Ameloblastin in Hertwig’s Epithelial Root Sheath Regulates Tooth Root Formation and Development
et al. (2013) Ameloblastin in Hertwig's Epithelial Root Sheath Regulates Tooth Root Formation
and Development. PLoS ONE 8(1): e54449. doi:10.1371/journal.pone.0054449
Ameloblastin in Hertwig's Epithelial Root Sheath Regulates Tooth Root Formation and Development
Naoto Hirose 0
Atsushi Shimazu 0
Mineo Watanabe 0
Kotaro Tanimoto 0
Souichi Koyota 0
Toshihiro Sugiyama 0
Takashi Uchida 0
Kazuo Tanne 0
Junming Yue, The University of Tennessee Health Science Center, United States of America
0 1 Department of Orthodontics, Applied Life Sciences, Hiroshima University Institute of Biomedical & Health Sciences , 1-2-3 Kasumi, Minami-ku, Hiroshima , Japan , 2 Department of Public Oral Health, Integrated Health Sciences, Hiroshima University Institute of Biomedical & Health Sciences , 1-2-3 Kasumi, Minami-ku, Hiroshima , Japan , 3 Department of Oral Biology, Basic Life Science, Hiroshima University Institute of Biomedical & Health Sciences , 1-2-3 Kasumi, Minami-ku, Hiroshima , Japan , 4 Department of Biochemistry, Akita University Graduate School of Medicine , 1-1-1 Hondo, Akita , Japan
Tooth root formation begins after the completion of crown morphogenesis. At the end edge of the tooth crown, inner and outer enamel epithelia form Hertwig's epithelial root sheath (HERS). HERS extends along with dental follicular tissue for root formation. Ameloblastin (AMBN) is an enamel matrix protein secreted by ameloblasts and HERS derived cells. A number of enamel proteins are eliminated in root formation, except for AMBN. AMBN may be related to tooth root formation; however, its role in this process remains unclear. In this study, we found AMBN in the basal portion of HERS of lower first molar in mice, but not at the tip. We designed and synthesized small interfering RNA (siRNA) targeting AMBN based on the mouse sequence. When AMBN siRNA was injected into a prospective mandibular first molar of postnatal day 10 mice, the root became shorter 10 days later. Furthermore, HERS in these mice revealed a multilayered appearance and 5-bromo-29deoxyuridine (BrdU) positive cells increased in the outer layers. In vitro experiments, when cells were compared with and without transiently expressing AMBN mRNA, expression of growth suppressor genes such as p21Cip1 and p27Kip1 was enhanced without AMBN and BrdU incorporation increased. Thus, AMBN may regulate differentiation state of HERS derived cells. Moreover, our results suggest that the expression of AMBN in HERS functions as a trigger for normal root formation.
After the completion of crown morphogenesis, tooth root
formation is initiated under a regulatory mechanism with an
interaction between inner and outer enamel epithelia, which forms
Hertwigs epithelial root sheath (HERS) . HERS proliferates
downward to the apical region and controls tooth root formation.
Epithelial cell rests of Malassez, derived from HERS, are located
in periodontal ligament tissues near the developing tooth root and
remain in periodontal ligaments throughout life [1,3]. Root
formation is characterized by a series of sequential interactions
between HERS and dental follicles. During this process, dental
follicular cells are differentiated into odontoblasts. However,
HERS cells undergo no calcification and diminish expression of
amelogenin and enamelin, except for ameloblastin (AMBN),
although HERS cells are derived from the enamel epithelium .
AMBN is an enamel matrix protein, also known as sheathelin or
amelin, is secreted by ameloblasts, and has the second highest
content among all enamel proteins in mature enamel .
Immediately after ameloblasts secrete AMBN for enamel
formation during crown morphogenesis, AMBN is cleaved into several
fragments  by its own proteolytic enzymes such as enamelysin
(matrix metalloproteinase - 20) and kallikrein-4 after secretion
from ameloblasts [6,7]. Fragments spread and localize in various
sites in newly formed enamel . These fragments may have
biological activity because AMBN contains the binding domains
for calcium [8,9], fibronectin  and heparin . Recent
studies indicate that AMBN knockout mice show abnormal
enamel structures and ameloblasts fail to adhere to immature
enamel layers, which then partially detach from the incisors [11
13]. In addition, ameloblasts in these mice lose their ability to
maintain normal polarization and exhibit marked proliferation,
suggesting that AMBN is required for growth and differentiation
of these cells and is necessary to constitute enamel structure.
Although AMBN has generally been believed to be located in
ameloblasts, recent studies report that AMBN is not a specific
protein in ameloblasts and is expressed in odontoblasts [14,15],
osteoblasts [16,17] and cementoblasts . AMBN also increased
proliferation in periodontal ligament cells  and osteoblasts
. HERS expresses AMBN, whereas other enamel proteins
including amelogenin, enamelin, and tufterin are eliminated in the
tooth root developmental process . It may be assumed that
AMBN is related to tooth root formation, but its role in root
formation remains unclear. To elucidate the role of AMBN in root
formation, we investigated the influence of AMBN downregulation
on HERS using small interfering RNA (siRNA) for AMBN.
F: GTG CCG GCA TTT CCT CAA CAA CCT G
R: CTG CAA GGG CAG CTG TCC
F: AAC TTT GGC ATT GTG GAA GG
R: GGG TTT CTT ACT CCT TGG AG
F: GAG AAC GGT GGA ACT TTG ACT T
R: CTC AGA CAC CAG AGT GCA AGA C
F: GAT ATG GAA GAA GCG AGT CAG C
R: GAG TTT GCC TGA GAC CCA ATT A
F: GGT ACT TAC GGT GTG GTG TAT AAG G
R: GAT GGA GTC CAG GTA CTT CTT GAG
F: GGT CAC CCT AGT GTT TGA GCA TAT AG
R: GTA GAT TCT AGC TAG GCC AAA GTC AG
F: GAC GGA CAG AGA AAC CAA GCT TAC
R: GAT CAC GAT GCA CTA CTC TGT GAG
Materials and Methods
Immuno-cyotochemical Analyses for Root Developmental Processes
C57BL/6 mice (Japan CLEA, Tokyo, Japan) were used
throughout this study. Permission for all experiments in this study
was granted by the Animal Experiment Committee of Hiroshima
University. Mandibles were dissected and immersed in 4%
paraformaldehyde (PFA) in 0.067 M phosphate buffer, pH7.4 at
4uC for 24 h and decalcified with 10% ethylenediaminetetraacetic
acid (EDTA) for about 1 week at 4uC. Specimens were embedded
in paraffin and cut into sections 5 mm thick along the mesiodistal
direction. These sections were mounted on MAS-GP coated glass
slides (Matsunami, Osaka, Japan) and stained with hematoxylin
(Sigma-Aldrich, St. Louis, MO) and eosin (Sigma-Aldrich).
Deparaffinized sections were rehydrated in 0.01 M phosphate
buffered saline (PBS) of pH 7.4. Specimens were dipped in 0.3%
H2O2 for 30 min to block endogenous peroxidase, and then
incubated with PBS containing 10% FCS for 30 min. Sections
were incubated overnight at 4uC with either one of the following
three antibodies. Mouse polyclonal antibody against AMBN (Y48)
was generated by immunization of rabbits with synthetic peptides
(NKAQQPQIKRDAWRF) , and used at a dilution of 0.2 mg/
ml. Mouse monoclonal antibody against cytokeratin 5 (Covance,
Princeton, NJ) was used at a dilution of 1:500, and mouse
monoclonal antibody against 5-bromo-29-deoxyuridine (BrdU,
Convance) was used at a dilution of 1:500. After rinsing with PBS,
sections were incubated with either biotinylated anti rabbit IgG
(Vector, Burlingame, CA) for AMBN or biotinylated anti mouse
IgG for cytokeratin 5 and BrdU for 30 min at room temperature.
Positive reactions were visualized with 3, 39-diaminobenzidine
solution. The experimental protocols were approved by the
Animal Care and Use Committee of Hiroshima University.
Isolation of Hertwigs Epithelial Root Sheath (HERS) Cells
Postnatal day 10 C57BL/6 mice have no erupted lower molars.
First molar crowns were removed from mandibles and HERS was
carefully dissected from the apical root under a stereomicroscope
(SZX10; Olympus Tokyo, Japan). Tissues were washed with PBS
followed by digestion with 0.05% trypsin in 0.53 mM EDTA for
10 min. Isolated HERS cells were placed in culture dishes with
MCDB153 medium (Wako, Osaka, Japan) supplemented with
10% fetal bovine serum (FCS, Life Technologies, Carlsbad, CA),
100 U/ml penicillin, and 100 mg/ml streptomycin
(Sigma-Aldrich). The medium was changed every 3 days and cells were
cultured at 37uC under 5% CO2 in air for 2 weeks. No
contaminated fibroblasts survived in this condition due to the
low calcium content of MCDB153 medium. For immuno-staining
of cultured cells, cells were fixed in PBS containing 4% PFA and
0.5% Triton X-100 for 5 min, and then immuno-staining was
Total RNA Isolation and Reverse Transcription
Polymerase Chain Reaction (PCR)
Total RNA was extracted using RNeasy (Qiagen, Venlo,
Netherlands) according to the manufacturers instructions. Total
RNA of 1.0 mg was reverse-transcribed in 50 mM Tris-HCl
(pH 7.4), 75 mM KCl, and 2.5 mM MgCl2, 25 mg/ml
oligo(dT)1218, 200 U of M-MLV reverse transcriptase (Toyobo,
Osaka, Japan), 2 mM dNTP, and 10 mM DTT at 37uC for
30 min. PCR was performed on 1 ml of cDNA using the primer
pairs in Table 1. Aliquots of PCR products were separated on a 1%
agarose gel containing ethidium bromide and visualized with
Knockdown of AMBN Expression by Small Interfering
A siRNA targeting AMBN was designed based on the mouse
sequence and synthesized by Integrated DNA Technologies
(Coralville, IA). The siRNA compounds were 27mer
Dicersubstrate duplexes optimized for Dicer processing and showed
increased potency when compared with 21mer duplexes. The
siRNA oligonucleotides used in this study were as follows; AMBN
siRNA: 59-AGAGCACUAAGCAUAUAUU AAUAAA-39.
HERS cells were transfected with 40 nM siRNA using
LipofectamineTM RNAiMAX (Life Technologies) according to
the manufacturers instructions. Forty-eight hours after
transfection, total RNA was isolated and RT-PCR was performed to
verify the knockdown of AMBN expression.
Figure 1. Light micrographs illustrating root development and Hertwigs epithelial root sheath (HERS) in postnatal day 15 mice. (A)
Hematoxylin and eosin staining of the mandible. (B) Immuno-staining with ameloblastin (AMBN). AMBN is localized in ameloblasts of the outer
enamel epithelium in the first and the second molars (arrow) and incisor (arrow-head). (C) Hematoxylin and eosin staining of the distal root of the
lower first molar. (D) Immuno-staining with cytokeratin 5. Ameloblasts, epithelial cell rests of Malassez, and HERS positively reacted against
cytokeratin 5 immunohistochemistry. (E) Immuno-staining with AMBN. Ameloblasts around enamel (asterisk) and in the basal portion of HERS (arrow)
reacted with AMBN antibody, but not in the tip (arrowhead). am, ameloblasts; od, odontoblasts; en, enamel; de, dentin; pl, periodontal ligament; erm,
epithelial call rests of Malassez; HERS, Hertwigs root sheath. Red circle marks the location of HERS. Scale bars: 500 mm in (A) and (B), and 100 mm in
For injecting siRNA into dental lamina of postnatal day 10
mice, an ultrafinest 33G needle (NanoPass33, Termo, Tokyo,
Japan), Hamilton syringe (Hamilton, Reno, NV), and 0.61 mm
polyethylene tube (Becton Dickinson, Franklin Lakes, NJ) were
used. Under SZX10 (Olympus), either 1 ml AMBN or negative
control siRNA solution was injected into the mesial hillside of the
mandibular first molar in postnatal day 10 mice. Ten days after
the injection, histological observation was performed for the distal
root of the first molar because the mesial root may have been
affected by surgical or physical damage.
Cell Preparation for Transient Expression of AMBN
ALC cells had oral epithelial origins and were established from
tooth germs of newborn C57BL/6 mice . These cells were
maintained in spinner modified minimum essential medium
(Sigma-Aldrich) supplemented with 10% FCS (Life Technologies),
100 U/ml penicillin, and 100 mg/ml streptomycin
(Sigma-Aldrich) at 37uC under 5% CO2 in air. Full length AMBN in the
pcDNA3.1 plasmid vector (Life Technologies) or only the vector
was transfected into cells using Lipofectamine 2000 (Life
Technologies) according to the manufacturers instructions.
Forty-eight hours after transfection, transient expression of AMBN
was confirmed with RT-PCR analyses.
All the experiments were carried out in triplicate. Results were
expressed as means and standard deviations (SD). Students t-test
was performed to examine significant difference between treated
and untreated groups. SSPS software, version 18 (IBM, Armonk,
NY) was used for all statistical analyses. Differences between
means were considered significantly different when values of
*p,0.05 and ** p,0.01.
Figure 2. Expression of AMBN in HERS derived cells and suppression of AMBN expression by treatment with AMBN siRNA. HERS
derived cells were isolated from postnatal day 10 mice and maintained in MCDB153 medium. (A) Immuno-staining with AMBN. (B) Immuno-staining
without AMBN. (C) Immuno-staining with cytokeratin 5. (D) Immuno-staining without cytokeratin 5. Scale bars: 10 mm. (E) Expression of AMBN was
analyzed with total RNA from cells treated with either AMBN siRNA or control siRNA. MW; 100 bp molecular weight marker; C, treated with control
siRNA; Si, treated with AMBN siRNA. Expression of AMBN was inhibited by 60% when treated with AMBN siRNA. (F) The BrdU incorporation rate of
control siRNA group was higher than that of the siRNA treated group two days after siRNA treatment. Statistical analysis was performed using
Students t-test, **; p,0.01.
To determine the presence and distribution of AMBN during
tooth root developmental processes, we first assessed expression of
AMBN in postnatal day 15 mice. As shown in Fig. 1, AMBN was
immuno-localized in ameloblasts in the first and second molars
and incisors in the mandible. In postnatal day 15 mice, the process
of root formation had already been initiated and HERS extended
along with dental follicular tissue for root formation. Under higher
magnification, HERS was confirmed by immuno-reactivity against
cytokeratin 5, indicating their epithelial origin (Fig. 1D), whereas
Figure 3. Histological observation of the lower first molar treated with siRNAs. Distal root length of the lower first molar in AMBN siRNA
treated was shorter than that of controls. (A) Stereomicroscopic appearance of the lower first molar treated with AMBN siRNA. (B) Hematoxylin and
eosin staining in control and AMBN siRNA. Treatment with AMBN siRNA caused irregularities in dentin form at the tip of the root. (C) Average root
length in control and AMBN siRNA treated groups. Root length in treatment with AMBN siRNA (n = 10) was shorter than that of control siRNA (n = 10)
(Two headed arrow indicates root length in Figure B.). Statistical analysis was performed using Students t-test, *; p,0.05.
AMBN was immuno-distributed in the basal portion of HERS, not
at the tip (Fig. 1E).
To assess the functional roles of AMBN localized in HERS in
root development, siRNA targeting AMBN mRNA was designed
based on the mouse sequence and HERS cells were isolated from
postnatal day 10 mice. These cells were maintained in MCDB153
medium and no contaminated fibroblasts survived in this
condition due to the low calcium content. These cells exhibited
typical epithelial morphology and revealed reactivity against
AMBN (Fig.2A) and cytokeratin 5 (Fig. 2C); however, proliferation
was very slow without any confluence. These cells expressed both
AMBN mRNA and protein confirmed by RT-PCR analysis and
immuno-staining. Treatment with siRNA showed a 60% partial
knockdown of AMBN mRNA in HERS cells over that of negative
control siRNA (Fig.2E). In addition, treatment with siRNA
exhibited an increase in the number of incorporated BrdU in
these cells (Fig.2F).
Ten days after the AMBN siRNA injection into dental lamina of
postnatal day 10 mice, histological observation was performed in
the distal region of the first molar root in order to avoid side effects
from surgical damage. AMBN siRNA injected mice displayed
shorter roots in the first molar. In addition, these mice presented
slightly irregular root dentin structures, as observed histologically
(Fig. 3). However, no inflammatory disorder, infiltration of
immune cells, or root resorption was observed in the surrounding
area of dentin and cementum layers. Under higher magnification
in HERS, AMBN siRNA injected mice revealed a multilayered
appearance in the basal portion of HERS; although control siRNA
injected mice showed two-layered HERS derived from inner and
outer enamel epithelia (Fig. 4). Furthermore, AMBN siRNA
injected mice revealed that BrdU positive cells increased in
number in the outer layer of HERS. This result is consistent with
the mitotic index which was significantly higher than in control
To examine increases in proliferation by the knockdown of
AMBN, we prepared cells transiently expressing AMBN mRNA.
These cells expressed AMBN at a high intensity whereas control
cells had no AMBN mRNA (Fig. 5). Furthermore, these AMBN
non-expressing cells were higher in ratio to BrdU incorporation
than AMBN expressing cells. In addition, control cells exhibited
weak expression levels in negative cell cycle regulatory factors,
such as p21Cip1 and p27Kip1, although AMBN expressing cells
expressed distinct signals for these negative factors. However, there
were no significant differences in expression of positive cell cycle
Figure 4. Light micrographs illustrating HERS treated with siRNAs. AMBN siRNA injected mice revealed a multilayered appearance in the
basal portion of HERS and increased BrdU positive cells in that of the outer layer. Treatment with control siRNA (AC) and AMBN siRNA (DF). (A) and
(D) Hematoxylin-eosin staining. (B) and (E) Immuno-staining with cytokeratin 5. Treatment with AMBN siRNA revealed a multilayered appearance in
the basal portion of HERS (arrow). (C) and (F) Immuno-staining with AMBN. Treatment with AMBN siRNA increased BrdU positive cells in the outer
layer of HERS (arrows). de, dentin; od, odontoblasts; pl, periodontal ligament; HERS, Hertwigs root sheath. Red dotted lines mark the location of HERS.
Scale bars: 100 mm. (G) The percentage of BrdU positive cells per total HERS cells was counted and compared between treatments with control and
AMBN siRNA. The percentage of BrdU positive cells was higher with treatment with AMBN siRNA (n = 10) than that with control siRNA (n = 10).
Statistical analysis was performed using Students t-test, *; p,0.05.
regulatory factors, including CDK1, CDK4, and CDK6, between
cells with and without AMBN. These results indicate that
elimination of AMBN promotes proliferation via inhibition of
negative cell cycle regulators.
Ameloblasts secrete an abundance of enamel matrix proteins
such as AMBN, amelogenin, enamelin, and tuftelin [20,21].
Various studies have been conducted to investigate the role of
AMBN in enamel formation. AMBN is abundantly present in
immature enamel during the matrix formation stage [5,8], and is
identified as a protein with different nucleic acid sequences from
other enamel proteins . AMBN has the distinctive function to
immediately cleave itself into several fragments after being
secreted from ameloblasts . These fragments spread and
localize to various sites of newly formed enamel during crown
morphogenesis. The short fragment from the n-terminal domain
distributes in whole enamel and gradually degrades from the
enamel matrix during the matrix formation stage, whereas that
from the c-terminal domain distributes in only the superficial layer
of enamel and then rapidly degrades from immature enamel.
AMBN may be related to formation and maturation of enamel
crystallization. In recent studies using knockout mice against
enamelin , enamelin mutations caused ameloblast
malfunctions in cell morphogenesis, detachment from the tooth surface,
apoptosis, and formation of ectopic calcifications. In contrast,
mutating AMBN, amelogenin, or both in mice caused enamel
hyperplasia, with no enamel defects irrespective of disordered
functions in ameloblasts . It may thus be considered that these
enamel related molecules play important roles in mineral
deposition and crystallization in the organization and regulation of
However, a number of observations indicate that AMBN is not
an enamel specific protein because AMBN exists in a wide variety
of cells including osteoblasts [16,17], odontoblasts [14,15], and
cementoblasts . AMBN may play an important role in dentin
and cementum formation during differentiation processes in the
periodontium. HERS extends along with dental follicular tissue
and AMBN is highly distributed in the basal areas of HERS. A
histological study showed that ameloblasts change expression
levels of AMBN and amelogenin during enamel formation .
Based on this evidence, constituted cells in HERS may modulate
expression levels of AMBN in a developing and/or site specific
manner for root development similar to enamel formation.
This study was designed to focus on root formation, unintended
consequences which eliminated AMBN in enamel formation were
undesired. Injection of siRNA into cells provided useful
information about the undefined functions and roles of various gene
molecules at a specific stage [25,26]. A method involving siRNA
in vitro is a more convenient and inexpensive technique than that
with knockout mutants. However, intravenous injection of siRNA
in vivo has disadvantages in cost and effect because it is hard to
deliver siRNA into target organs through the circulation and it is
necessary to inject large amounts of siRNA. On the other hand,
local administration of siRNA into living organisms has been
shown to be beneficial, but it is necessary to apply to a confined
space in order to avoid leaking reagent solution over a long period,
such as lumbar vertebra, retina, and the brain .
In this study, microinjection of AMBN siRNA into the mesial
side of a prospective mandibular first molar was performed in
postnatal day 10 mice. Ten-day-old mice have no erupted and
developing molars in jaws and their molars are surrounded by
alveolar bone with a confined space where reagent solution can be
kept. In local administration, AMBN siRNA is reliably able to
Figure 5. Comparison between AMBN expressing and non expressing cells. Full length AMBN cDNA transfected cells expressed AMBN at
a high intensity whereas control cells had no AMBN mRNA. (A) and (B) Positive cell cycle regulatory factors, CDK1, CDK4 and CDK6, and negative cell
cycle regulatory factors, p21Cip1 and p27Kip1, were examined by RT-PCR analysis. AMBN non expressing cells had lower expression levels of negative
cell cycle regulators than those of AMBN expressing cells. (C) BrdU positive cells were counted and compared between AMBN expressing and
nonexpressing cells. Cell numbers in the AMBN non-expressing group was lower than those of the AMBN expressing group. Statistical analysis was
performed using Students t-test, **; p,0.01.
reach the tip of the tooth root. AMBN siRNA injection revealed
shorter roots and slightly irregular root dentin formation in mice
and HERS cells showed abnormal proliferation. Meanwhile,
a previous study using AMBN knockout mice demonstrated an
influence on enamel formation, but not root formation [11,12]. It
has recently been shown that previously-believed AMBN knockout
mice still produce a short truncated form of AMBN, although this
is not the full length of AMBN . Such a short truncated form
of AMBN still contains the binding domains for calcium,
fibronectin, and heparin. The truncated form of AMBN influences
enamel formation, but quantitative depletion of AMBN in this
study may be important for root formation. Elimination of AMBN
may cause modulation of the differentiation status of HERS cells
as well as proliferation. A disparity in AMBN expression in HERS
may result in the modulation of reciprocal influences to the
peripheral local environment including odontoblasts.
A number of genes are involved in cell division regarding
positive and negative cell cycle regulatory factors. CDK1, CDK4,
and CDK6 are positive regulators, and p21Cip1 and p27Kip1 are
negative regulators in the cell cycle . In this study, there was
a considerable discrepancy between cells expressing AMBN and
their proliferation. Omitting AMBN in cells diminished the
inhibitory effects of p21Cip1 and p27Kip1, thereby cells increased
their ratio of BrdU incorporation. A previous study revealed that
overexpression of AMBN in human ameloblastoma inhibited
proliferation through suppression of negative cell cycle regulators
including p21Cip1 and p27Kip1 . However, overexpression of
AMBN with a defect in heparin binding domains had relatively
little effect on proliferation. Therefore, heparin binding domains
in AMBN may be important for regulating cell growth. From these
findings, it may be presumed that suitable expression of AMBN in
HERS may function as a certain trigger of root formation and the
progress essential for optimal root development.
Conceived and designed the experiments: NH AS MW KT TU KT.
Performed the experiments: NH AS TU. Analyzed the data: NH AS.
Contributed reagents/materials/analysis tools: NH SK TS. Wrote the
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