R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/beta-catenin pathway.

Abstract

R-Spondin1 (RSpo1) is a novel secreted protein that augments canonical Wnt/beta-catenin signaling. We injected recombinant RSpo1 protein into transgenic Wnt reporter TOPGAL mice and have identified the oral mucosa as a target tissue for RSpo1. Administration of RSpo1 into normal mice triggered nuclear translocation of beta-catenin and resulted in increased basal layer cellularity, thickened mucosa, and elevated epithelial cell proliferation in tongue. We herein evaluated the therapeutic potential of RSpo1 in treating chemotherapy or radiotherapy-induced oral mucositis in several mouse models. Prophylactic treatment with RSpo1 dose-dependently overcame the reduction of basal layer epithelial cellularity, mucosal thickness, and epithelial cell proliferation in tongues of mice exposed to whole-body irradiation. RSpo1 administration also substantially alleviated tongue mucositis in the oral cavity of mice receiving concomitant 5-fluorouracil and x-ray radiation. Furthermore, RSpo1 significantly reduced the extent of tongue ulceration in mice receiving a single fraction, high dose head-only radiation in a dose-dependent manner. Moreover, combined therapy of RSpo1 and keratinocyte growth factor resulted in complete healing of tongue ulcers in mice subjected to snout-only irradiation. In conclusion, our results demonstrate RSpo1 to be a potent therapeutic agent for oral mucositis by enhancing basal layer epithelial regeneration and accelerating mucosal repair through up-regulation of Wnt/beta-catenin pathway.

Full text

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R-Spondin1 protects mice from chemotherapy or
radiation-induced oral mucositis through the
canonical Wnt/?-catenin pathway
Jingsong Zhao1,2, Kyung-Ah Kim1, Josephine De Vera, Servando Palencia, Marie Wagle, and Arie Abo2
Department of Research, Nuvelo, Inc., 201 Industrial Road, Suite 310, San Carlos, CA 94070
Edited by Michael Karin, University of California at San Diego School of Medicine, La Jolla, CA, and approved December 9, 2008 (received for review
May 28, 2008)
R-Spondin1 (RSpo1) is a novel secreted protein that augments canon-
icalWnt/?-cateninsignaling.WeinjectedrecombinantRSpo1protein
into transgenic Wnt reporter TOPGAL mice and have identified the
oralmucosaasatargettissueforRSpo1.AdministrationofRSpo1into
normalmicetriggerednucleartranslocationof?-cateninandresulted
in increased basal layer cellularity, thickened mucosa, and elevated
epithelial cell proliferation in tongue. We herein evaluated the ther-
apeuticpotentialofRSpo1intreatingchemotherapyorradiotherapy-
induced oral mucositis in several mouse models. Prophylactic treat-
ment with RSpo1 dose-dependently overcame the reduction of basal
layer epithelial cellularity, mucosal thickness, and epithelial cell pro-
liferation in tongues of mice exposed to whole-body irradiation.
RSpo1 administration also substantially alleviated tongue mucositis
in the oral cavity of mice receiving concomitant 5-fluorouracil and
x-ray radiation. Furthermore, RSpo1 significantly reduced the extent
of tongue ulceration in mice receiving a single fraction, high dose
head-only radiation in a dose-dependent manner. Moreover, com-
bined therapy of RSpo1 and keratinocyte growth factor resulted in
complete healing of tongue ulcers in mice subjected to snout-only
irradiation. In conclusion, our results demonstrate RSpo1 to be a
potent therapeutic agent for oral mucositis by enhancing basal layer
epithelial regeneration and accelerating mucosal repair through up-
regulation of Wnt/?-catenin pathway.
RSpo1 ? tongue ulceration ? head-only radiation ? TOPGAL ? 5-FU
T
way both in vitro and in vivo (1–4). All four RSpo family members
contain an N-terminal signal peptide, two furin-like domains,
followed by a thrombospondin type1 domain, and a C-terminal
region rich with positively charged amino acids (5, 6). The Wnt/?-
catenin signaling plays a pivotal role in both embryonic develop-
ment and homeostatic self-renewal of adult tissues, including
gastrointestinal (GI) and oral mucosa (7). Abdominal distension
along the entire intestinal tract with a correlation of nuclear
translocation of ?-catenin in mucosal epithelial cells was seen in
both RSpo1 transgenic mice and normal mice injected with
recombinant RSpo1 protein (1, 8, 9). Additionally, mucosal
injury in mouse models of enterocolitis could be ameliorated
through RSpo1-mediated stimulation of epithelial cell growth in
GI mucosa (8).
Oral mucositis characterized by mucosal damage and inflamma-
tion in the oral cavity is a common complication of chemotherapy
and/or radiotherapy for cancer patients with head and neck tumors
and hematological malignancies (10). Impairment of the regener-
ative capacity of the oral mucosal epithelium leads to atrophy,
ulceration, and loss of barrier function during pathogenesis and
progressionoforalmucositis(11).Asaconsequence,oralmucositis
is frequently associated with pain, increased risk of infection, and
canleadtoimpairedqualityoflifeinaffectedindividuals(12).Oral
mucositis also limits the ability of patients to tolerate optimal
anti-tumor treatment regimens, thus compromising cancer therapy
outcomes and overall patient survival (13).
he R-Spondin (RSpo) family of secreted proteins is implicated
in the amplification of canonical Wnt/?-catenin signaling path-
Mucosal epithelial growth factors such as keratinocyte growth
factor (KGF) have been shown to be efficacious in treating exper-
imental animal models of chemotherapy or radiotherapy-induced
oral mucositis, and is the first approved biologic therapy for human
cancer patients with oral mucositis (14–20). However, KGF has
been shown to enhance the growth of human epithelial tumor cell
linesinvitroandtoincreasetherateoftumorcellgrowthinhuman
carcinoma xenograft models, probably by binding to its receptor
present on the surface of these cells (21). In comparison, we have
recently demonstrated that RSpo1 acts as a GI mucosal prolifer-
ative agent by triggering the physiological regenerative mechanism
throughamplificationoftheWnt/?-cateninpathway(4,8,9).While
the biological outcome is the growth of the GI mucosal epithelium,
RSpo1 activity appears to be dependent on Wnt ligands (4, 8, 9).
To explore the in vivo role of RSpo1 in the augmentation of the
canonical Wnt/?-catenin pathway, we chose the Wnt reporter
TOPGAL mice (22) and identified oral mucosa as a target tissue
for RSpo1 responsiveness. Here, we show that RSpo1 stimulates
epithelial cell growth in the tongue mucosa and significantly
reduces oral mucositis induced by either chemotherapy or radio-
therapyinmice.RSpo1regeneratesmousetonguemucosathrough
augmentation of Wnt/?-catenin pathway in squamous epithelium.
Moreover, RSpo1 attenuates head-only irradiation-induced overt
tongueulcerationinmicebothaloneandincombinationwithKGF.
Taken together, our results support RSpo1 as a novel approach for
potential treatment of oral mucosal damage in human cancer
patients receiving intensive chemotherapy and/or radiotherapy.
Results
RSpo1 Augments the Canonical Wnt/?-Catenin Signaling in TOPGAL
Mouse Tongue. To explore the in vivo role of RSpo1 in the
augmentation of canonical Wnt/?-catenin signaling in target tis-
sues, we used Wnt reporter TOPGAL mice. Recombinant RSpo1
protein was injected into TOPGAL mice and multiple tissues were
analyzed including tongue, stomach, lung, liver, small intestine,
colon,skin,andreproductiveorgans.While?-galactosidaseactivity
was broadly detected in dorsal tongue with whole-mount Bluo-Gal
staining (Fig. 1Aii), an increase in ?-galactosidase staining was also
detected in TOPGAL mice injected with RSpo1 (Fig. 1Aiii). To
further identify RSpo1 responsive cells, mice tongues were sec-
tionedandexaminedfor?-galactosidaseactivity.ElevationofLacZ
Author contributions: J.Z., K.-A.K., and A.A. designed research; J.Z., J.D.V., S.P., and M.W.
performed research; J.Z. contributed new reagents/analytic tools; J.Z. analyzed data; and
J.Z. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1J.Z. and K.-A.K. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: jingsongzhao@yahoo.com or
arieabo@hotmail.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0805159106/DCSupplemental.
© 2009 by The National Academy of Sciences of the USA
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gene expression was detected in the dorsal mucosal epithelium of
TOPGAL mice injected with RSpo1, especially at sites underneath
filliform papillae (Fig. 1A e and f). No ?-galactosidase activity was
found on the TOPGAL mouse tongue besides dorsal mucosa.
There was no detectable ?-galactosidase activity in wild-type con-
trol mice (Fig. 1 Ai and Aiv).
Administration of RSpo1 protein also enhanced basal layer
epithelial cell proliferation, as revealed by bromodeoxyuridine
(BrdU) immunohistochemistry, in the dorsal mucosa in TOPGAL
mouse tongue (Fig. 1Avii–ix). A similar effect was also found in the
mucosal basal layer of the ventral tongue in TOPGAL mice,
although ?-galactosidase activity was not detected in the ventral
tongue (data not shown).
To confirm the augmentation of canonical Wnt/?-catenin path-
way by RSpo1, we performed ?-catenin immunohistochemical
staining on tongue sections from TOPGAL mice. In control
TOPGAL mice, ?-catenin reactivity was detected predominately
on the cell surface of the mucosal epithelium in both dorsal and
ventral tongue (Fig. 1 Bi and Biii). In comparison, activation of the
Wnt pathway was evident in RSpo1-treated TOPGAL mice as
indicated by nuclear ?-catenin staining in mucosal epithelium in
both dorsal and ventral tongue (Fig. 1 Bii, Biv, and Biv).
Taken together, our data indicate that augmentation of the Wnt
pathway by RSpo1 in mouse tongue is associated with proliferation
of the tongue mucosa.
RSpo1 Induces Mucosal Hyperplasia in Mouse Tongue by Antagonizing
Dickkopf-1(Dkk1).WehavepreviouslyshownthatRSpo1modulates
the canonical Wnt/?-catenin signaling by relieving the Dkk1 inhi-
bition imposed on the Wnt pathway, and subsequently increasing
cell surface levels of the Wnt pathway coreceptor LRP6 (4). To
further define the physiological mechanism of RSpo1 in regulating
mouse tongue mucosal homeostasis, we treated mice with RSpo1
aloneorincombinationwithanadenoviraladministrationofDkk1.
WhilemucosalhyperplasiawasdetectedinRSpo1injectedanimals
(Fig. 2Aii), administration of adenoviral Dkk1 to mice resulted in
mucosal hypoplasia and reduction of mucosal thickness in the
tongue (Fig. 2Aiii), suggesting that Wnt signaling plays a role in
regulating the growth of the tongue mucosa. Furthermore, RSpo1
treatment antagonized Dkk1-mediated mucosal damage, thus par-
tially restoring the integrity of the tongue mucosa (Fig. 2Aiv).
Administration of RSpo1 significantly increased basal layer
epithelial cell density (Fig. 2B, P ? 0.05), mucosal thickness (Fig.
2C, P ? 0.05), and mitotic cell number in the basal layer epithelium
(Fig. 2A v–viii, and D, P ? 0.05) of the mouse ventral tongue
mucosa. In contrast, adenoviral Dkk1 administration significantly
reduced basal layer epithelial cellularity (Fig. 2B, P ? 0.05),
mucosal thickness (Fig. 2C, P ? 0.05), and basal layer cell prolif-
erative index (Fig. 2D, P ? 0.05) in the mucosa of mouse ventral
tongue. In comparison, addition of RSpo1 partially rescued Dkk1-
induced inhibition of basal layer cellularity (Fig. 2B, P ? 0.05),
mucosal thickness (Fig. 2C, P ? 0.12), and basal layer cell prolif-
eration (Fig. 2D, P ? 0.05) in mouse ventral tongue mucosa.
Immunohistochemistry of ?-catenin in mouse ventral tongue
mucosa (Fig. 2A ix–xii) also demonstrated that RSpo1 induced
nuclear localization of ?-catenin (Fig. 2Ax) in mice that received
adenoviralDkk1(Fig.2Axii).Theseresultsconfirmedourprevious
findings that RSpo1 acts as an antagonist of the Wnt pathway
inhibitor Dkk1 and suggest that RSpo1-mediated tongue mucosal
proliferation is achieved through up-regulation of the canonical
Wnt/?-catenin pathway.
A
i
B
ii
iii
iv
v
vi
vii
viii
ix
i
ii
iii
iv
Fig. 1.
mucosa in TOPGAL mice. (A) (i–iii)
Whole-mount
mousedorsaltongues.(Scalebar,0.5
mm.)(iv–vi)CoronalsectionsofBluo-
Gal-stained mouse dorsal tongues
with nuclear fast red counterstain.
(Scale bar, 100 ?m.) (vii–ix) BrdU im-
munocytochemistry of mouse dorsal
tongue mucosa. (Scale bar, 100 ?m.)
(i, iv, and vii) WT, (ii, v, and viii)
TOPGAL treated with saline, (iii, vi,
and ix) TOPGAL treated with RSpo1.
(B) ?-catenin immunohistochemical
study in dorsal (i and ii) and ventral
(iii and iv) tongues of TOPGAL mice.
(i and iii) Saline; (ii and iv) RSpo1.
(Scale bar, 100 ?m; Insets, 25 ?m.)
Effect of RSpo1 on tongue
Bluo-Gal-stained
A
i
ii
iii iv
v
vivii
viii
ix
x
xixii
B
D
C
100
150
200
250
Basal layer cellularity
(cells/mm)
*
*
#
25
50
75
100
125
Naïve RSpo1Dkk1RSpo1+Dkk1 Naïve RSpo1Dkk1 RSpo1+Dkk1
Naïve RSpo1Dkk1RSpo1+Dkk1
Mucosal thickness (µ µm)
*
*
0
2
4
6
8
10
12
Basal layer BrdU label index (%)
*
*
#
Fig.2.
(i–iv) BrdU immunohistochemistry, (v–viii) and ?-catenin immunohistochemistry
(ix–xii) of mouse ventral tongue mucosa. i, v, and ix, naïve; ii, vi, and x, RSpo1; iii,
vii, and xi, Dkk1; viii, and iv, RSpo1?Dkk1. (Scale bar, 100 ?m; Insets, 25 ?m .) (B)
Basal layer epithelial cellularity in mouse ventral tongue mucosa. (C) Mucosal
thickness in mouse ventral tongue. (D) Basal epithelial layer BrdU labeling index
in mouse ventral tongue.*P ? 0.05 (RSpo1 vs. naïve, Dkk1 vs. naïve);#P ? 0.05
(RSpo1?Dkk1 vs. Dkk1).
EffectofRSpo1and/orDkk1onmousetonguemucosa.(A)H&Estaining
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The hyperplastic effect of RSpo1 in the oral mucosa is not
restricted to a single mouse strain. Similar biological activity was
also seen in BALB/c [supporting information (SI) Fig. S1], BDF-1,
and CD-1 mice (data not shown). As a Wnt/?-catenin pathway
modulator,RSpo1appearstoplayakeyroleinregulatingepithelial
cell proliferation and regeneration in mouse tongue mucosa.
RSpo1 potentially plays a central role in enhancing the endog-
enous canonical Wnt signaling pathway required for tissue ho-
meostasis and repair. Therefore, RSpo1 could be used for treating
tongue mucosal hypoplasia, a hallmark of oral mucositis. To
evaluate the therapeutic potential of RSpo1, we tested its ability to
reduce experimental oral mucositis in several commonly-used
mouse models.
RSpo1 Treatment Inhibits Whole-Body Irradiation-Induced Oral Mu-
cositis in Mice. To test the protective effect of RSpo1 treatment
against ionizing radiation-induced oral mucositis, we induced oral
mucositis by exposing BDF-1 mice to whole-body radiation at a
singlefractionof13Gy.Micewerepretreatedwith3dailyinjections
of RSpo1 (5 to 20 mg/kg) before irradiation, and mouse tongue
tissue was harvested 4 days after radiation and examined for extent
of mucosal damage. As shown in Fig. 3A, while radiation caused
extensive mucosal injury characterized by reduced mucosal basal
layer epithelial cellularity and mucosal thickness in ventral tongue
of saline-treated mice (Fig. 3Ai and Aii), RSpo1 treatment pro-
tected mice from whole-body radiation-induced ventral tongue
mucosal erosion in a dose-dependent manner (Fig. 3A iii–v).
Likewise, BrdU immunostaining showed that RSpo1 administra-
tion overrode irradiation-induced inhibition of ventral tongue mu-
cosal basal layer epithelial cell proliferation in a dose-dependent
fashion (Fig. 3A vi–x).
Morphometric analysis of hematoxylin and eosin (H&E) or
BrdU-stained ventral tongue sections confirmed the protective
effect of RSpo1 in treating whole-body radiation-induced oral
mucositis in mice. While 13 Gy radiation reduced mucosal basal
layer epithelial cellularity in ventral tongue from 195 cells/mm in
naïve mice to 140 cells/mm (P ? 0.05), RSpo1 treatment signifi-
cantly increased basal layer epithelial cellularity in ventral tongue
mucosa to 166 (P ? 0.05), 181 (P ? 0.05), and 203 cells/mm (P ?
0.05) at respective doses of 5, 10, and 20 mg/kg in mice receiving 13
Gy whole-body radiation (Fig. 3B). Likewise, while whole-body
radiation thinned the ventral tongue mucosa in mice from 73.3 ?m
in naïve to 48.4 ?m (P ? 0.05), administration of RSpo1 (5, 10, and
20 mg/kg) dose-dependently restored mucosal thickness to 65.6
(P ? 0.05), 70.1 (P ? 0.05), and 94.6 ?m, respectively (P ? 0.05)
(Fig. 3C). Consistent with the above observations, RSpo1 also
reversed the radiation-induced inhibition of basal layer epithelial
cell mitosis in mouse ventral tongue mucosa (Fig. 3D).
A similar protective effect of RSpo1 was also seen in dorsal
tongue mucosa and cheek mucosa in the oral cavity of irradiated
mice (data not shown). These results demonstrate that RSpo1 acts
as a potent epithelial regenerative protein and is an effective
therapeuticagentinreducingirradiation-inducedmucosalinjuryin
mouse oral cavity.
To further assess the therapeutic effects of RSpo1 during oral
mucosal injury, we also established an oral mucositis model using
the chemotherapeutic agent CPT-11. Similar to the irradiation-
induced oral mucositis model, injection of RSpo1 alleviated CTP-
11-inducedmucosaldamagetothemousetongue(Fig.S2).Wealso
found that RSpo1 reduced the severity of oral mucositis in mice
receiving 5-fluorouracil (5-FU), another cytoablative agent (data
not shown). Together, our data indicate that augmentation of the
canonical Wnt/?-catenin pathway by RSpo1 triggers epithelial
proliferation and subsequently restores the mucosal barrier in mice
exposed to either radiotherapy or chemotherapy.
Administration of RSpo1 Suppresses Concomitant Chemotherapy and
Radiation-Induced Oral Mucositis in Mice. To further study the
therapeutic potential of RSpo1 in healing tongue mucosa, we next
developed an oral mucositis mouse model that is induced by
concomitant chemotherapy and radiotherapy, a preclinical setup
that is more representative of human cancer patients. Oral mu-
cositis was induced in mice using 5-FU (50 mg/kg, qdx2) followed
by a relatively low dose of whole-body radiation (10 Gy, single
fraction).
Saline control mice with 5-FU and 10 Gy radiation developed
severeoralmucositisintheventraltonguecharacterizedbyreduced
mucosalbasallayercellularity,thinnedmucosallayer,andinhibited
mucosal basal layer epithelial cell proliferation (Fig. 4A). Concom-
itant 5-FU and 10 Gy radiation significantly reduced the mucosal
B
C
D
i
ii
iii
iv
v
vi
vii
viii
ix
x
A
Fig.3.
tongue mucosa in mice. i and vi, naïve; ii and vii, 13 Gy/saline; iii and viii, 13 Gy/RSpo1 (5 mg/kg); iv and ix, 13 Gy/RSpo1 (10 mg/kg); v and x, 13 Gy/RSpo1 (20 mg/kg).
(Scale bar, 100 ?m.) (B) Basal layer epithelial cellularity in ventral tongue mucosa in mice. (C) Mucosal thickness in ventral tongue in mice. (D) BrdU labeling index in
basal epithelial layer of mouse ventral tongue.*P ? 0.05 (13 Gy/saline vs. naïve);#P ? 0.05 (13 Gy/RSpo1 vs. 13 Gy/saline).
EffectofRSpo1ontonguemucosainwhole-bodyirradiatedmice.(A)MicroscopicimagesofH&Estaining(i–v)andBrdUimmunohistochemistry(vi–x)ofventral
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basallayercellularityinmouseventraltonguefrom221cells/mmin
naïve mice to 140 (P ? 0.05) (Fig. 4B). However, RSpo1 partially
increasedmucosalbasallayercellularityinventraltongueupto184
cells/mm (P ? 0.05) in mice treated with both 5-FU and 10 Gy
irradiation (Fig. 4B). While combined 5-FU and 10 Gy radiation
significantly eroded mucosal thickness to 43.4 ?m (P ? 0.05) in
comparison to 65.3 ?m in naïve mice, RSpo1 treatment markedly
increased mucosal thickness in ventral tongue to 56.1 ?m in mice
receiving concomitant 5-FU and 10 Gy radiation (Fig. 4C). BrdU
morphometry also revealed that RSpo1 stimulated the basal layer
epithelialcellproliferationintheventraltongueofmiceexposedto
both 5-FU and 10 Gy irradiation (Fig. 4D). These results demon-
strate that RSpo1 ameliorates oral mucositis in mice induced by
concomitant chemotherapy and radiotherapy, by repopulating ep-
ithelial cells within the tongue mucosa.
RSpo1ReducesOvertTongueUlcerationinMiceInducedbyHighDose,
Head (Snout)-Only Radiation. Tongue ulceration represents the mu-
cosal damage seen in high grade oral mucositis in cancer patients.
To evaluate the therapeutic potential of RSpo1 during ulcerative
oral mucositis, we have used a mouse model that recapitulates the
clinical features of severe oral mucositis. Herein, we designed
custom-made mouse jigs with lead shields (1 inch thick) so that the
radiation could be limited to the snout region of mouse heads (see
Fig. 5A for illustration). In preliminary studies, we demonstrated
the correlation of radiation dose to degree of tongue mucosal
damageanddeterminedthatasinglefractionofhighlevelradiation
at 30 Gy directly delivered to the mouse heads (snout area) reliably
resulted in overt tongue mucosal ulceration which peaked at 9 days
afterirradiation(datanotshown).Verylowmortalityrates(?10%)
of mice were seen during the experimental period following 30 Gy
irradiation restricted to mouse heads (data not shown). In the
currentstudy,asinglefraction30Gysnout-onlyirradiationresulted
in 38.7% tongue ulceration with completely denuded mucosal
epithelium in saline control mice (Fig. 5B). In contrast, adminis-
tration of RSpo1 to mice significantly reduced tongue ulceration in
a dose-dependent fashion to 22.1% (P ? 0.05), 15.6% (P ? 0.05),
and 10.7% (P ? 0.05) at 5, 10, and 20 mg/kg, respectively (Fig. 5B).
Furthermore, while KGF (6.25 mg/kg) alone significantly reduced
tongue ulceration to 12.4% (P ? 0.05), combined use of RSpo1 (10
mg/kg) and KGF resulted in complete closure of tongue ulceration
(0%, P ? 0.05) (Fig. 5B). Besides reduction of overt tongue
ulceration, administration of RSpo1 also increased the mucosal
thicknessandbasallayercellularityinnon-ulceratedareasofmouse
tongue (Fig. 5C).
To further evaluate the implication of RSpo1 in the Wnt/?-
catenin signaling process during repair of oral mucositis, we next
examined the RSpo1 effect on ?-catenin localization in mouse
tongue following head-only irradiation (Fig. 5D). While ?-catenin
was detected mainly on the cell surface in normal tongue mucosa,
reduced ?-catenin immunostaining was found in both dorsal and
ventral tongue mucosa of mice that received 30 Gy head-only
irradiation (Fig. 5D i, ii, iv, andv). In contrast, RSpo1 treatment led
to an increase in both immunostaining intensity and nuclear
localization of ?-catenin in stratified mucosal epithelial cells in
irradiated mice (Fig. 5D iii and vi).
Taken together, RSpo1 treatment significantly suppresses high
dose,head-onlyirradiation-inducedoverttongueulcerationinmice
by amplifying the Wnt/?-catenin signaling pathway and stimulates
the proliferative response required for the regeneration of mucosal
barrierfunction.WealsoshowthatRSpo1maypotentiatetheeffect
of KGF in inhibiting oral mucosal damage and ulceration in mice
receiving irradiation.
Discussion
RSpo family proteins play a key role in modulating the Wnt
pathway (1, 4). We have previously demonstrated that the
implication of RSpo1 in GI epithelial proliferation and its
potential therapeutic application for repairing damaged GI
mucosa (8, 9). Here we have extended this observation and have
shown that RSpo1 also stimulates epithelial proliferation in the
oral mucosa via augmentation of the Wnt/?-catenin signaling,
thereby amplifying the physiological regenerative mechanism to
restore the damaged oral mucosa. Since Wnt ligands are indis-
pensable for RSpo function, RSpo proteins act as Wnt modu-
lators not as bona fide growth factors (4).
In the current study, we initiated a comprehensive approach to
explore the in vivo role of RSpo1 in regulating the canonical Wnt
pathway using TOPGAL mice. TOPGAL mice were previously
developed to monitor canonical Wnt/?-catenin activity by engi-
neering the transgene containing the LacZ gene under the control
of a regulatory sequence consisting of three consensus LEF/TCF-
binding motifs upstream of a minimal c-fos promoter (22). The
TOPGAL transgenic mouse model was extensively used to analyze
the canonical Wnt signaling in vivo (23–26).
We injected TOPGAL mice with RSpo1 protein and examined
multiple tissues for in vivo augmentation of the Wnt/?-catenin
pathway as detected by the LacZ reporter gene. Among har-
vested tissues, tongue was found to have substantially enhanced
?-galactosidase signal in mucosal epithelium. We also observed
?-galactosidase activity in various other tissues in both saline and
RSpo1-treated TOPGAL mice including lung, liver, hair follicles,
and stomach, suggesting constitutive activity of the Wnt signaling
pathway in these tissues (data not shown). RSpo1-mediated signal
was seen in the stratified epithelium of the oral mucosa in TOP-
GAL mice. In agreement with this result, Wnt/?-catenin signaling
A
B
C
D
i
ii
iii
iv
v
vi
Fig. 4.
bined treatment of 5-FU and whole-body radiation. (A) Representative micro-
scopicimagesofH&Estaining(i–iii)andBrdUimmunohistochemistry(iv–vi)from
mouseventraltonguemucosa.(iandiv)Naïve,(iiandv)(5-FU?10Gy)/saline,(iii
and vi) (5-FU ? 10 Gy)/RSpo1. (Scale bar, 100 ?m.) (B) Epithelial cellularity of
ventraltonguemucosalbasallayerinmice.(C)Ventraltonguemucosalthickness
in mice. (D) Epithelial cell BrdU labeling index in mouse ventral tongue mucosal
basal layer.*P ? 0.05 [(5-FU ? 10 Gy)/saline vs. naïve];#P ? 0.05 [(5-FU ? 10
Gy)/RSpo1 vs. (5-FU ? 10 Gy)/saline].
Characterization of RSpo1 on tongue mucosa in mice receiving com-
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hasrecentlybeenshowntobecriticalforfungiformpapillaandtaste
bud formation during tongue development in mice (25, 27). Our
data indicate that RSpo1 may play an essential role in regulating
tongue mucosa during homeostasis and pathogenesis.
The TOPGAL mouse system has its own limitations. Multimer-
ized TCF reporter is necessary but may not be sufficient to give a
complete and definitive readout of Wnt signaling in vivo (22). The
discrepancies between TOPGAL reporter activity and Wnt signal-
ing have been described previously (28, 29) and may be due to
differences in the regulation of transgene expression in specific
tissue contexts. We observed that the Wnt response cells, via either
TOPGAL reporter activity or nuclear localization of ?-catenin, do
not completely correlate with mitotic cells. Therefore, we cannot
exclude the possibility that the induction of cell proliferation in
tongue mucosa may be an indirect result of enactment of the
canonical Wnt pathway in distal cells.
To further investigate the role of the Wnt pathway during oral
mucosal homeostasis, we examined the effect of the Wnt inhibitor
Dkk1. We demonstrated that overexpression of Dkk1 resulted in a
mucositis-like hypoplastic phenotype in mouse tongue, indicating a
pivotal role of Wnt signaling in normal homeostasis of tongue
mucosa.Inaddition,Dkk1-inducedmucosaldamageandreduction
of ?-catenin expression was efficiently reversed by RSpo1 treat-
ment, suggesting that RSpo1-mediated mucosal hyperplasia in the
oral cavity is an outcome of the up-regulation of the Wnt/?-catenin
pathway. These observations are consistent with our previously
proposed mechanism that RSpo1 modulates the Wnt signaling
pathway by interfering with Dkk1 function (4). As we recently
demonstrated, RSpo1 plays a central role in regulating cell surface
levels of the coreceptor LRP6, by interfering with Dkk1/Kremen-
mediated LRP6 internalization (4). Based on the molecular mech-
anism of action of RSpo1, we proposed that RSpo1 could be an
effective therapeutic agent in treating diseases that are dependent
on Wnt signaling for tissue repair.
Usingexperimentalcolitismodels,wehavepreviouslyshownthat
RSpo1 can effectively regenerate the GI mucosa, a tissue that is
fully dependent on canonical Wnt/?-catenin signaling for its self-
renewal (8, 30). We herein provided further evidence that demon-
strates the therapeutic potential of RSpo1 in tissue regeneration.
We used several mouse models of experimental oral mucosal
damage induced by chemotherapy and/or radiation to demonstrate
that enhancement of the Wnt/?-catenin pathway by RSpo1 accel-
erates the oral mucosal epithelial cell regeneration. Furthermore,
RSpo1, either alone or together with the growth factor KGF,
significantly inhibits overt tongue ulceration in mice that received
high dose, snout-only irradiation.
The mucosal epithelium lining the oral cavity undergoes rapid
self-renewal and is thus susceptible to the non-specific toxicity that
occurs during radiation therapy and chemotherapy (13, 31). Oral
mucositis is a common side-effect among patients who receive
aggressive myeloablative chemotherapy for hematologic malignan-
cies and in patients who get radiotherapy as a treatment for cancers
of the oral cavity, oropharynx, and nasopharynx (11). In its mild
form, oral mucositis presents an erythematous, atrophic lesion in
which the mucosal lining still remains intact. In contrast, patients
with more severe oral mucositis develop visible ulcerations that
penetrate into the submucosal lamina propria and cause impair-
ment on the quality of life (32). Oral mucositis also markedly
increases the risk of infection and compromises the efficacy of
treatment plans, by necessitating breaks and reductions in doses
used in chemotherapy and radiotherapy for affected patients (12).
Pathogenesis of oral mucositis, induced by chemotherapy and
radiotherapy, in mice is similar to that seen in humans and have
thus been widely used in proof-of-concept studies for preclinical
testing of therapeutic candidate agents (33, 34). As a Wnt
modulator, RSpo1 significantly reduces both chemotherapy and
radiotherapy-induced damage to the oral mucosa by amplifying
the Wnt/?-catenin signaling and subsequently triggering epithe-
lial cell growth to accelerate mucosal healing in mice. Moreover,
RSpo1 decreases the extent of overt tongue ulceration by
repairing disintegrated mucosa in mice exposed to high dose
radiation delivered to head region only. As we have shown in
severalanimalmodels,RSpo1appearstobeaneffectivemucosal
repair agent for regenerating tongue mucosa damaged by inten-
sive chemotherapy or radiotherapy.
Oral mucositis closely follows the paradigm of an acute
mucosaldamagephasecharacterizedbyinflammation,epithelial
cell apoptosis, and ulcerative lesions, followed by a self-healing
phase with restoration of mucosal epithelium and barrier func-
tion (10). We thus assessed the time-dependent effect of RSpo1
in reducing oral mucositis in irradiated mice (Fig. S3). We found
that RSpo1 is efficacious in both injury and healing phases of
oral mucositis in mice by increasing the basal layer epithelial cell
density in tongue (Fig. S3). Moreover, RSpo1 treatment is also
effective in reducing oral mucositis activity in mice during
courses of both non-ulcerated and ulcerative mucosal damages
A
B
C
D
i
ii
iii
iv
v
vi vii
i
ii
iii
iv
v
vi
Fig. 5.
ulceration in mice treated with high
dose head-only irradiation. (A) Sche-
matic illustration of setup of head-only
radiation for mice. Red triangle repre-
sents passage of x-ray irradiation. (B)
Overt ulceration in mouse tongue mu-
cosa.*P ? 0.05 (30 Gy/RSpo1 or 30
Gy/KGF vs. 30 Gy/saline);#P ? 0.05
[30Gy/(RSpo1?KGF)vs.30Gy/saline,30
Gy/RSpo1, or 30 Gy/KGF]. (C) Histologi-
cal representations of mouse dorsal
tongue (H&E staining). Red lines do-
nate tongue ulceration with complete
mucosal denudation. (i) Naïve, (ii) 30
Gy/saline, (iii) 30 Gy/RSpo1 (5 mg/kg),
(iv) 30 Gy/RSpo1 (10 mg/kg), (v) 30 Gy/
RSpo1(20mg/kg),(vi)30Gy/KGF,(vii)30
Gy/(RSpo1?KGF). (Scale bar, 100 ?m.)
(D) ?-catenin immunohistochemical
staining in mouse dorsal (i–iii) and ven-
tral (v and vi) tongues. (i and iv) Naïve,
(ii and v) 30 Gy/saline, (iii and vi) 30
Gy/RSpo1. (Scale bar, 100 ?m; Insets,
25 ?m.)
Activity of RSpo1 on tongue
Zhao et al.
PNAS ?
February 17, 2009 ?
vol. 106 ?
no. 7 ?
2335
MEDICAL SCIENCES