineage, ALPactivity is a marker of osteoprogenitor cell differentiation (26, 27),and we found elevated ALP activity both in bone extracts (Fig. 1F)and on the endosteal bone surfaces of Axin2LacZ/LacZ mice relative towild-type controls (Fig. 1, G and H).

Using tartrate-resistant acid phosphatase (TRAP) activity as amarker for bone-resorbing osteoclast activity (28), we found no differ-ences in either the amount of TRAP activity (Fig. 1I) or the distribu-tion (Fig. 1, J and K) of osteoclasts in wild-type or Axin2LacZ/LacZ bones.These data indicated that osteoprogenitor cells from Axin2LacZ/LacZ

mice showed an earlier commitment toward the osteoblastic pheno-type (29) when they were compared to osteoprogenitor cells fromwild-type mice.

A prolonged Wnt response in Axin2 mutant mice results inaccelerated skeletal healingThe normal bone morphology of the Axin2LacZ/LacZ mice stands outagainst the pathologically thickened bones of mice carrying activatingmutations in other components of Wnt signaling (9). This normalbone morphology may be attributable to functional compensationof Axin2 by its homolog Axin (30), but it could also mean thatAxin2LacZ/LacZ cells behave the same as wild-type cells in the absenceof an acute Wnt stimulus (for example, Fig. 1A). One method toinduce an acute Wnt stimulus in vivo is to injure the skeleton (5).Using Western blot analysis, we showed that both activated andtotal b-catenin were expressed at higher levels in injured bone re-lative to intact bone (Fig. 2A). We verified that the Wnt pathwaywas elevated in response to injury using quantitative reverse transcrip-tion polymerase chain reaction (qRT-PCR): The Wnt target Axin2was not significantly up-regulated by injury in wild-type mice (Fig. 2,B and C), but injury caused a significant increase in messenger RNA(mRNA) transcripts for Axin2 (exon 1) in Axin2LacZ/LacZ mice (Fig.2B). Using X-galactosidase (X-Gal) staining, we found that both thedistribution and the number of X-Gal–positive cells in Axin2LacZ/LacZ

mice were increased after injury (Fig. 2, D and E). This injury-inducedincrease in Wnt signaling was restricted to the area of damage; we de-tected no obvious changes in Wnt signaling at sites distant to the in-jury (fig. S3).

This region of increased Wnt signaling coincided with enhancedcell proliferation. Using 5-bromo-2′-deoxyuridine (BrdU) incorpora-tion and immunostaining for the proliferation markers proliferatingcell nuclear antigen (PCNA) and Ki67 (31), we found more dividingcells in injury sites than in intact bones (fig. S4). In addition, wefound more dividing cells in the Axin2LacZ/LacZ injury sites than in

poG

ALP

activ

ity

en

cb

HAxin2 LacZ/LacZWild-type

TRAP

act

ivity

Kpo

J

en

cb

10 h 30 h 50 h 70 h

1

2

5

15

25

0

A

(lucif

eras

e ac

tivity

x 1

0 )3

Wnt

resp

onsiv

enes

s

Wnt3a added Removed

Wild-typeAxin2 LacZ/+

Axin2 LacZ/LacZ

BMDC BVF

Tota

l bv/

ROI

0

0.05

0.10

0.15

0

100

200

300

FemurFemur Tibia

mg

(HyA

p)/c

m3

Femur Tibia

Femur Tibia

0

2

4

6

8

TRAP

act

/tota

l DNA

B

E Axin2 LacZ/LacZ

cb

D

en

poWT

Axin2 LacZ/LacZWild-type

0246

14

ALP

act/t

otal

DNA

81012

*

*

F

I

Axin2 LacZ/LacZWild-type

Tibia

Fig. 1. Wnt signaling is amplified in Axin2LacZ/LacZmice. (A) In vitro analysisof wild-type (WT), Axin2LacZ/+, and Axin2LacZ/LacZ preosteoblasts transduced
with a 7xTcf-Luc Wnt-activated reporter construct. Luciferase activity wasmonitored for 70 hours. Cells were exposed to Wnt3a protein from 0 to 4hours (green arrow), after which time the Wnt was removed and mediumwas replaced (red arrow). Data are presented as the mean ± SEM. Lines rep-resent modeled exponential decay from peak Wnt responsiveness at t = 25hours, shown in red for Axin2LacZ/LacZ, blue for Axin2LacZ/+, and green forWT cells. (B) mCT reconstruction to detect structural differences in WT andAxin2LacZ/LacZ bones. (C) Bonevolume fraction (BVF) (n=5) andbonemineraldensity (BMD) (n = 5) calculated from mCT data ± SEM (fig. S2); ROI denotesentire skeletal element (tibia or femur). (D and E) WT (D) and Axin2LacZ/LacZ (E)cortical bones stained with pentachrome. cb, cortical bone; en, endosteum;po, periosteum. (F) ALP activity in femurs and tibiae of WT mice is signif-icantly lower than in femurs and tibiae of Axin2LacZ/LacZmice (n = 6 for eachgenotype; P = 0.017 for femurs, P = 0.0043 for tibiae). (G andH) Distributionof ALP activity in bones fromWT (n=4) (G) andAxin2LacZ/LacZ (n=3) (H)mice.(I) TRAP activity in femurs and tibiae from WT mice is not significantly dif-ferent than in femurs and tibiae of Axin2LacZ/LacZ mice (n = 6 for each geno-type). (J andK) Distribution of TRAP activity in bones fromWT (n = 3) (J) andAxin2LacZ/LacZ (n = 9) (K) mice. Scale bar, 100 mm.



on

May

2, 2

010

wild-type sites (Fig. 2, F to H, and fig. S4). The proliferating cells werelargely restricted to the injury site (fig. S4).

Bone healing was accelerated in Axin2LacZ/LacZ mice. Compared towild-type mice, the osteoblast markers Runx2, Collagen type I, andOsteocalcin were expressed sooner in the injury sites of Axin2LacZ/LacZ

mice (Fig. 3, A to F). Relative to wild-type controls, ALP activity wasalso detectable at an earlier time point in injury sites of Axin2LacZ/LacZ

mice (Fig. 3, G and H). By day 7, Axin2LacZ/LacZ injury sites were filledwith new bone regenerate, far in advance of new bone generation ininjuries of wild-type bones (Fig. 3, I and J). We found no differencesin the pattern or level of TRAP activity within the injury sites ofAxin2LacZ/LacZ and wild-type mice (fig. S5). The regenerative advan-tage conferred by the Axin2LacZ/LacZ mutation was maintained for atleast 2 weeks (Fig. 3K), and the appearance of the Axin2LacZ/LacZ

bony regenerate was indistinguishable from the bony regenerate inwild-type mice at later stages of healing (fig. S6).

Cumulatively, our data demonstrated that injury transiently acti-vated endogenous Wnt signaling and that Axin2LacZ/LacZ cells re-sponded more robustly to this Wnt stimulus (Fig. 1). In the injurysite, Axin2LacZ/LacZ cells showed enhanced proliferation (Fig. 2) andalso adopted an osteoblastic fate sooner than wild-type cells, whichresulted in a robust regenerative response and faster skeletal healing(Fig. 3). This regenerative response was self-limiting: Within 3 weeksof injury, endogenous Wnt signaling gradually returned to a pre-

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

injury level (fig. S7). Collectively, our dataraised the possibility that early, transientexposure to additional Wnt signals couldenhance cell proliferation and acceleratebone healing after skeletal damage.

Liposomal Wnt enhances bonetissue regenerationTo test whether an exogenous Wnt stim-ulus could promote bone regeneration,we packaged the purified Wnt proteininto liposomal vesicles (32) and deliveredthem, or control (PBS) liposomes, to theskeletal injury site by injection.

Skeletal defects treated with liposomalWnt healed faster. We found that theonset of mineralization (Fig. 4, A andB) and new osteoid deposition (Fig. 4,C and D) was accelerated by liposomalWnt treatment. Histomorphometric mea-surements demonstrated that within 3days of a single treatment, injury sitestreated with liposomal Wnt had 3.5times as much new bone as injury sitestreated with PBS (Fig. 4E). We com-pared the pro-osteogenic effects of lipo-somal Wnt with bare Wnt protein andfound that only liposomal Wnt effec-tively enhanced bone regeneration: De-fects treated with bare Wnt showed thesame amount of bone regeneration asthose treated with PBS (Fig. 4, F to H).Liposomal Wnt-treated injuries also un-derwent the final stages of remodeling

www.S

sooner than PBS-treated defects, resulting in more organized, maturebone matrix at earlier time points (Fig. 4, I and J).

We gained mechanistic insights into the basis for the pro-osteogeniceffects of liposomal Wnt by carrying out a series of molecular andcellular analyses at earlier time points during the healing process.In the first 24 hours after injection, we saw no apparent histologicaldifferences between injuries treated with liposomal PBS and thosetreated with liposomal Wnt (Fig. 5, A and B), but by using X-Galstaining, we detected an obvious increase in the number of Wnt-responding cells after liposomal Wnt treatment (Fig. 5, C and D).Liposomal Wnt-treated sites also had significantly more proliferat-ing cells than those treated with liposomal PBS (n = 6 for each con-dition; Fig. 5, E and F, and fig. S8). Cells in injury sites treated withliposomal Wnt also expressed the osteogenic genes Runx2, Collagentype I, and Osteocalcin sooner than did cells in injury sites treatedwith PBS (Fig. 5, G to L). Bisphosphonates promote bone accrualprimarily by inhibiting osteoclast function (33), but by using TRAPstaining as an indicator of osteoclast activity, we found that lipo-somal Wnt did not enhance bone accrual by blocking bone re-sorption (Fig. 5, M and N). Thus, the bone-promoting effects ofWnt liposomal treatment were achieved via the proliferation ofskeletal progenitor cells, which rapidly adopted an osteogenic fateand gave rise to abundant new bone regenerate specifically in theinjury site.

cbF

G

is

Axin

2ZcaL/ZcaLZcaL/ZcaL

Wild

-type

H

0

0.1

0.2

0.3

Cel

l pro

lifer

atio

n(P

CN

A c

ells

/tota

l cel

ls)

Wild-typeAxin2 LacZ/LacZ *

ED

Intact Injured

A

Totalβ catenin

Activatedβ catenin

0

0.2

0.4

0.6

0.8

1.0

1.2Intact

Injured

Fold

cha

nge

in β-

cate

nin

norm

aliz

ed to

β-a

ctin

β -Actin

Total β -cat

Activ. β -cat

Intact Injured

Rel

ativ

e Ax

in2

(exo

n 1)

exp

ress

ion

B

0

1.0

1.5

*

0.5

#

Wild-typeAxin2 LacZ/LacZ

Axin2

Exon 1,Axin2

β -Actin

Intact

Axin2 LacZ/LacZ

Injured

Wild-typeInjuredIntact

C

Axin

2LacZ

/Lac

Z

+

Fig. 2. Injury increases Wnt signaling and skeletal progenitor cell proliferation in Axin2LacZ/LacZ mice.(A) Immunoblot for total b-catenin and activated b-catenin protein, with quantification of bands
shown normalized against b-actin protein. (B) Validation of injury-induced Wnt pathway up-regulationusing qRT-PCR for exon 1 of the Axin2 gene. Injury sites in Axin2LacZ/LacZ mice show a significant increasein mRNA transcripts for Axin2 (exon 1) compared to sham-treated controls (#) (n = 6; P < 0.05) and to WTmice (*). (C) Representative gel from RT-PCR analyses using primers to detect Axin2, Axin2 (exon 1), andb-actin in Axin2LacZ/LacZ and WT intact and injured bones. (D and E) X-Gal staining of the endosteum ofAxin2LacZ/LacZ intact bones (D) and injured bones (n = 6 for each condition) (E). (F and G) PCNA immu-nostaining identifies proliferating cells in the injury sites (is) of WT (n = 4) (F) and Axin2LacZ/LacZ (n = 5) (G)mice. (H) Quantification of cell proliferation in injury sites of WT and Axin2LacZ/LacZ mice. Data representmean ± SD (P < 0.01). Scale bar, 100 mm.



on

May

2, 2

010

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

DISCUSSION

In this and in previous work, we have shown that Wnt-responsivecells are located on the endosteal surfaces of bone, where they con-stitute a pool of skeletal stem or progenitor cells that participate inbone homeostasis (Fig. 2) (34). Skeletal trauma activates endogenousWnt signaling and leads to an expansion in this skeletal stem or pro-genitor pool. Eventually, skeletal progenitor cells express the osteogenicgenes Runx2 and Collagen type I, which indicates their commitment toan osteogenic fate (Fig. 3).

If Wnt signaling is blocked, then both the proliferative effect andosteogenic commitment are impeded in skeletal progenitor cells(5, 23). Here, we have shown that increasing the Wnt signal has anopposite, and beneficial, effect on bone repair. We demonstrate thatloss of the negative regulator Axin2 results in a prolonged, amplifiedWnt signal, which has a potent effect on cells in the injury site,causing them to proliferate to a greater degree. Axin2LacZ/LacZ skeletalprogenitor cells eventually cease their proliferation and differentiateinto osteoblasts.

The transient nature of the Wnt signal in Axin2LacZ/LacZ mice isimportant because other mutations (for example, Lrp5G171V, glycogensynthase kinase 3, activated b-catenin) that either produce con-stitutive activation of the Wnt pathway or disrupt other signalingpathways in addition to Wnt (17, 35–37) maintain skeletal stem orprogenitor cells in a proliferative state, delaying their differentiationinto osteoblasts and impeding the repair process (5). Thus, a balancebetween osteoprogenitor proliferation and osteoblast differentiation isrequired for timely bone regeneration, and Wnt signaling is a keymediator of this equilibrium.

On the basis of these findings, we developed a biochemical ap-proach to transiently increase Wnt signaling after injury. Previously,the use of Wnts as bioactive reagents had been limited because ofinherent difficulties in purifying and delivering the hydrophobic pro-tein in vivo. By packaging the lipid-modified Wnt into lipid vesicles(32), we could test the therapeutic efficacy of Wnts. When we sup-plied liposomal Wnt after the initial inflammatory period, cells in theinjury site proliferated more and accelerated their differentiation intoosteoblasts. Thus, liposomal Wnt treatment effectively prolonged theendogenous Wnt signal that is initiated by injury and, in doing so,accelerated bone healing.

Bone morphogenetic proteins (BMPs) have been proposed as bonehealing reagents (38). Recombinant BMP-2 (rBMP-2) treatment in-duces cartilage formation, and thus, repair takes place via endo-chondral ossification (39). In contrast, the same injuries treated withliposomal Wnt heal via intramembranous ossification. In both hu-mans (40) and our mouse model (41), rBMP-2 treatment inducesheterotopic ossifications; in contrast, we have never observed ectopicbone formation associated with liposomal Wnt3a treatment. There-fore, liposomal Wnt and rBMPs differentially affect the process ofskeletal repair. BMP and Wnt signals are integrated at the level ofSmad1 (42), and in skeletal injury, rBMP-2 both induces phospho-rylation of Smad1/Smad5/Smad8 and represses b-catenin–dependentWnt signaling (41). As a consequence, Wnt-dependent Sox9 inhibi-tion (18) is relieved and skeletal stem or progenitor cells in theinjured periosteum adopt a chondrogenic lineage (41). RecombinantBMP-2 treatment also represses Wnt signaling in cells that populatethe injured bone marrow cavity (41). In vivo, these skeletal stem or pro-genitor cells have only one fate—to differentiate into osteoblasts. Byrepressing Wnt signaling in this cell population, bone formation is

www.S

inhibited within the marrow cavity. Collectively, these experimentalresults are in keeping with clinical observations associated withrBMPs (43).

Cells in the injured periosteum and bone marrow cavity are re-sponsive to a liposomal Wnt stimulus but, beyond the fact that theycan differentiate into osteoblasts, the identities of these cells are stillunknown. Multiple cell populations contribute to the bone healingprocess, including inflammatory cells, muscle satellite cells, pericytes,and angioblasts. Most of these cell types migrate into the wound sitesduring the first 72 hours after injury and participate to varying de-grees in bone regeneration. Many of these populations contain Wnt-responsive cells (44–47) and could be targets for the liposomal Wntsignal. A genetic strategy whereby the progeny of Axin2-expressingcells in the injury site could be permanently labeled and thenfollowed throughout the bone healing program would provide valu-able insights.

In conclusion, we have demonstrated that liposomal Wnt de-livered to an injury site expedites the healing process. We elucidatethe basis for their pro-osteogenic effects by showing that liposomalWnt stimulates skeletal stem or progenitor cell proliferation and also

C

Osteocalcin

D

E

F

cb

Collagen type I

is

Axin

2LacZ

/Lac

Z

Histomorphometry

7 140

4

8

12

16

Aniline blue

cbis

I

J

KALP activity

G

H

A

B

Runx2

is is

cbcb

cb

is

Axin

2LacZ

/Lac

ZW

ild-ty

peW

ild-ty

pe

4R

egen

erat

ed b

one

(pix

els

x 10

)

Days after injury

Axin2Wild-type

LacZ/LacZ

**

Fig. 3. Loss of Axin2 accelerates bone regeneration. (A to F) Three daysafter injury, Runx2 expression is evaluated by in situ hybridization in the
injury sites of WT (n = 6) (A) and Axin2LacZ/LacZ (n = 6) (B) mice. Collagentype I expression in the injury sites of WT (n = 5) (C) and Axin2LacZ/LacZ
(n = 6) (D) mice. Osteocalcin expression in the injury sites of WT (n = 3)(E) and Axin2LacZ/LacZ (n = 6) (F) mice. (G to J) After 7 days, ALP activitymarks the onset of osteoprogenitor cell differentiation in the injury sitesof WT (n = 3) (G) and Axin2LacZ/LacZ (n = 6) (H) mice. After 7 days, aniline bluestaining indicates new osteoid matrix in WT (n = 3) (I) and Axin2LacZ/LacZ

(n = 6) (J) mice. (K) Histomorphometric measurements are used to quan-tify new bone formation on day 7 (P < 0.05) and day 14 (WT, n = 6;Axin2LacZ/LacZ, n = 14; P < 0.01). Scale bars, 100 mm.




on

May

2, 2

010

stm

.sci

ence

mag

.org

nloa

ded

from

accelerates the differentiation of progenitor cells into mature osteo-blasts. In invertebrates, Wnts regulate tissue regeneration (48, 49),and in vertebrates, Wnts are implicated in the repair of multipleorgans and tissues (50–55). Given the highly conserved nature ofthe Wnt pathway, and its critical role in maintaining embryonicand adult stem cell self-renewal and proliferation (56), liposomalWnts may have therapeutic applications far beyond skeletal tissueapplications.

Dow

MATERIALS AND METHODS

In vitro quantification of Wnt responsivenessA DNA fragment containing seven Tcf/lymphoid enhancer factor–binding sites, the minimal promoter, and the 5′ untranslated regionof the pSuperTOPflash reporter plasmid (57) was amplified by PCRand inserted upstream of the firefly luciferase (FFluc) gene in theself-inactivating lentivirus TOP-FFluc (58). The 7xTcf-FFluc//SV40viral construct also contained a SV40-puro cassette for puromycinselection of transfected cells. Lentivirus was produced by transienttransfection in 293T cells.

Primary calvarial osteoprogenitor cells were harvested from post-natal day 2 Axin2LacZ/+, Axin2LacZ/LacZ, and wild-type (WT) mice. Pas-sage 1 cells were transfected with 7xTcf-FFluc//SV40-puro lentivirus for24 hours at a concentration of one transfecting agent per cell. Cultureswere treated with puromycin (4 mg/ml) for 2 days to select for trans-fected cells. Cells were trypsinized, plated in 96-well plates, and treatedwith purified Wnt3a protein (100 ng/ml) to examine the Wnt respon-

www.ScienceTranslationalMedicine.or

siveness. Cells were collected at differenttime points after treatment, and luciferaseactivity was measured with Luc-ScreenFirefly Luciferase Reporter Gene AssaySystem (Applied Biosystems). Linear re-gression was calculated with Excel.

In vivo Wnt responsivenessAxin2LacZ/+ mice were bred to generateAxin2LacZ/LacZ offspring (24). TOPgaltransgenic mice were also used (Fig. 5,C and D) as bred as described (59). TheLacZ product, b-galactosidase, was de-tected by X-Gal staining. Tissues wereembedded in optimum cutting tempera-ture compound followed by cryosection-ing, then fixed with 0.2% glutaraldehydefor 15 min, and stained with X-Gal over-night at 37°C.

mCT analysisAfter anesthesia, male, 3-month-old WT,Axin2LacZ/+, and Axin2LacZ/LacZ mice weresubjected to mCT analysis with a GEMedical Systems eXplore RS MicroCTSystem (General Electric Healthcare).Mice were scanned in a prone positionat 98-mm resolution. Tape was used to se-cure limbs and minimize distortion frombreath motion. Individual CT slices were

reconstructed with GE reconstruction software, and data were analyzedwith GE MicroView. A “phantom” density standard containing air,water, and hydroxyapatite (synthetic bone) was scanned alongside eachmouse to calibrate and standardize the density of the scanned tissue.MicroView software was used to calculate bone volume fraction andbone mineral density. A standard region of interest (ROI) was specifiedfor either tibiae or femurs of all mice before analysis and was notchanged among samples.

Generation of skeletal injuriesAll procedures followed protocols approved by the Stanford Commit-tee on Animal Research. Adult mice (males, between 3 and 5 monthsold) were anaesthetized with an intraperitoneal injection of ketamine-xylazine. A 5-mm incision was made over the anterior-proximal tibia,and the tibial surface was exposed while carefully preserving the peri-osteum. A 1.0-mm hole was drilled through the anterior cortex with ahigh-speed dental drill. Wounds were closed with size 6-0 vicryl su-tures. After surgery, mice received subcutaneous injections of carprofenfor analgesia and were allowed to ambulate freely. Mice were eutha-nized at days 1, 2, 3, 4, 5, 6, 7, 14, and 28 after surgery.

Western blot and qRT-PCRForty-eight hours after skeletal injury (or sham surgery, where ac-cess to the bone was made without producing a monocortical de-fect), tissues were collected by making two transverse cuts 1.0 mmproximal and distal to the injury site. Extreme care was taken toensure that the same segment of the tibiae was isolated from injuredand sham controls.

B

A

L-PB

SL-

Wnt

3a

C

D

Aniline blue (day 3)

E8

4

0

*L-Wnt3a

is

is

G

H

FPentachrome (day 3)ALP activity (day 2)

cbis

is

is

Wnt3a protein

J

IAniline blue (day 28)

L-PBS

is

is

L-Wnt3a

cbcb

4R

egen

erat

ed b

one

(pix

els

x 10

) L-PBS

Fig. 4. Liposomal Wnt treatment accelerates bone regeneration. Skeletal defects were generated in WTCD1 mice and treated with PBS liposomes (10 ml) of liposomal Wnt3a (10-ml volume with an effective
Wnt3a concentration of 0.5 mg/ml). (A and B) After 2 days, ALP activity detects the onset of osteoprogenitorcell commitment to an osteoblastic lineage in PBS (A) and liposomal Wnt3a (L-Wnt3a)–treated (B) injuries.(C and D) After 3 days, aniline blue staining identifies new osteoid matrix in injury sites (is) treated withPBS (n = 5) (C) and liposomal Wnt3a (D) (n = 6). (E) Histomorphometric measurements (see Materials andMethods) demonstrate a factor of 3.5 increase in new bone in liposomal Wnt3a–treated sites. (F to H) After3 days, pentachrome staining identifies new bone matrix (greenish yellow) in injury sites treated withliposomal PBS (L-PBS) (n = 5) (F), liposomal Wnt3a (n = 6) (G), or bare Wnt3a protein (n = 4) (H). (I and J)After 28 days, aniline blue staining illustrates the remodeled bone matrix in injuries treated with PBS(n = 5) (I) and liposomal Wnt3a (J). Boxed areas indicate former injury site. Scale bar, 100 mm.
g 28 April 2010 Vol 2 Issue 29 29ra30 5



on

May

2, 2

010

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

For gene expression analyses, tissues were homogenized in TRIzol(Invitrogen), and RNA was isolated with RNeasy mini column (Qiagen).Reverse transcription was performed with SuperScript III First-StrandSynthesis SuperMix for qRT-PCR (Invitrogen). Quantitative PCR re-actions were performed and monitored with StepOnePlus Real-TimePCR System. Normalized expression levels reported were calculatedon the basis of differences between threshold cycles for the gene ofinterest and the housekeeping gene b-actin. The following primer setswere used: b-actin, 5′-GGAATGGGTCAGAAGGACTC-3′ (sense) and5′-CATGTCGTCCCAGTTGGTAA-3′ (antisense) (110). In Axin2LacZ/LacZ

mice, exon 2 is replaced by the LacZ gene, which results in a trun-cated, nonfunctional Axin2 protein (24). Exon 1 is unaffected by theLacZ insertion, and therefore, the following primers were used to detectAxin2 expression in Axin2LacZ/LacZ mice: 5′-TTGATAAGGTCCTGG-CAACTC-3′ (sense) and 5′-GCGAACGGCTGCTTATTT-3′ (antisense).

For Western blot analyses, the isolated tissue was snap-frozen withliquid nitrogen and homogenized. Proteins were lysed in buffercontaining 50 mM tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40,and 0.1% SDS supplemented with protease inhibitor cocktail (Sigma)and phosphatase inhibitors (1 mM NaF and 1 mM Na3VO4). Pro-

www.ScienceTranslationalM

teins were fractionated by 10% SDS–polyacrylamidegel electrophoresis, transferred to nitrocellulose mem-brane, and incubated with respective primary anti-bodies. Bound primary antibodies were detected withhorseradish peroxidase–conjugated secondary anti-bodies, visualized by enhanced chemiluminescence(Amersham), and autoradiographed. Rabbit poly-clonal total b-catenin (Themo Fisher Scientific),mousemonoclonal antibodies to activated b-catenin,which is dephosphorylated on Ser37 or Thr41 (60),andpan–b-actin (ThemoFisher Scientific)were used.

To quantify the bands obtained via Westernblots and RT-PCR, we performed densitometricanalysis by using the public domain Java imageprocessing program ImageJ (developed at theNational Institutes of Health). The software cal-culates the area and pixel value statistics of de-fined selections on the image. Relative change wascalculated by between the target proteins and b-actin,which served as an internal control.

Cell quantificationTo quantify PCNA immunostaining, we used4′,6-diamidino-2-phenylindole (DAPI) to coun-terstain cell nuclei. Using Adobe Photoshop, weused the magic wand tool to highlight browncells (tolerance = 30). Nonspecific brown areaswere manually deselected. These highlighted cellswere cut out. Publicly available ImageJ was usedto convert these pictures to binary, and the “An-alyze Particles” function was used to count dis-crete particles. To quantify DAPI, we used ImageJto count discrete nuclei. The “watershed” func-tion was used to approximate tightly packed nu-clei. Data were expressed as positive cells pertotal cells.

X-Gal–positive cells were quantified with ImageJ(see above). For intact bones, X-Gal–positive cells

were counted along a 3-mm section of the endosteum and then nor-malized against the cross-sectional area of the marrow cavity. Toquantify X-Gal cells in the tibial injury, we took an image of the totalcross-sectional area of the injury and counted the total number ofblue cells within the injury site and normalized against the totalcross-sectional area of the injury.

Molecular and cellular assaysUnder ribonuclease-free conditions, tibiae were harvested, the skinand outer layers of muscle were removed, and the tissues were washedin 1× phosphate-buffered saline (PBS) at 4°C and then fixed in 4%paraformaldehyde. Tissues were decalcified in 19% EDTA for 10 to14 days at 4°C and then prepared for paraffin embedding. Paraffinembedding followed standard protocols, and sections were generatedat an 8-mm thickness. For in situ hybridization, the relevant digoxigenin-labeled mRNA antisense probes were prepared from complementaryDNA templates for Runx2, Collagen type I, and Osteocalcin. Sectionswere dewaxed, treated with proteinase K, and incubated in hybridiza-tion buffer containing the relevant RNA probe. Probe was added atan approximate concentration of 0.25 mg/ml. Stringency washes of

cb

is

Collagen type I (day 2)G

H

I

J

Osteocalcin (day 3)K

cb

L

is

cb

is

Runx2 (day 2)

L-PB

SL-

Wnt

3a

B

APentachrome

L-PB

SL-

Wnt

3a

M

N

TRAP activity (day 3)

cb

is

BrdU (day 1)Wnt response (day 1)

is

E

F

iscb

D

C

Fig. 5. Liposomal Wnt stimulates skeletal stem or progenitor cell proliferation and enhancesosteogenic differentiation. (A and B) Pentachrome staining is used to assess organization of
the injury site 1 day after treatment with liposomal PBS (A) or liposomal Wnt3a (B). (C and D)X-Gal staining on similar sections reveals Wnt-responsive cells in the injury sites treated withliposomal PBS (n = 6) (C) or liposomal Wnt3a (n = 6) (D). (E and F) BrdU incorporation indicatesskeletal stem or progenitor cell proliferation in injury sites 1 day after treatment with liposomalPBS (n = 6) (E) or liposomal Wnt3a (n = 6) (F). (G andH) Runx2 expression is evaluated in the injurysite by in situ hybridization 2days after delivering liposomal PBS (n=4) (G) or liposomalWnt3a (n=7) (H). (I and J)Collagen type I is evaluated in injury sites 2days after delivering liposomal PBS (n=4)(I) or liposomalWnt3a (n = 7) (J). (K and L)Osteocalcin expression is evaluated in injury sites 3 daysafter treatmentwith liposomal PBS (n=5) (K) or liposomalWnt3a (n=5) (L). (M andN) TRAPactivityreveals osteoclast function in injury sites 3 days after treatment with liposomal PBS (n = 4) (M) orliposomal Wnt3a (n = 7) (N). Scale bars, 100 mm [(A), (B), (E), and (F)], 10 mm (all other panels).
edicine.org 28 April 2010 Vol 2 Issue 29 29ra30 6



on

May

2, 2

010

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

saline sodium citrate solution were done at 52°C and further washedin maleic acid buffer with 1% Tween 20. Slides were treated with anantibody to digoxigenin (Roche). For color detection, slides were in-cubated in nitro blue tetrazolium chloride (Roche) and 5-bromo-4-chloro-3-indolyl phosphate (Roche). After developing, the slides werecoverslipped with aqueous mounting medium. For immunostaining,tissue sections were dewaxed followed by immersion in H2O2-PBS,washed in PBS, incubated in ficin (Zymed), treated with 0.1 M glycine,washed further, and then blocked in ovalbumin (Worthington) and1% whole donkey immunoglobulin G (Jackson ImmunoResearch).Appropriate primary antibody was added and incubated overnight at4°C and then washed in PBS. Samples were incubated with peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) for anhour, and a DAB substrate kit (Vector Laboratories) was used to de-velop the color reaction. Some commonly used antibodies includePCNA (Zymed) and platelet endothelial cell adhesion molecule1 (BD Biosciences). For TRAP staining, tissue sections were dewaxedand then treated with a TRAP staining kit (Sigma).

For whole-bone ALP and TRAP activity, tibiae and femurs fromWT and Axin2LacZ/LacZ mice were collected immediately after eu-thanasia. The bone was snap-frozen and homogenized in 400 ml of0.1% Triton X-100 solution. Sample ALP and TRAP activity wasdetermined with an enzymatic assay on the basis of hydrolysis ofp-nitrophenyl phosphate (pNP-PO4) to pNP. Briefly, for ALP activ-ity, aliquots of the sample homogenate (5 ml) were added to 95 ml of10 mM pNP-PO4 in 0.1 M Na2CO3 and 2 mMMgCl2 (pH 10.0). Thesamples were then incubated at 37°C for 15 min, and the reaction wasterminated by addition of 100 ml of 0.1 N NaOH. For TRAP activity,aliquots of the sample (5 ml) were added to 95 ml of 0.1 M pNP-PO4

in 0.2 M sodium citrate, 0.2 M sodium chloride, and 80 mM sodiumL-(+)-tartrate. The samples were then incubated at 37°C for 1 hour,and the reaction was terminated by addition of 100 ml of 1 N NaOH.Absorbance for both assays was measured at 410 nm with a micro-plate reader.

Histology and histomorphometric analysesPentachrome and aniline blue staining were performed; slides weremounted with Permount after dehydration in a series of ethanol andxylene. To quantify new bone, we represented the 1.0-mm circularmonocortical defect across about forty 8-mm-thick tissue sections.Of those 40 sections, we used a minimum of 8 sections to quantifythe amount of aniline blue–stained new osteoid matrix. Tissue sectionswere photographed with a Leica digital imaging system (5× objective).The resulting digital images were analyzed with Adobe PhotoshopCS2 software. We chose a fixed, rectangular ROI that in all imagescorresponded to 106 pixels. The injury site was always representedinside this ROI by manually placing the box in the correct positionon each image. Aniline blue–positive pixels were automatically se-lected with the magic wand tool set to a color tolerance of 60. Thistolerance setting resulted in highlighted pixels with a range of bluethat corresponded precisely with the histological appearance of newosteoid tissue in the aniline blue–stained sections. Cortical surfaces,or bone fragments resulting from the drill injury, were manually de-selected. The total number of aniline blue–positive pixels for each sec-tion was then recorded. The pixel counts from individual sectionswere averaged for each tibia sample, and the differences within andamong treatment groups were calculated on the basis of theseaverages.

www.S

Liposomal preparation and deliveryLiposomal Wnt3a was prepared as described (32). Briefly, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC; Sigma) in chloroform was driedto a thin film in a 10-ml round bottom flask. Purified Wnt3a with aconcentration of 1 to 1.3 mg/ml was mixed with dried DMPC. The lipid-Wnt3a solution was extruded 40 times through a 100- to 200-nm polycar-bonate membrane in a thermobarrel extruder, keeping the temperatureconstant at 30° to 32°C (Avanti Polar Lipids). The supernatant was re-moved and the liposome pellet was resuspended in 1× Dulbecco’s mod-ified Eagle’s medium (Mediatech). Liposomes were stored at 4°C andused within 10 days of preparation. The liposomal preparation hadan effective Wnt3a concentration of 0.5 mg/ml, and a single (10 ml) doseof this solution was delivered to the injury site by injection.

Monocortical tibial defects were treated with liposomal Wnt3apreparation by injecting 10 ml of liposomal Wnt3a into the injury siteon postsurgical day 3.

Statistical analysesResults are presented as the mean ± SD, with n equal to the number ofsamples analyzed. Both Student’s t test and nonparametric Wilcoxontest were used to test for significant differences between data sets. Sig-nificance was attained at P < 0.05, and all statistical analyses wereperformed with the JMP software (SAS).

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/2/29/29ra30/DC1Fig. S1. Constitutively active Wnt signaling, but not Axin2LacZ/LacZ mutation, causes pathologicalbone accrual.Fig. S2. Bone volume fraction, bone mineral density, and cortical bone thickness in wild-typeand Axin2LacZ/LacZ mice.Fig. S3. Wnt signaling is elevated only at the site of injury.Fig. S4. Cell proliferation is enhanced in Axin2LacZ/LacZ mice at the site of injury.Fig. S5. TRAP activity is similar between wild-type and Axin2LacZ/LacZ mice.Fig. S6. The bony regenerate in Axin2LacZ/LacZ mice appears sooner but is histologically in-distinguishable from wild-type mice.Fig. S7. Wnt signaling is transiently elevated by skeletal injury.Fig. S8. Liposomal Wnt3a stimulates cell proliferation in skeletal injury sites.

REFERENCES AND NOTES

1. K. A. Moore, I. R. Lemischka, Stem cells and their niches. Science 311, 1880–1885 (2006).2. P. Leucht, S. Minear, D. Ten Berge, R. Nusse, J. A. Helms, Translating insights from development

into regenerative medicine: The function of Wnts in bone biology. Semin. Cell Dev. Biol. 19,434–443 (2008).

3. K. Willert, J. D. Brown, E. Danenberg, A. W. Duncan, I. L. Weissman, T. Reya, J. R. Yates III,R. Nusse, Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature423, 448–452 (2003).

4. R. Takada, Y. Satomi, T. Kurata, N. Ueno, S. Norioka, H. Kondoh, T. Takao, S. Takada, Mono-unsaturated fatty acid modification of Wnt protein: Its role in Wnt secretion. Dev. Cell 11,791–801 (2006).

5. J. B. Kim, P. Leucht, K. Lam, C. Luppen, D. Ten Berge, R. Nusse, J. A. Helms, Bone regenerationis regulated by Wnt signaling. J. Bone Miner. Res. 22, 1913–1923 (2007).

6. C. N. Bennett, K. A. Longo, W. S. Wright, L. J. Suva, T. F. Lane, K. D. Hankenson, O. A. MacDougald,Regulation of osteoblastogenesis and bone mass by Wnt10b. Proc. Natl. Acad. Sci. U.S.A. 102,3324–3329 (2005).

7. Y. Gong, R. B. Slee, N. Fukai, G. Rawadi, S. Roman-Roman, A. M. Reginato, H. Wang, T. Cundy,F. H. Glorieux, D. Lev, M. Zacharin, K. Oexle, J. Marcelino, W. Suwairi, S. Heeger, G. Sabatakos,S. Apte, W. N. Adkins, J. Allgrove, M. Arslan-Kirchner, J. A. Batch, P. Beighton, G. C. Black,R. G. Boles, L. M. Boon, C. Borrone, H. G. Brunner, G. F. Carle, B. Dallapiccola, A. De Paepe,B. Floege, M. L. Halfhide, B. Hall, R. C. Hennekam, T. Hirose, A. Jans, H. Jüppner, C. A. Kim,K. Keppler-Noreuil, A. Kohlschuetter, D. LaCombe, M. Lambert, E. Lemyre, T. Letteboer,L. Peltonen, R. S. Ramesar, M. Romanengo, H. Somer, E. Steichen-Gersdorf, B. Steinmann,




on

May

2, 2

010

stm

.sci

ence

mag

.org

Dow

nloa

ded

from

B. Sullivan, A. Superti-Furga, W. Swoboda, M. J. van den Boogaard, W. Van Hul, M. Vikkula,M. Votruba, B. Zabel, T. Garcia, R. Baron, B. R. Olsen, M. L. Warman; Osteoporosis-PseudogliomaSyndrome Collaborative Group, LDL receptor-related protein 5 (LRP5) affects bone accrualand eye development. Cell 107, 513–523 (2001).

8. F. Morvan, K. Boulukos, P. Clément-Lacroix, S. Roman Roman, I. Suc-Royer, B. Vayssière,P. Ammann, P. Martin, S. Pinho, P. Pognonec, P. Mollat, C. Niehrs, R. Baron, G. Rawadi,Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation andbone mass. J. Bone Miner. Res. 21, 934–945 (2006).

9. R. D. Little, J. P. Carulli, R. G. Del Mastro, J. Dupuis, M. Osborne, C. Folz, S. P. Manning,P. M. Swain, S. C. Zhao, B. Eustace, M. M. Lappe, L. Spitzer, S. Zweier, K. Braunschweiger,Y. Benchekroun, X. Hu, R. Adair, L. Chee, M. G. FitzGerald, C. Tulig, A. Caruso, N. Tzellas,A. Bawa, B. Franklin, S. McGuire, X. Nogues, G. Gong, K. M. Allen, A. Anisowicz, A. J. Morales,P. T. Lomedico, S. M. Recker, P. Van Eerdewegh, R. R. Recker, M. L. Johnson, A mutation inthe LDL receptor–related protein 5 gene results in the autosomal dominant high–bone-mass trait. Am. J. Hum. Genet. 70, 11–19 (2002).

10. L. M. Boyden, J. Mao, J. Belsky, L. Mitzner, A. Farhi, M. A. Mitnick, D. Wu, K. Insogna,R. P. Lifton, High bone density due to a mutation in LDL-receptor–related protein 5. N. Engl.J. Med. 346, 1513–1521 (2002).

11. P. Babij, W. Zhao, C. Small, Y. Kharode, P. J. Yaworsky, M. L. Bouxsein, P. S. Reddy, P. V. Bodine,J. A. Robinson, B. Bhat, J. Marzolf, R. A. Moran, F. Bex, High bone mass in mice expressing amutant LRP5 gene. J. Bone Miner. Res. 18, 960–974 (2003).

12. T. Gaur, C. J. Lengner, H. Hovhannisyan, R. A. Bhat, P. V. Bodine, B. S. Komm, A. Javed,A. J. van Wijnen, J. L. Stein, G. S. Stein, J. B. Lian, Canonical WNT signaling promotesosteogenesis by directly stimulating Runx2 gene expression. J. Biol. Chem. 280, 33132–33140(2005).

13. P. V. Bodine, W. Zhao, Y. P. Kharode, F. J. Bex, A. J. Lambert, M. B. Goad, T. Gaur, G. S. Stein,J. B. Lian, B. S. Komm, The Wnt antagonist secreted frizzled-related protein-1 is a negativeregulator of trabecular bone formation in adult mice. Mol. Endocrinol. 18, 1222–1237(2004).

14. S. L. Holmen, C. R. Zylstra, A. Mukherjee, R. E. Sigler, M. C. Faugere, M. L. Bouxsein, L. Deng,T. L. Clemens, B. O. Williams, Essential role of b-catenin in postnatal bone acquisition.J. Biol. Chem. 280, 21162–21168 (2005).

15. D. A. Glass II, P. Bialek, J. D. Ahn, M. Starbuck, M. S. Patel, H. Clevers, M. M. Taketo, F. Long,A. P. McMahon, R. A. Lang, G. Karsenty, Canonical Wnt signaling in differentiated osteoblastscontrols osteoclast differentiation. Dev. Cell 8, 751–764 (2005).

16. T. P. Hill, M. M. Taketo, W. Birchmeier, C. Hartmann, Multiple roles of mesenchymal b-cateninduring murine limb patterning. Development 133, 1219–1229 (2006).

17. T. F. Day, X. Guo, L. Garrett-Beal, Y. Yang, Wnt/b-catenin signaling in mesenchymal progenitorscontrols osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell8, 739–750 (2005).

18. D. ten Berge, S. A. Brugmann, J. A. Helms, R. Nusse, Wnt and FGF signals interact tocoordinate growth with cell fate specification during limb development. Development135, 3247–3257 (2008).

19. Q. Zhang, M. B. Major, S. Takanashi, N. D. Camp, N. Nishiya, E. C. Peters, M. H. Ginsberg,X. Jian, P. A. Randazzo, P. G. Schultz, R. T. Moon, S. Ding, Small-molecule synergist of theWnt/b-catenin signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 104, 7444–7448 (2007).

20. J. Liu, X. Wu, B. Mitchell, C. Kintner, S. Ding, P. G. Schultz, A small-molecule agonist of theWnt signaling pathway. Angew. Chem. Int. Ed. Engl. 44, 1987–1990 (2005).

21. J. Shan, D. L. Shi, J. Wang, J. Zheng, Identification of a specific inhibitor of the dishevelledPDZ domain. Biochemistry 44, 15495–15503 (2005).

22. L. Meijer, M. Flajolet, P. Greengard, Pharmacological inhibitors of glycogen synthase kinase 3.Trends Pharmacol. Sci. 25, 471–480 (2004).

23. Y. Chen, H. C. Whetstone, A. C. Lin, P. Nadesan, Q. Wei, R. Poon, B. A. Alman, b-Cateninsignaling plays a disparate role in different phases of fracture repair: Implications for therapyto improve bone healing. PLoS Med. 4, e249 (2007).

24. B. Lustig, B. Jerchow, M. Sachs, S. Weiler, T. Pietsch, U. Karsten, M. van de Wetering,H. Clevers, P. M. Schlag, W. Birchmeier, J. Behrens, Negative feedback loop of Wnt sig-naling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell.Biol. 22, 1184–1193 (2002).

25. A. Y. Zeng, R. Nusse, Wnt proteins are self-renewing factors for mammary stem cells andpromote their long-term expansion in culture. Cell Stem Cell 10.1016/j.stem.2010.03.020 (2010).

26. J. S. Blum, R. H. Li, A. G. Mikos, M. A. Barry, An optimized method for the chemiluminescentdetection of alkaline phosphatase levels during osteodifferentiation by bone morphogeneticprotein 2. J. Cell. Biochem. 80, 532–537 (2001).

27. K. H. Wlodarski, A. H. Reddi, Alkaline phosphatase as a marker of osteoinductive cells.Calcif. Tissue Int. 39, 382–385 (1986).

28. N. Udagawa, N. Takahashi, T. Akatsu, H. Tanaka, T. Sasaki, T. Nishihara, T. Koga, T. J. Martin,T. Suda, Origin of osteoclasts: Mature monocytes and macrophages are capable of differ-entiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc. Natl. Acad. Sci. U.S.A. 87, 7260–7264 (1990).

www.S

29. G. I. Im, S. A. Qureshi, J. Kenney, H. E. Rubash, A. S. Shanbhag, Osteoblast proliferation andmaturation by bisphosphonates. Biomaterials 25, 4105–4115 (2004).

30. J. Behrens, B. A. Jerchow, M. Würtele, J. Grimm, C. Asbrand, R. Wirtz, M. Kühl, D. Wedlich,W. Birchmeier, Functional interaction of an axin homolog, conductin, with b-catenin, APC,and GSK3b. Science 280, 596–599 (1998).

31. M. J. Iatropoulos, G. M. Williams, Proliferation markers. Exp. Toxicol. Pathol. 48, 175–181 (1996).32. N. T. Morrell, P. Leucht, L. Zhao, J. B. Kim, D. ten Berge, K. Ponnusamy, A. L. Carre, H. Dudek,

M. Zachlederova, M. McElhaney, S. Brunton, J. Gunzner, M. Callow, P. Polakis, M. Costa,X. M. Zhang, J. A. Helms, R. Nusse, Liposomal packaging generates Wnt protein with invivo biological activity. PLoS One 3, e2930 (2008).

33. D. Heymann, Y. Fortun, F. Rédini, M. Padrines, Osteolytic bone diseases: Physiologicalanalogues of bone resorption effectors as alternative therapeutic tools. Drug Discov. Today10, 242–247 (2005).

34. V. Krishnan, H. U. Bryant, O. A. Macdougald, Regulation of bone mass by Wnt signaling.J. Clin. Invest. 116, 1202–1209 (2006).

35. K. Tamai, M. Semenov, Y. Kato, R. Spokony, C. Liu, Y. Katsuyama, F. Hess, J. P. Saint-Jeannet,X. He, LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535(2000).

36. K. I. Pinson, J. Brennan, S. Monkley, B. J. Avery, W. C. Skarnes, An LDL-receptor-relatedprotein mediates Wnt signalling in mice. Nature 407, 535–538 (2000).

37. W. Y. Kim, X. Wang, Y. Wu, B. W. Doble, S. Patel, J. R. Woodgett, W. D. Snider, GSK-3 is amaster regulator of neural progenitor homeostasis. Nat. Neurosci. 12, 1390–1397 (2009).

38. O. P. Gautschi, S. P. Frey, R. Zellweger, Bone morphogenetic proteins in clinical applications.ANZ J. Surg. 77, 626–631 (2007).

39. M. R. Urist, Bone: Formation by autoinduction. Science 150, 893–899 (1965).40. V. Joseph, Y. R. Rampersaud, Heterotopic bone formation with the use of rhBMP2 in posterior

minimal access interbody fusion: A CT analysis. Spine 32, 2885–2890 (2007).41. S. Minear, P. Leucht, S. Miller, J. A. Helms, rBMP represses Wnt signaling and influences skeletal

progenitor cell fate specification during bone repair. J. Bone Miner. Res. 10.1002/jbmr.29 (2010).42. L. C. Fuentealba, E. Eivers, A. Ikeda, C. Hurtado, H. Kuroda, E. M. Pera, E. M. De Robertis,

Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal.Cell 131, 980–993 (2007).

43. K. R. Garrison, S. Donell, J. Ryder, I. Shemilt, M. Mugford, I. Harvey, F. Song, Clinical effec-tiveness and cost-effectiveness of bone morphogenetic proteins in the non-healing offractures and spinal fusion: A systematic review. Health Technol. Assess. 11, 1–150 (2007).

44. M. D. Gordon, M. S. Dionne, D. S. Schneider, R. Nusse, WntD is a feedback inhibitor ofDorsal/NF-kB in Drosophila development and immunity. Nature 437, 746–749 (2005).

45. I. B. Lobov, S. Rao, T. J. Carroll, J. E. Vallance, M. Ito, J. K. Ondr, S. Kurup, D. A. Glass, M. S. Patel,W. Shu, E. E. Morrisey, A. P. McMahon, G. Karsenty, R. A. Lang, WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature. Nature 437, 417–421(2005).

46. F. Le Grand, A. E. Jones, V. Seale, A. Scimè, M. A. Rudnicki, Wnt7a activates the planar cellpolarity pathway to drive the symmetric expansion of satellite stem cells. Cell Stem Cell 4,535–547 (2009).

47. A. Blumenthal, S. Ehlers, J. Lauber, J. Buer, C. Lange, T. Goldmann, H. Heine, E. Brandt,N. Reiling, The Wingless homolog WNT5A and its receptor Frizzled-5 regulate inflammatoryresponses of human mononuclear cells induced by microbial stimulation. Blood 108,965–973 (2006).

48. C. P. Petersen, P. W. Reddien, Smed-bcatenin-1 is required for anteroposterior blastemapolarity in planarian regeneration. Science 319, 327–330 (2008).

49. C. P. Petersen, P. W. Reddien, A wound-induced Wnt expression program controls planarianregeneration polarity. Proc. Natl. Acad. Sci. U.S.A. 106, 17061–17066 (2009).

50. W. Goessling, T. E. North, S. Loewer, A. M. Lord, S. Lee, C. L. Stoick-Cooper, G. Weidinger,M. Puder, G. Q. Daley, R. T. Moon, L. I. Zon, Genetic interaction of PGE2 and Wnt signalingregulates developmental specification of stem cells and regeneration. Cell 136, 1136–1147 (2009).

51. M. Ito, Z. Yang, T. Andl, C. Cui, N. Kim, S. E. Millar, G. Cotsarelis, Wnt-dependent de novo hairfollicle regeneration in adult mouse skin after wounding. Nature 447, 316–320 (2007).

52. V. Greco, T. Chen, M. Rendl, M. Schober, H. A. Pasolli, N. Stokes, J. Dela Cruz-Racelis, E. Fuchs,A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4,155–169 (2009).

53. M. V. Plikus, J. A. Mayer, D. de la Cruz, R. E. Baker, P. K. Maini, R. Maxson, C. M. Chuong,Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration. Nature451, 340–344 (2008).

54. B. I. Jugdutt, Limiting fibrosis after myocardial infarction. N. Engl. J. Med. 360, 1567–1569(2009).

55. R. Jagasia, H. Song, F. H. Gage, D. C. Lie, New regulators in adult neurogenesis and theirpotential role for repair. Trends Mol. Med. 12, 400–405 (2006).

56. T. Reya, H. Clevers, Wnt signalling in stem cells and cancer. Nature 434, 843–850 (2005).57. M. T. Veeman, D. C. Slusarski, A. Kaykas, S. H. Louie, R. T. Moon, Zebrafish prickle, a mod-

ulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 13,680–685 (2003).




58. T. Reya, A. W. Duncan, L. Ailles, J. Domen, D. C. Scherer, K. Willert, L. Hintz, R. Nusse,I. L. Weissman, A role for Wnt signalling in self-renewal of haematopoietic stem cells.Nature 423, 409–414 (2003).

59. R. DasGupta, H. Rhee, E. Fuchs, A developmental conundrum: A stabilized form of b-cateninlacking the transcriptional activation domain triggers features of hair cell fate in epidermalcells and epidermal cell fate in hair follicle cells. J. Cell Biol. 158, 331–344 (2002).

60. M. van Noort, M. van de Wetering, H. Clevers, Identification of two novel regulated serinesin the N terminus of b-catenin. Exp. Cell Res. 276, 264–272 (2002).

61. Acknowledgments: We thank S. Rooker for technical assistance with the experimentswith wild-type and Axin2LacZ/LacZ mice and L. Zhao for the fabrication of liposomal Wnt3a.Funding: CIRM TR1-01249 and FA9550-04-1-0075. S.M. was supported by a HowardHughes Medical Institute medical scholars fellowship. J.A.H. is a Hagey Faculty Scholar.Author contributions: P.L.: experiments associated with liposomal Wnt3a in the bone healingexperiments; S.M.: experiments associated with injuries in Axin2LacZ/+ and Axin2LacZ/LacZ mice;J.J.: in vitro and in vivo analyses on Axin2LacZ/+ and Axin2LacZ/LacZ mice; B.L.: Western analyses;

www.S

Y.A.Z. provided Axin2LacZ/+ and Axin2LacZ/LacZ mice; C.F.: development of lentiviral Wnt reporterconstruct; R.N. contributed to experimental design, discussions, data interpretation, andwriting of the study; J.A.H. contributed to the experimental design, data interpretation, analy-ses, and writing of the study. Competing interests: A patent application has been filed byJ.A.H., R.N., and P.L. for the composition, production, and therapeutic use for Wnt lipo-somes. U.S. Patent Application Serial No. 12/074,766 for “WNT Compositions and Methodsof Use Thereof.”

Submitted 26 June 2009Accepted 9 April 2010Published 28 April 201010.1126/scitranslmed.3000231

Citation: S. Minear, P. Leucht, J. Jiang, B. Liu, A. Zeng, C. Fuerer, R. Nusse, J. A. Helms, Wntproteins promote bone regeneration. Sci. Transl. Med. 2, 29ra30 (2010).


on

May

2, 2

010

stm

.sci

ence

mag

.org

Dow

nloa

ded

from


Documents

Wnt proteins promote bone regeneration