Ameloblastin Inhibits Cranial Suture Closure by Modulating Msx2 Expression and Proliferation
et al. (2013) Ameloblastin Inhibits Cranial Suture Closure by Modulating Msx2 Expression and
Proliferation. PLoS ONE 8(4): e52800. doi:10.1371/journal.pone.0052800
Ameloblastin Inhibits Cranial Suture Closure by Modulating Msx2 Expression and Proliferation
Phimon Atsawasuwan 0 1
Xuanyu Lu 1
Yoshihiro Ito 1
Youbin Zhang 1
Carla A. Evans 0
Xianghong Luan 0 1
Chi Zhang, University of Texas Southwestern Medical Center, United States of America
0 Department of Orthodontics University of Illinois College of Dentistry , Chicago, Illinois , United States of America
1 Brodie Laboratory for Craniofacial Genetics, Department of Oral Biology University of Illinois College of Dentistry , Chicago, Illinois , United States of America
Deformities of cranial sutures such as craniosynostosis and enlarged parietal foramina greatly impact human development and quality of life. Here we have examined the role of the extracellular matrix protein ameloblastin (Ambn), a recent addition to the family of non-collagenous extracellular bone matrix proteins, in craniofacial bone development and suture formation. Using RT-PCR, western blot and immunohistochemistry, Ambn was localized in mouse calvarial bone and adjacent condensed mesenchyme. Five-fold Ambn overexpression in a K14-driven transgenic mouse model resulted in delayed posterior frontal suture fusion and incomplete suture closure. Moreover, Ambn overexpressor skulls weighed 13.2% less, their interfrontal bones were 35.3% thinner, and the width between frontal bones plus interfrontal suture was 14.3% wider. Ambn overexpressing mice also featured reduced cell proliferation in suture blastemas and in mesenchymal cells from posterior frontal sutures. There was a more than 2-fold reduction of Msx2 in Ambn overexpressing calvariae and suture mesenchymal cells, and this effect was inversely proportionate to the level of Ambn overexpression in different cell lines. The reduction of Msx2 expression as a result of Ambn overexpression was further enhanced in the presence of the MEK/ERK pathway inhibitor O126. Finally, Ambn overexpression significantly reduced Msx2 down-stream target gene expression levels, including osteogenic transcription factors Runx2 and Osx, the bone matrix proteins Ibsp, ColI, Ocn and Opn, and the cell cycle-related gene CcnD1. Together, these data suggest that Ambn plays a crucial role in the regulation of cranial bone growth and suture closure via Msx 2 suppression and proliferation inhibition.
Funding: This study has been generously supported by grant DE19155 from the National Institute of Dental and Craniofacial Ressearch (http://www.nidcr.nih.
gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
During skull development, calvarial bones expand from initial
ossification centers toward the suture interface between adjacent
]. Developmentally, this gradual intramembranous
ossification is accomplished by the differentiation of
ectomesenchymal cells into calvarial osteoblasts [
]. On a molecular level,
membranous bone ossification is regulated by a number of growth
and transcription factors as well as extracellular matrix proteins,
including fibroblast growth factor receptors FGFR1 and FGFR2
], transcription factors CBFA1/RUNX2 [
], MSX2 [
], and matrix proteins osteopontin and tenascin
]. Abnormal changes in these factors not only affect bone
thickness and mineralization, but also the expansion of bone along
the ossification center/suture axis, resulting in either synostosis or
From a clinical perspective, disturbances in normal skull
ossification result in a number of pathologies with often dramatic
consequences for children’s health. Premature ossification of one
or several sutures of the skull leads to an abnormal skull shape or
retarded skull growth (Craniosynostosis, CS). The most common
type of CS involves disturbances in the formation of a single suture
(simple or non syndromic CS), and its etiology remains
unexplained. Multiple suture synostoses (syndromic CS) are often
associated with syndromes of genetic origin i.e. Apert syndrome,
Crouzon syndrome, Pfeiffer syndrome and Saethre-Chotzen
]. The incidence of non-syndromic CS in United
States is about 34.3/100,000 live births while syndromic CS is
rare; their incidence is about 1.5/100,000 [
]. The treatment of
CS is composed of multiple surgical interventions to correct the
abnormal shape of the head, give space for the brain to grow in a
normal fashion and prevent the development of intracranial
pressure leading to neurological and brain damage complications
or death [
]. Complications from surgical treatment can lead to
infection, re-ossification, and death from hemorrhage [
Cranial osteogenesis and the differentiated state of the bone/
suture interface are regulated by extracellular matrix molecules
that transmit signals across the cell membrane into the cytoplasm,
activating a number of signaling pathways, which subsequently
affect gene expression [
]. One of the recently discovered
members of the bone extracellular matrix is ameloblastin (AMBN),
a glycoprotein previously only associated with the extracellular
enamel matrix [
]. Recent studies have noted Ambn
expression also in dentin, cementum, pulp, cranial bones, and
primary osteoblasts [
]. Moreover, Ambn has been shown to
regulate Msx2 expression in ameloblasts  [
Msx2 mRNA expression was dysregulated in Ambn deficient mice
]. In the present study, we hypothesized that Ambn might
affect the growth of craniofacial bones and the patency of cranial
sutures through its effect on Msx2 and possibly other mechanisms.
Here we have tested this hypothesis using Ambn transgenic mice
and in vitro models. Our studies reveal a distinct effect of Ambn on
craniofacial bone growth and suture closure, and suggests possible
mechanisms of action.
Materials and Methods
Transgene constructs and transgenic mice
Two transgenic constructs were generated using a modified
pSKII-trans vector in which the K14 promoter, the polyA signal (a
generous gift from Dr. Elaine Fuchs, Rockefeller University), the
b-globulin intron, the mouse Ambn coding region, or the LacZ gene
was inserted. The b-globulin intron was used to ensure that the
transgenes were transcripted properly. The transgenic fragments
were freed from pSKII-K14-AMBN or pSKII-K14-LacZ by
digesting the constructs with Sac I and Hind III, gel purified and
microinjected into mouse zygotes [
]. Mice used in the present
study included human keratin 14 (K14) promoter-driven Ambn
transgenic mice (AmbnTg, E18.5, P1, 20, 35 and 60 days of age,
n = 14) and K14 promoter-driven LacZ transgenic mice (P1, n = 3).
This study was carried out in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
protocol was approved by the Committee on the Ethics of Animal
Experiments of the University of Illinois at Chicago (Permit
Number: 11–077). For further analysis, mice were euthanized in a
CO2 chamber followed by cervical dislocation, and calvarial bone
and suture tissues were collected for fixation or RNA/protein
Genotyping. Genotyping was carried out using tails collected
from AmbnTg or LacZ transgenic heterozygous litters [
tails were lysed in DirectPCR (Tail) buffer (Viagen, Los Angeles,
CA) at 50uC overnight and then 85uC for 40 min. PCR was
performed using a set of specific primers for K14 promoters: a
forward primer 59GCTTAGCCAGGGTGACAGAG 39 and a
reverse primer 59CACAGAGGCGTAAATGCAGA39. After 35
reaction cycles, aliquots of the PCR products were separated on a
2.0% TAE (Tris acetate EDTA) agarose gel, stained with ethidium
bromide, and photographed under UV light [
Dry skull preparation, whole mount X-gal staining and alcian blue/alizarin red staining
Dry skulls of three adult mice from wild-type and Ambn
transgenic mice at postnatal day 60(n = 3) were prepared using
Dermestes beetles. Briefly, the crude muscle and soft tissues were
removed from the animal heads and left cool-dry for several days.
The dry skulls were gently placed in a container with Dermestes
beetles for several days at 30uC under close observation. Once the
soft tissues of skulls were completely removed, the dry skulls were
collected and cleaned meticulously with a soft brush. The dry skull,
and especially the cranial suture of each animal was examined and
photographed under a stereo microscope. Width of the posterior
frontal suture of each animal was measured with a digital caliper
and recorded in mm units. For whole mount X-gal staining,
deskinned animal heads were fixed with 4% paraformaldehyde in
PBS at 4uC overnight. The samples were rinsed with PBS at room
temperature 3 times for 15 minutes each and then incubated in
the dark with a staining buffer containing 0.05 mM K3Fe(CN)6,
0.05 mM K4Fe(CN)6, 1 mM MgCl2 and 1 mg/ml X-gal at 37uC
for 7 hours. For whole mount alcian blue/alizarin red staining,
three embryos from the wild type and Ambn transgenic groups at
embryonic day 18.5(n = 3) were fixed and dehydrated with 70%,
100% ethanol and acetone. The samples were stained with
saturated alcian blue (Sigma, St Louis, MO) in 95% ethanol for
2 days, destained with 95% ethanol, and rehydrated.
Subsequently, samples were then stained with saturated alizarin red S(Sigma)
in 0.5% potassium hydroxide (KOH) solution for 2 days,
destained in 0.5% KOH solution until all soft connective tissue
turned clear and then stored in 80% glycerol.
Calvaria and cranial sutures from wildtype, Ambn or LacZ
transgenic mice at age of embryonic 18 and postnatal 35 days
(n = 3) were dissected and fixed with 10% formalin at 4uC and
demineralized in a solution containing 45% EDTA, 4.5% NaOH
and 1% formalin at 4uC until the demineralization was completed.
The demineralized samples were embedded in paraffin, cut in
5 mm sagittal sections and mounted on glass slides. Thereafter, the
samples were deparaffinized, rehydrated, and stained with
Hematoxylin and Eosin or subjected to immunohistochemistry.
Sections were deparaffinized, rehydrated, and treated with 6%
peroxide and methanol followed by a brief incubation in 10 mM
sodium citrate buffer with 0.05% Tween 20 at pH 6.0 for antigen
retrieval. Sections were then incubated in 2% bovine serum
albumin (BSA) for 30 minutes at room temperature to block
nonspecific binding of the antibody. After blocking, sections were
first incubated with affinity purified anti-Ambn antibody [
at dilution of 1:200, and then with anti-rabbit secondary antibody
(Abcam, Cambridge, MA) at dilution of 1:2000. The expression of
Ambn protein was visualized with a Histomouse Broad Spectrum
AEC kit (Invitrogen, Carlsbad, CA) under a light microscope. As a
negative control, non-immune rabbit serum was used instead of
the primary antibody.
BrdU and TUNEL staining
For BrdU staining, pregnant mice at E18 were injected with
BrdU (100 mg/kg) intraperitoneally prior to euthanization.
Calvaria and cranial sutures were dissected and processed for
paraffin sections. The sections were deparaffinated, rehydrated,
and stained with biotinylated anti-BrdU antibody. The BrdU
positive cells were detected with the streptavidine-biotin system
(Invitrogen). For TUNEL staining, sections were first exposed to
proteinase K(20 mg/ml) at ambient temperature for 10 min, and
then incubated with TdT staining solution at 37uC for 1 hour
according to the manufacturer’s instruction (Promega, Madison,
WI). The fluorescence-dUTP-labeled DNA was visualized using a
The mouse suture mesenchymal cells were isolated from the
underlying dura mater and overlying pericranium of posterior
frontal and sagittal sutures of wildtype and Ambn transgenic mice at
postnatal 3 days, and maintained in a-minimum essential medium
supplemented with 10% fetal bovine serum, 100 U/ml penicillin,
100 mg/ml streptomycin and 25 ng/ml Amphotericin B in a 5%
CO2 atmosphere at 37uC. The medium was changed twice a
week. To study the effects of Ambn on cell proliferation and
differentiation, suture mesenchymal cells from wildtype and Ambn
transgenic mice were subjected to regular culture medium or a
mineralization induction medium containing 50 mg/ml ascorbic
acid and 2 mM b-glycerophosphate, and cultured for 2–21 days.
Upon terminating the culture, cells were used for MTT assay,
alkaline phosphatase activity test, alizarin red staining, or RNA
and protein preparation.
Alkaline phosphatase and in vitro mineralization assay
After 7 days of culture in osteogenic media, cells were washed
and stained with alkaline phosphatase substrate (Roche
Diagnostic, Indianapolis, IN) to verify early osteogenic activity. After
21 days of culture in osteogenic media, the cells were fixed with
methanol, stained with 10% alizarin red solution, and mineralized
nodules were identified as red spots on the culture dish.
Cell proliferation assay
Prior to termination of culture, cells were incubated in MTT
solution (2 mg/ml of MTT in DMEM with 2% FBS) for 4 hours.
To quantify proliferative activity, the MTT stained cells were lysed
in HCL/Isopropanol, and the absorbance was detected at 570 nm
with background subtraction at 630 nm [
RNA extraction and RT-real time PCR
Total RNAs were isolated from mouse suture tissues or cultured
cells using the TRIZOL LS Reagent (Invitrogen) according to the
manufacturer’s instructions. Two micrograms of total extracted
RNA was applied toward cDNA generation with the Sprint RT
Complete kitH (Clontech, Mountain View, CA). To quantify the
mRNA expression levels of transcription factors and bone marker
genes, real-time PCR primers were designed based on EMBL/
GenBank searches (shown in Table I). Real-time PCR was
performed using sequence specific sybergreen primers and the ABI
Prism 7000 Sequence detection system (Applied Biosystems, Foster
City, CA). Reaction conditions were as follows: 2 min at 50uC
(one cycle), 10 min at 95uC (one cycle), and 15 sec at 95uC, and
1 min at 60uC (40 cycles). Samples were normalized using
GAPDH. The analyses were performed in triplicate for three
independent experiments to confirm reproducibility of the results.
Relative expression levels were calculated using the 2–DDCt method
], and values were graphed as the mean expression level 6
Protein extraction and western blot analysis
Calvaria from 3 transgenic and 3 control animals at age P20
were collected and homogenized under liquid N2. Equal amounts
of protein extracts in a lysis buffer containing 100 mM Tris HCl
pH 9.0, 200 mM KCl, 25 mM EGTA, 36 mM MgCl2, 2%
deoxycholic acid and 10% DTT v/v were subjected to SDS–
polyacrylamide gel electrophoresis (Biorad, Hercules, CA), and the
separated proteins were transferred to a PVDF (Polyvinylidene
Difluoride) membrane (Immobilon PH, Millipore, Billerica, MA).
The membrane was incubated with rabbit anti-mouse Ambn
], Msx2, or CcnD1 antibodies (Abcam). Immune
complexes were detected with goat anti-rabbit IgG horseradish
peroxidase-conjugated secondary antibody (Molecular ProbesH,
Carlsbad, CA) and enhanced chemiluminescence reagents (Pierce
Biotechnology, Rockford, IL). The amount of protein expression
was compared after normalization with the amount of b-actin as
an internal calibrator in each lane.
Quantitative data were presented as means 6 SD from three
independent experiments and compared using Kruskall-Wallis
one-way analysis of variance. The difference between groups was
considered statistically significant at P,0.05.
AMBN is expressed in calvarial bone
Previous studies have demonstrated that the extracellular matrix
protein Ambn is expressed in cranial tissues including enamel [
], and cranial bones [
]. To examine the
function of Ambn in cranial bone and suture formation, Ambn
expression in mouse posterior frontal suture structures was
characterized. RT-PCR analysis of samples ranging between
postnatal days 1–20 revealed Ambn mRNA expression in
postnatal cranial sutures (Fig. 1A). Western blotting recognized
50 and 55 kDa bands positive for Ambn in calvarial bone extracts
(Fig. 1B). Using immunohistochemistry, Ambn protein was
localized in calvarial bone, dura mater, and adjacent condensed
mesenchyme (Fig. 1C).
Five-fold AMBN overexpression in a K14-driven transgenic mouse model
Gain of function mice are useful models to mimic syndromes of
genetic origin such as suture synostoses (syndromic CS) [
therefore generated Ambn transgenic mice using the human K14
promoter to drive Ambn gene expression [
]. In addition,
K14LacZ transgenic mice were created to verify the location of the
enforced Ambn gene expression [
]. Whole mount b-gal staining
of the K14-driven LacZ transgenic skull revealed dark blue staining
in parietal, frontal, maxillary, ethmoid and occipital bones and in
calvarial sutures (Fig. 2B), when compared to wildtype mice
(Fig. 2A). Histological analysis demonstrated b-gal blue staining in
calvarial osteoblasts in transgenic mouse sections (Fig. 2D), but not
in control mouse sections (Fig. 2C). When compared on western
blots and after normalization with b-actin, the amount of Ambn
protein from calvarial bones and posterior frontal (PF) sutures was
5 times higher in the Ambn transgenic mice than in wildtype
controls (Figs. 2E and F).
Posterior frontal suture fusion was delayed in Ambn transgenic mice
To determine the effect of Ambn on cranial suture closure,
skulls from Ambn transgenic and wildtype mice were compared
using alcian blue/alizarin red staining for cartilage and bone
mineralized tissue. Ambn transgenic mice at embryonic day 18.5
exhibited wider gaps of interfrontal and interparietal spaces
(Figs. 3B and D) compared to those of their controls (Figs. 3A and
C) (n = 3). There was less bone density and ossification along the
suture (Fig. 3D), and the osteogenic margins of frontal bones were
disorganized and not well defined (Fig. 3D), when compared to the
wildtype control (Fig. 3C). This phenotype was indicative of a
delayed development of the frontal bones. Skull samples of adult
Ambn transgenic animals (age 60 days postnatal) revealed the
patency of the posterior frontal sutures, revealing spanned gaps
between the frontal bones (Figs. 3F and H), while the controls
exhibited complete suture closure (Figs. 3E and G). The posterior
frontal suture gap width in the Ambn transgenic mice was 0.12–
0.42 mm (Fig. 3J), compared to 0.02–0.04 mm in controls (Fig. 3I)
(n = 3).
Ambn overexpressor skulls weighed less, and their interfrontal bones were thinner and wider
To further characterize the phenotypes of Ambn transgenic mice,
dried skull samples from wildtype and Ambn transgenic mice at age
of postnatal day 60 were compared, resulting in at least three
significant differences. First, there was a significant 13.2%
reduction in weight of Ambn transgenic skulls compared to WT
controls (Fig. 4A). Second, skull width of Ambn transgenic animals
was 14.3% increased when measured at the interfrontal bone/
coronal suture interface (Figs. 4B). Third, there was a significant
35.3% reduction in interfrontal bone thickness in transgenic mice
(Fig. 4D). Interestingly, there was no significant difference in the
length of skulls (data not shown), and in the length of the posterior
frontal suture, when comparing wildtype and transgenic groups
Incomplete posterior frontal suture closure and reduced proliferation rates in Ambn transgenic mice
Histomorphological analysis demonstrated complete closure of
the posterior frontal suture in control mice (Fig. 5A) in contrast to
incomplete closure in transgenic mice (Fig. 5B). The micrograph
was a fused bony bridge formation on the telencephalic aspect of
the calvaria in control mice (Fig. 5A), while the frontal bone arch
was interrupted by a soft tissue interface in the Ambn transgenic
mice (Fig. 5B). The interrupting soft tissue extended from the
periosteum to the dura mater in vertical direction (Fig. 5B). BrdU
staining of the developing posterior frontal suture (E18) revealed
29% less proliferating cells in Ambn transgenic mice. The number
of BrdU positive cells per 0.01 mm2 in the control group was
124+/25.6 while the number in Ambn TG was 88+/22.8. In
contrast, the number of TUNEL positive cells was 1.3-fold higher
in Ambn overexpressors (Figs. 5D and F) when compared to
wildtype mice (Figs. 5C and E).
Ambn inhibited mouse suture mesenchymal cell proliferation and differentiation in vitro
To determine how Ambn affects suture development, suture
mesenchymal cells were isolated from underlying dura mater and
overlying pericranium of posterior-frontal sutures of wildtype and
transgenic mice. Suture mesenchymal cells from wildtype mice
expressed basal Ambn protein levels (Fig. 6A), while the cells from
two different transgenic mice over-expressed Ambn protein at
about 4.05(TG1) and 2.5(TG2) times higher levels (Figs. 6A and
7B). To further examine whether Ambn expression levels affect
cell function, proliferation of suture mesenchymal cells was
analyzed using the MTT assay. There was an inverse relationship
between Ambn expression levels and cell proliferation capacity,
with highest MTT OD value in wildtype cells, followed by TG2
and then TG1 cells (Fig. 6B). In addition, there was a substantial
reduction in alkaline phosphatase activity and Alizarin red staining
in the two cell lines from Ambn overexpressor mice, TG1 and
TG2 (Figs. 6C and D). Remarkably, the high Ambn
overexpressing cell line TG1 had a stronger effect on Alizarin red-detected
mineralization inhibition than the less Ambn overexpressing cell
line TG2, suggesting that Ambn over-expression inhibited suture
mesenchyme osteogenic cell differentiation in a dose-dependent
manner (Figs. 6D).
Ambn regulated Msx2 and downstream gene expression
Ambn overexpressor mice displayed posterior frontal suture
patency, a phenotype previously reported in Msx2 deficient mice
]. In addition, Ambn over-expressing suture mesenchymal cells
displayed lower proliferative capacity and delayed differentiation
similar to the effects of Msx2 deficiency on mesenchymal cell
]. Therefore, Msx2 expression in suture tissues as well
as suture mesenchymal cells from Ambn transgenic and wildtype
mice was examined. Western blot analysis revealed that Msx2 and
CcnD1 expression in calvarial bones and PF suture tissues of Ambn
transgenic mice were downregulated (Fig. 7A). Msx2 mRNA
expression in suture mesenchymal cells from wildtype and
transgenic mice was reverse proportional to Ambn mRNA
expression, when comparing cells from two different transgenic
lines (Fig. 7B and C). To determine whether Ambn affects
expression of Msx2 down-stream target genes, expression levels of
osteogenic transcription factors Runx2 and Osx, bone matrix
proteins Ibsp, ColI, Ocn and Opn, as well as the cell cycle-related
gene CcnD1 were detected using quantitative RT real time PCR.
Our data demonstrated that Ambn overexpression resulted in
significant decreases in bone transcription factor and bone
mineralization marker gene expression in a dose-dependent
manner (Figs. 7D and E). Similarly, CcnD1 expression was
downregulated in TG1 and TG2 cells. Together, these studies
indicate that Ambn inhibits cell proliferation and reduces Msx2
and downstream gene expression in vivo and in vitro.
Transgenic animal models are ideal biological systems to test the
effect of forced gene expression on the development of a
phenotype. In the present study we have used the human Keratin
14 promoter to overexpress the extracellular matrix protein Ambn
in calvarial osteoblasts and suture mesenchymal cells. Keratin
14(K14, Cytokeratin 14 = CK14) is commonly known as an
epithelial protein encoded by the KRT14 gene. However, K14
expression in calvarial osteoblasts has been previously reported
and confirmed by Western blot [
]. In addition, reports of
keratins in non-epithelial cell lineages have become more frequent
in the literature, including mouse studies related to keratins
involved in endodermal and mesoderm adhesion in early
] (Vijayaraj et al. 2010, reports on K19 in
dental papilla and dental pulp [
] (Webb et al. 1995), microarray
and western blot detection of K18 in cementoblasts [
et al. 2011), findings of K19 and K8 in mature striated muscle [
(Shah et al. 2012), and K8 and K18 expression in mesenchymal
progenitors of regenerating limbs [
] (Cocoran and Ferretti
1997). Here, K14 expression in calvarial osteoblasts was further
confirmed by b-gal staining for the K14 LacZ transgene in K14
LacZ transgenic mice using both histology and whole mount
staining. The K14 promoter system has become a promoter of
choice in our and other laboratories because of its efficient
expression of many transgenes [
overexpression of Ambn in mouse calvaria resulted in a robust 5-fold
enhancement of Ambn levels in calvarial tissue extracts.
Our study indicates that Ambn was expressed in developing
calvarial bone and sutures. This finding matches other reports of
Ambn expression during bone formation [
] and expands its
original concept as an ameloblast-specific gene product [
However, when compared to the developing enamel matrix,
expression levels were less, suggesting that Ambn may not act
directly on bone development by controlling crystal growth as it
does in the enamel matrix [
], but rather indirectly as a
signaling molecule affecting the expression of transcription factors
and extracellular matrix signaling pathways [
concept is supported by findings reported in the present study that
already small differences in Ambn concentration have significant
effects on cell behavior, including cell proliferation and
Our human K14-driven Ambn overexpressor mice suffered from
delayed and/or incomplete cranial suture closure and displayed
patency of the posterior frontal suture. Moreover, frontal bones of
Ambn overexpressors weighed less and were thinner, suggestive that
Ambn was involved in tissue growth or mineralization or both. We
interpret these findings to indicate that Ambn plays an important
role in the growth and development of cranial bones and
subsequent suture closure. Our concept that Ambn affects
mineralized tissues outside of teeth is supported by earlier studies
related to the effect of Ambn on bone [
]. In our study, the
phenotype of incomplete suture closure in Ambn overexpressing
mice is explained by two related in vitro findings, namely (i) a
significant reduction in suture mesenchymal cell proliferation and
(ii) a downregulation of the cell proliferation marker cyclin D1
(CcnD1). In addition, studies in our and other laboratories have
also provided evidence for the inhibitory effect of Ambn on cell
proliferation, e.g. an increase of the cell proliferation inhibitors
p21 and p27 in Ambn overexpressing ameloblasts [
contrast, there have also been reports suggesting that Ambn
promotes bone healing by enhancing progenitor cell proliferation
] (Tamburstuen et al. 2011) and regulates osteogenic
] (Iizuku et al.). We propose that such findings may be
due to different concentrations of Ambn acting either as a
signaling molecule or a structural matrix protein [
Another factor responsible for the inhibition of suture closure by
Ambn overexpression is the transcription factor Msx2, a causative
gene involved in human cranial suture pathologies such as CS and
]. Our finding that Msx2 is downregulated by Ambn
is supported by earlier studies reporting upregulation of Msx2 in
ameloblasts from Ambn deficient mice [
] and downregulation of
MSX2 in human ameloblastoma cells overexpressing AMBN [
Notably, Msx2 deficient mice featured a patency of the posterior
frontal suture [
], a phenotype resembling the Ambn
overexpressor phenotype described in the present study. Underscoring
the severe clinical impact of defects caused by changes in Msx
levels and suture development, Msx1/2 double mutant embryos
were lacking frontal bones and died at E17 to E18 [
these studies indicate that changes in Ambn affect craniofacial
growth and suture closure via Msx2.
Evidence from previous studies suggests that the effects of Ambn
on Msx2 suppression and calvarial osteoblast proliferation
inhibition may be related, as it has been shown that Msx2
increases the expression of CcnD1, inhibits cell differentiation
], and influences the number of proliferative osteogenic cells in
growth centers of the developing mouse skull [
]. Others have
reported that calvarial osteoblasts derived from Msx2 deficient
mice had a lower rate of proliferation and demonstrated
accelerated osteoblastic differentiation at a later stage when
compared to osteoblasts derived from wildtype mice [
finding that suture mesenchymal cells from transgenic mice
reduced CcnD1 and osteogenic gene expression under
subconfluent condition supported a potential connection between
the role of Ambn in osteogenic differentiation and its role in cell
proliferation inhibition. Together, our data indicate that Ambn
affects calvarial osteoblast proliferation and differentiation through
suppression of the transcription factor Msx2.
These data suggested that the extracellular matrix protein
Ambn plays a crucial role in cranial suture closure. The effect of
Ambn on suture closure may be explained by the inhibitory effect
of Ambn on the transcription factor Msx2 and its effect on cell
proliferation, resulting in reduced growth and differentiation of
calvarial osteoblasts as well as delayed suture closure in mice.
Conceived and designed the experiments: X.Luan. Performed the
experiments: PA YI X.Lu YZ. Analyzed the data: PA X.Lu. Wrote the
paper: PA CE X.Luan.
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