Critical Reviews™ in Eukaryotic Gene Expression, 18(4):345–360 (2008)

The Amelogenin “Enamel Proteins” and Cells in the

Periodontium
Carolyn W. Gibson*
Department of Anatomy and Cell Biology, University of Pennsylvania School of Dental Medicine,
Philadelphia, PA 19104, USA

* Author to whom all correspondence should be addressed; Gibson@biochem.dental.upenn.edu

ABSTRACT: This overview will examine the multifunctional nature of a group of proteins known as the am-
elogenins. These secreted proteins were named in the 1960s because of their expected role during development
of dental enamel [Eastoe JE. Adv Fluorine Res. 1965;21:5–17]. As gene expression assays became more sensitive,
expression was also noted in tissues not involved with enamel formation leading to hypotheses concerning ad-
ditional roles for these proteins. In vitro approaches led to the discovery that some of the amelogenins are able to
regulate gene expression and to participate in cellular signaling. An extract containing predominately amelogenins
has been used clinically in treatment of certain forms of periodontal disease with regenerative results noted
originally in animal models, but later in human patients as well. Much literature has been devoted to the roles
of amelogenins during mineral formation, and therefore this topic will be covered primarily in the Introduction.
The goal of this review will be to focus on strategies that have been used to uncover roles of amelogenins related
to gene expression and development apart from the roles in enamel mineral, and the possible functions that these
proteins could have if delivered to normally nonexpressing tissues for therapeutic approaches.

KEY WORDS: multifunctional proteins, moonlighting proteins, enamel extracellular matrix, alternative splicing,
ameloblasts, odontoblasts, cementoblasts

I. INTRODUCTION bone, which forms the tooth “socket” and holds

The developing tooth has long been considered
to be an excellent model for studies of epithelial-
mesenchymal interactions,1–4 since tooth organs
are able to develop in culture, allowing histological
changes to be easily monitored. The tooth also
holds major interest for studies of mineralized
tissue because it includes three different types of
hard tissue: the enamel covers the crown of the
tooth, which projects into the oral cavity; the
dentin underlies the enamel in the crown and also
forms the bulk of the root; and the cementum,
which is a thin layer of mineralized tissue cover-
ing the root and attaching the root to a layer of
soft tissue, the periodontal ligament (Fig. 1). This
ligament is attached peripherally to the alveolar
the tooth in place within the jaw.
The three mineralized tissues are distinct in
origin and structure. Enamel is a unique mineral-
ized tissue since it is an epithelial product made
by cells referred to as ameloblasts. When the tooth
erupts into the oral cavity, the ameloblasts are lost
and thus biological repair of the enamel layer is
not possible. Dentin is produced by an internal
layer of ectomesenchymal cells, the odontoblasts,
which remain in the pulp region throughout the
life of the tooth. Odontoblasts are organized as
a single layer of columnar cells with long cellular
extensions through the dentin layer to the dentin-
enamel junction. Cementoblasts are also mesen-
chymal cells, but are thought to have a different
origin in an external structure called the dental

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Volume Begell4House, Inc. 345
FIGURE 1. Structures found within and surrounding an incisor tooth. Ameloblasts produce enamel, but these
cells are lost as the tooth erupts into the oral cavity. Odontoblasts secrete dentin, and the mineralized cementum
layer is made by cementoblasts and cementocytes. The periodontal ligament attaches the root of the tooth to
the alveolar bone.

follicle. Some cementum-forming cells are located
within lamellae of cementum similar to osteocytes
in bone and are referred to as cementocytes, while
cementum-producing cells on the surface of the
root are called cementoblasts.
In the organic matrix of cementum, as in bone,
collagen type I forms the major structural compo-
nent.5 Enamel has a quite different composition,
since collagen is not a component of enamel but
the enamel proteins provide the structural func-
tion. The enamel extracellular matrix provides an
environment for the enamel to develop into the
hardest tissue in the body.
Several groups of enamel proteins are found
in developing enamel, including the amelogenins,
enamelins, ameloblastins, and various proteases,
which break down the organic material in a
regulated manner as the mineral crystals grow.6–8
This review will focus on the amelogenins, since
they represent approximately 90% of the enamel
matrix.9 Although originally and for many years
amelogenins were thought to be completely enamel
specific, it is now accepted that amelogenins
are also produced by odontoblasts or other cells
in the pulp of the tooth, but at a much lower
abundance.10,11 A similar phenomenon has been
shown for many of the enamel and dentin matrix
proteins, where a high level is seen in one miner-
alizing tissue, but a much lower level is measured
in the adjacent one.12,13 In addition, expression
of amelogenins has been reported in the tissues
adjacent to the root. While this location for ex-

346 Critical Reviews™ in Eukaryotic Gene Expression

pression may be a species-specific phenomenon,
it has led to much discussion and new avenues
of research (see Sections III and V).
II. MULTIFUNCTIONAL NATURE OF
AMELOGENINS
Amelogenins were originally characterized as
structural proteins that guide the growth of enamel
mineral crystals, which become much larger than
crystals in the other mineralized tissues, such as
dentin, cementum, or bone.14,15 The secreted am-
elogenins assemble into nanometer-scale structures
referred to as nanospheres when expressed and
studied in vitro, and similar structures can be
observed in developing enamel in vivo in rodent
models.16,17 During development the amelogenin
proteins are degraded by the enamel proteases,18,19
and the biochemical properties of the amelogenins
are thought to change. Only small amounts re-
maining in the mature enamel layer.
The importance of the evolutionarily conserved
amelogenins to enamel development has been
shown by patients with amelogenin gene muta-
tions who have an enamel defect,20–22 and by the
generation of an Amelx null mouse, which has an
enamel defect similar to affected patients.23
Determination of amelogenin function is
complicated by a high level of alternative splicing
of the amelogenin primary transcript, described in
a number of species including human and mouse.
More than 15 X-chromosomal amelogenin mes-
sages have been described for mouse and at least
three for human thus far.24–26 It is likely that most
of these mRNAs are translated into proteins, but
the amount varies from highly abundant to only a
trace amount. The 180 amino acid amelogenin is
able to rescue the Amelx null hypoplastic enamel
phenotype in mice, and therefore this amelogenin
seems to be involved in determining enamel thick-
ness and organization (Fig. 2). However, this single
amelogenin is unable to entirely rescue the complex
phenotype of normal enamel,27 and it is assumed
that other amelogenins must have additional
complementary functions during development of
enamel. Multiple activities within single amelogenin
proteins are possible because of the domainlike
structure, including a cell binding function.28–30
Two additional observations lend credence
to the concept of amelogenins as multifunctional
proteins. Alternative splicing of the primary am-
elogenin transcript can lead to inclusion of exons
1,2,3,5, part of 6 and 7, and the translated protein
is relatively abundant, but only 59 amino acids in
length (Fig. 2). This small amelogenin, referred to
as leucine-rich amelogenin peptide or LRAP, was
isolated from bovine teeth by Fincham,31 testifying
to its abundance as a translated protein. The cDNA
was cloned supporting the concept that LRAP
is a product of an alternatively spliced cDNA.32
LRAP as well as a slightly longer amelogenin
including exon 4 also can interact with various
cell types in vitro or in vivo, leading to changes
in gene expression.33 LRAP also seems to have a
role in enamel mineral formation.34

FIGURE 2. Amelogenin alternative splicing. (A) The amelogenin gene includes seven exons and intervening introns.
Abundant amelogenins include a 180 amino acid protein (B), and the 59 amino acid leucine–rich amelogenin
peptide, LRAP (C). The three amelogenins depicted, including the 79 amino acid amelogenin (D), have been
implicated in cellular signaling or communication. Filled exons indicate they are skipped.

Volume 18 Number 4 347

 A second observation is that a porcine enamel
matrix extract has been used clinically to repair
diseased periodontal tissues, leading to parital
regeneration of an intact periodontium. The active
reagent(s) in this extract has not been ascertained,
but the product includes primarily amelogenin
proteins. The healing effect involves changes in
gene expression required for regeneration of the
cementum, periodontal ligament, and alveolar
bone, and it is assumed that proliferation, dif-
ferentiation, and perhaps migration of different
cell types are required.
This review will discuss the strategies that
have been used to dissect the functions of am-
elogenin proteins related to gene expression, but
will omit for the most part functions related to
enamel mineral development. The focus will be
on understanding potential roles of amelogenins
within the developing and regenerating periodon-
tium, and will include hypotheses related to the
multifunctional nature of matrix proteins, the role
of alternative splicing in generating additional
functions, and how therapeutic approaches have
led to new ideas related to amelogenin function.
III. EXPRESSION OF AMELOGENINS
DURING DEVELOPMENT
A. By Ameloblasts Producing Enamel
Amelogenin messages and proteins are produced
by ameloblasts while the tooth is developing within
the jaw.1,35 Amelogenin expression was first evident
using RT-PCR at embryonic day E15 of murine
embryonic development and alternative splicing
could be detected by day E18.36 Alternative splic-
ing of the primary transcript can be seen as an
important mechanism in generating amelogenin
protein diversity. Each message has all motif ele-
ments required for translation, but the different
amelogenin messages and proteins are present in
developing enamel in different abundances. The
most abundant 180 amino acid amelogenin and the
59 amino acid LRAP seem both to have important
roles in development of dental enamel, yet both
also have the capacity to alter gene expression in
vitro, and in some cases in vivo as well. Expression
of an LRAP mRNA that includes exon 4 was also
reported in the layer adjacent to the ameloblasts,
the stratum intermedium.37
348
B. By Cementoblasts and Other
Periodontal Cells
The cellular structure that anchors the root of
the tooth within the jaw is the periodontium.
This region includes a cementum layer, which
covers the root dentin, and the periodontal
ligament, which is the unmineralized connective
tissue attaching the cementum of the root to
the third structure, the alveolar bone or socket
(Fig. 1). In addition, a transient epithelial layer,
Hertwig’s epithelial root sheath (HERS), covers
root dentin prior to cementum formation. The
HERS begins initially as an extension of the
ameloblast and outer enamel epithelial layers,
and grows downward into the region where the
root will develop after the crown has formed. This
double cell layer fenestrates and forms a cellular
network, and has been the topic of many studies
because its developmental fate is not entirely
clear. Unanswered questions are whether the in-
ner layer of HERS can secrete some enamel or
enamel-like proteins since this is the layer that
extends downward from the ameloblast layer,
and whether the inner HERS layer is able to
undergo epithelial-mesenchymal transforma-
tion leading to a phenotypic switch of HERS
cells to cementoblasts.5,38,39 This is considered an
alternative hypothesis to a dental follicle origin
for cementoblasts (see Introduction).
Through use of immunohistochemistry,
amelogenins have been detected in the murine
periodontium, the apical end of developing hu-
man tooth roots, and in human HERS.40–43 Im-
munolocalization was used to detect amelogenin
on epithelial remnants in the rat periodontium
or in the cervical region of the porcine root.44,45
However, others have used similar methodology
and did not detect amelogenins on pig tooth
roots.38 Using a sensitive RT-PCR procedure,
two amelogenin alternative splice products that
encode M180 and LRAP were identified in the
molar periodontium from six-month-old wild-
type mice, in which ameloblasts are no longer
present.46
The OCCM.30 cell line is an SV40-immor-
talized murine cementoblast cell line, which has
been the topic of many studies, and responds to a
variety of mediators.47 Sonoyama et al.43 detected
amelogenin proteins in human cementoblasts and
HERS cells from adult impacted third molars,
Critical Reviews™ in Eukaryotic Gene Expression
although the source of expression of these pro-
teins was not evaluated. The Somerman group
was unable to detect any amelogenin message
in HERS, follicle cells, or cementoblasts during
tooth root formation using in situ hybridization
of mandibles obtained from CD-1 mice. Fur-
thermore, on culture of the latter cells (follicle
cells and cementoblasts), regardless of whether
or not cells were stimulated with specific growth
factors or unstimulated, amelogenin message was
not detected using RT-PCR (M. Somerman,
personal communication). Amelogenin also could
not be detected in HERS by in situ hybridization
or in epithelial cells isolated from the human
periodontium.48,49
These differences in experimental results
could be due to a combination of the antibodies
employed, species variations, developmental age
of the animal model, and of course abundance of
the message or protein.
C. By Odontoblast and Pulp Cells
A protein extracted from dentin that had chon-
drogenic-inducing activity was identified by
amino acid sequence determination as a small
amelogenin.50 Expression of amelogenins by rat,
pig, or human dentin-producing odontoblast
cells was later described using various methods,
including RT-PCR, through construction and
screening of cDNA libraries, in situ hybridization,
and immunohistochemistry.10,11,13,51
D. Other Sites Where Amelogenin Has
Been Localized
Amelogenin was detected in brain, eye, calvariae,
and hematopoietic cells using RT-PCR or immu-
nohistochemistry.52,53 By immunohistochemistry,
in situ hybridization or Western blot, amelogenin
message or protein was detected in cells within long
bone, cartilage, the epiphyseal growth plate, and in
precursors such as marrow stromal cells.54 RNAs
encoding the M180 amelogenin and LRAP were
also detected in differentiating RW4 embryonic
stem cells.55 Expression in soft tissues could be
interpreted to mean an inhibitory role in mineral
formation, or a new function unrelated to hard
tissue formation.
Volume 18 Number 4
IV. REGULATION OF AMELOGENIN
EXPRESSION
Most work has been done using ameloblasts for
studies of regulation of expression because the
level is very high in these cells. However, early
in development, from embryonic day E15 to
E18, a low level of RNA is detectable by RT-
PCR,56 but protein is not easily detectable at
these early stages. The transgene used to express
various amelogenins in ameloblasts is also active
at embryonic day E15.57 Although these low-
abundance amelogenins may not organize into
nanospheres, they may instead function in cellular
communication.
Another mechanism for regulation has been
reported through uptake of amelogenin protein
into ameloblasts. This internalized protein has been
reported to interact with amelogenin mRNA in
the cytoplasm, leading to message stabilization in
a cultured dental epithelial cell line, HAT-7.58,59
A. Identification of the Requirements
for Expression of the Amelx Gene Using
Transgenic Mice
The initial Amelx transgenic mice were generated
to identify gene regulatory regions, and included
3.5 kb of bovine upstream amelogenin gene region
linked to the reporter β-galactosidase.57 Expression
was mostly limited to the ameloblasts, but some
X-gal stain in the adjacent stratum intermedium
layer was noted. Other mice were generated using
2263 bp of the murine upstream region linked
to luciferase, in which ameloblasts expressed the
reporter gene in the appropriate temporal and
spatial pattern.60
In order to attain the endogenous levels of
expression in transgenic mice, vectors were sub-
sequently generated that included up to 5.5 kb
of the upstream region, plus intron 1 and some
with 3’ genomic DNA, and these mice faithfully
replicated the expression pattern of the endogenous
gene during enamel formation.61–63 The periodontal
region was not described for these mice.
By mating a mouse that expressed Cre under
control of the keratin 14 regulatory region, which
is active in ameloblasts, with a mouse with a floxed
CCAAT/enhancer binding protein α (C/EBPα)
gene, Xu et al.64 were able to show reduced C/EBP
349
mRNA levels as well as a decrease in amelogenin
message. However, Cre deletion of both alleles no
longer led to reduction in amelogenin expression,
and therefore it was assumed that C/EBPδ may
functionally substitute for C/EBPα in regulation of
amelogenin expression in vivo in this situation.
B. Amelx Promoter Analysis—
Identification of Regulatory Transcription
Factors Using Cultured Cells
The C/EBPα was shown to transactivate the mu-
rine Amelx gene in cultured ameloblastlike LS8
cells.65,66 NF-Y and the C/EBPα act synergistically,
Msx2 interaction results in functional antagonism,
and factor YY1 also modulates expression in LS-
8 cells.67–69
C. Inducers and Repressors
A number of in vivo or organ culture models have
provided information on factors that can regulate
amelogenin expression. For example, biglycan and
FGF-2 repress expression while IGF I, IGF II,
and vitamin D3 treatment led to an induction of
amelogenin expression.70–74
D. Regulation of Alternative Splicing
The amelogenin RNA alternative splice pattern
changes during development,36,75,76 and therefore
it was expected that the splicing pattern could be
regulated by the cells. As described below, regula-
tion can also be due to species-specific differences
in gene structure.
In order to test whether exon 4, which is
invariably skipped in the bovine amelogenin mes-
sages, is also skipped in an unrelated cell type,
a green fluorescent protein (GFP) expression
vector was constructed that included in-frame
amelogenin exon 3, intron 3, exon 4, intron 4,
and exon 5, which was in frame with the C-
terminus of the GFP protein. This vector was
transfected into CHO cells, which are from a
different species and tissue origin compared to
bovine ameloblasts. Although the compound
message was highly expressed, inclusion of exon
4 could not be detected by RT-PCR. Extension
350
of the length of the unusually short 63 nt intron
4 by between 6 and 1000 nts increased inclusion
of exon 4, indicating that the physical property
of intron length was responsible for exon 4 skip-
ping in bovine amelogenin transcripts,77,78 and
revealed a species-specific difference. Intron 4
in mouse and human is sufficiently long to allow
incorporation of exon 4 into messages in these
species. A CMV-based minigene vector also
has been constructed to include Amelx exons 2
through 7 to study alternative splicing in various
cell lines.79
E. Summary
These approaches are in vivo and in vitro
methods for identification of transcriptional or
posttranscriptional regulation of the Amelx gene
in ameloblasts or when an amelogenin vector
is expressed in other cell types. Because of the
low level of expression outside of ameloblasts,
little information is available on regulation of
endogenous expression in other tissues where low
levels have been observed. Alternative splicing
is likely to be quite important since both the
M180 and LRAP proteins have been reported
to be expressed in the periodontal region and
in stem cells, and these amelogenins are likely
to have different, perhaps complementary, func-
tions in both locations. Finally, the Amelx gene
is physically localized within intron 1 of the
Arhgap6 gene, in the opposite orientation, and
such “nested” genes frequently have regulatory
relationships in common.80
V. FUNCTIONAL STUDIES OF EFFECTS
OF AMELOGENIN PROTEINS ON
PERIODONTAL CELLS
Because work in the 1980s and 1990s suggested
that some enamel-related proteins are expressed
in the periodontal region during develop-
ment,81–83 it was postulated that a role during
development could be utilized in a regenerative
approach following a disease process.84 An extract
containing enamel proteins from developing
porcine teeth has been studied for effects on a
number of nonameloblast cells using a variety
of strategies.
Critical Reviews™ in Eukaryotic Gene Expression
A. Emdogain and Treatment of
Periodontal Disease
Emdogain (EMD; Biora, Malmo, Sweden) is an
enamel organ extract rich in amelogenins, sus-
pended in a propylene glycol alginate vehicle.83
This product has been evaluated in animal models
and now is used clinically for patients seeking
periodontal regeneration. Hundreds of articles
have been published to evaluate effectiveness in
patients and in clinical trials, a topic too extensive
for this review. In brief, EMD is thought to have
healing properties, and to foster repopulation of
the periodontium with cementoblasts, periodon-
tal ligament fibroblasts, and alveolar osteoblasts,
each in their proper position and with partially
restored function.85–87 Some studies have shown
EMD treatment is associated with decrease in
discomfort following pulpal or periodontal surgery,
perhaps due to the modulation of inflammatory
mediators.88–90 Although EMD contains mostly
amelogenins, this extract made from pig enamel
organs includes much lesser amounts of proteases
and nonamelogenin enamel proteins.91,92
B. In Vitro Studies Using Emdogain
EMD has been widely used to test effects of
enamel proteins on gene expression in cultured
cells. EMD has been shown repeatedly to be able
to alter expression of a number of genes encoding
proteins related to bone mineral formation, as well
as rate of proliferation, formation of mineral foci,
and attachment to the external support. EMD
has recently been used for microarray approaches,
where it was shown to up- and downregulate
many gene families in MG-63 osteoblastlike or
periodontal ligament cells.93,94
These approaches provided insight into the
activities of EMD when used clinically, but
the causal relationships in these experiments
are quite difficult to interpret since EMD
contains amelogenin proteins, some of which
are likely proteolytically processed, plus other
enamel proteins and signaling factors such as
BMP or TGFβ-like proteins,92,95,96 which also
are expected to alter gene expression in the
cultured cells.
Because it has not been possible to determine
which proteins are the active components, some
Volume 18 Number 4
investigators have purified individual amelogenins
to evaluate their functions using cultured cells.
C. Response of Tissues or Cultured Cells
to LRAP
The small amelogenin LRAP was identified in
dentin extracts as a small polypeptide with chon-
drogenic-inducing activity.50,97 In vitro studies have
indicated effects of LRAP on gene expression,
cellular behavior, mineralization, and signaling.
Although LRAP is abundantly expressed by
ameloblasts, and seems to have a role in enamel
mineral formation,34 enamel abnormalities are
not observed in LRAP-overexpressing transgenic
mice.62
LRAP (A-4) and LRAP, which includes exon
4 (A+4; or M73 in Fig. 2) were expressed in vitro
and added to cultured rat muscle fibroblasts or
embedded into a scaffold and inserted into rat
thigh muscle.98 In both cases changes in gene
expression or increased levels of bone matrix
proteins including BSP were observed.
Changes in gene expression were reported
when A-4 or A+4 were added to cultured murine
teeth.99,100 LRAP treatment of human enamel
organ cells induced expression of amelogenin
and LAMP-1 (lysosome associated membrane
protein-1), but downregulated Notch1.101 When
either A-4 or A+4 was implanted into rat dental
pulp, an inflammatory reaction was first described
followed by osteoblast recruitment by A+4, but
cell differentiation by A-4, with increased BSP
expression.102 When implanted into murine oral
mucosa, again an inflammatory response was
initially observed, followed by expression of os-
teogenic and chondrogenic markers, but without
induction of mineralization.103
In immortalized murine cementoblast
OCCM.30 cells, LRAP had no effect on prolif-
eration, downregulated OCN, upregulated OPN,
and inhibited mineral nodule formation, similar to
the effect described above.104 Microarray analysis
of the effects of LRAP treatment of OCCM.30
cementoblasts revealed an increased expression
of inflammatory mediators including the ligands
CXC L and CC L (Fig. 3, M. Somerman and
B. Rutherford, unpublished). These ligands func-
tion to establish a concentration gradient for
migrating cells during inflammation,105 and may
351
FIGURE 3. LRAP-enhanced expression of chemokines in cementoblasts. Immortalized murine cementoblasts
(OCCM.30) were maintained in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum (FBS),
antibiotics and L-glutamine. On reaching confluence, FBS was reduced to 5% and experimental treatments
were added. Cells were exposed to 2 μM recombinant LRAP or vehicle (75 mM HEPES, 150 mM NaCl, 10 mM
reduced glutathione, 5 mM DTT, 2% N-octyl glucoside) (n = 3 per condition). Total RNA was isolated by Trizol at
12 and 24 h after treatment. GE Codelink whole mouse genome microarrays were used for expression analysis
of cementoblast +/− LRAP. Differentially expressed genes at each time point were determined using a modified
t-test followed by a correction for false discovery rate. Lists of significantly affected genes were analyzed by
GenMAPP and MaPPFinder using > +/− twofold selection criteria to assemble gene ontology (GO) groups of
functional interest. LRAP upregulated expression of multiple genes in the two subfamilies of the GO chemokine
activity group, (A) the CXC ligands (CXC L 1, 5, 7)and (B) the CC ligands (CC L 2, 5, 7).

have a role in the pulp implantation experiments
described above.
Coculture of wild-type and Amelx null ce-
mentoblast/periodontal ligament cells and osteo-
clast progenitor cells showed decrease in migration
of null cells and elevation in the number of cells
positive for TRAP, a marker for osteoclast cells.
These results were reversed in the presence of
LRAP.106
Finally, LRAP expression was detected in
RW4 ES cells during osteogenic differentiation.
Exogenously added LRAP also induced bone
sialoprotein, osterix, and mineral nodule forma-
tion in these cells.107
It is concluded that the effects of the LRAP
peptides vary according to cell and tissue type,
the presence or absence of exon 4, and potentially
a specific receptor or other as-yet-undiscovered
pathway within the responding cells. Expression
in the periodontal region, in stem cells, and other
tissues and the observed responses of changes in
gene expression suggest several additional func-
tions outside of the formation of dental enamel,
including a role in inflammation.
D. The Effects of Amelogenin In Vitro
and In Vivo
The 26 kDa (180 amino acid) amelogenin has
been added to cultured cells to ask questions
about gene expression similar to those that were
asked for LRAP. Amelogenin added to OCCM.30
cells (immortalized murine cementoblasts) slightly
increased BSP at low concentrations, but higher
concentrations decreased both BSP and mineral.

352 Critical Reviews™ in Eukaryotic Gene Expression

Expression of BSP in Amelx null mice was reduced
along the root surfaces and in alveolar bone.108 A
mechanism for amelogenin stimulation of BSP
expression was found to reside in the FGF2
response element and the TGFβ-1 activation
element using a rat osteoblast cell line.109
In vitro experiments in which TRAP, which
is the N-terminal cleavage fragment of amelo-
genin (45 amino acids), was added to OCCM.30
cementoblast cells led to an increase in OPN
but a decrease in OCN and in mineral nodule
formation.110
Amelx null mice developed periodontal defects,
with an increase in multinuclear cells including
osteoclasts. Cementicles, which are abnormal calci-
fied bodies, and resorptive lacunae deep into the
cementum or underlying dentin both increased
by six months to one year of age in the null mice
(46). In agreement, it was shown that amelogenin
inhibited osteoclastogenesis dramatically, includ-
ing a decrease in expression of several of the genes
in the resorptive pathway.111
Preosteoblasts plated on an amelogenin/apa-
tite–coated titanium surface expressed an increased
amount of osteocalcin and alkaline phosphatase
compared to cells plated without amelogenin in
the coating layer.112
Periodontal ligament cells derived from
Immortomouse treated with amelogenin led to
increases in proliferation, attachment, and expres-
sion of BMPs, OCN, and BSP.113
The periodontium includes the transient
epithelial structure, HERS. Human HERS cells,
which were shown to express amelogenin, were
removed from impacted third molars, placed
into culture, and were able to form mineralized
matrix. They also promoted cemento/osteogenic
differentiation of periodontal ligament stem cells
in coculture conditions. HERS treated with TGFβ
underwent epithelial-mesenchymal transition with
an increase in vimentin and N-cadherin, and when
transplanted subcutaneously to immunocompro-
mised mice formed a cementumlike structure.43
E. Search for an Amelogenin Receptor
It has been assumed that a cell surface receptor
for amelogenin exists on at least some cell types,
and efforts to identify such a receptor have led
in several directions.
Volume 18 Number 4
Using a yeast two-hybrid system, amelogenin
was shown to bind CD63, a member of the
tetraspanin family of cell surface proteins that
mediate signal transduction within the cell, as well
as LAMP-1 and LAMP-2 (lysosome-associated
membrane proteins114,115). By affinity binding
assays, far Western blotting, and immunohisto-
chemistry, LRAP was found to bind LAMP-1 on
C2C12 mouse fetal myoblast cells.116 Amelogenin
added to cultured MC3T3-E1 preosteoblasts
localized first to LAMP-1–positive vesicles, and
later to the perinuclear region,117 which illustrates
a likely pathway for removal of organic debris,
yet also points to a potential signaling pathway
toward the nucleus.
F. Summary
Approaches designed to better understand the
function of the enamel proteins during enamel
mineral development are continuing at the same
time as novel functions are being uncovered. An
evaluation of the role of “moonlighting” enamel
proteins has taken diverse approaches, from
traditional biochemistry and gene expression ex-
periments to yeast-two-hybrid experiments and
generation of novel animal models. Unfortunately,
it is not possible to cite all of the numerous pa-
pers dealing with these questions in this general
overview and the reader is referred to several
recent reviews.118,119
VI. CONCLUSIONS
Although there is disagreement concerning ex-
pression of the amelogenins in the periodontium
during development, expression would explain the
effectiveness of EMD for periodontal regeneration,
which opened a window for studies of the multi-
functional nature of amelogenin proteins. But in
addition to the question of whether amelogenins
have a developmental role apart from enamel, the
question can be asked whether amelogenins could
have a therapeutic role regardless of expression
pattern.
Emdogain is a mixture and it is not clear
whether the principal activity resides in one
or more amelogenins, or if some other protein
present in trace amounts is responsible. The
353
experiments where cultured cells were incu-
bated with purified amelogenins reveal that gene
expression changes in the presence of several
of the amelogenins, and a pathway has been
identified for amelogenin regulation of BSP.
Injecting amelogenins into various tissue types
clearly has an effect, leading to either migration
of new cells or change in expression pattern in
resident cell types. Receptors identified thus far
have a role in uptake of amelogenins linked to
lysosomal degradation, although it is possible
that a separate pathway could provide access to
the nucleus, which would explain some of the
effects relative to gene expression.
Many questions remain only partially an-
swered. Recent studies that identified amelogenin
protein in stem cells of long bones and mandibles
are intriguing because a role in stem cells could be
consistent with observations of regenerative ability.
A number of dentally important proteins are large
and multifunctional, but the smaller 26 kDa and
less amelogenins seem to generate much of their
multifunctional nature through alternative splicing
to produce many individual peptides. This group
of peptides can lead to the differences observed
in enamel of different species, and extraenamel
expression of various mRNAs will yield proteins
that can interact with cells with receptors, lead-
ing to new functional strategies. This fascinating
group of proteins is likely to continue to provide
surprises as greater understanding evolves and new
concepts materialize in future work.
ACKNOWLEDGMENTS
The author thanks M. Somerman, B. Foster, and
B. Rutherford for many productive discussions
and for sharing unpublished results; S. Akintoye,
M. Pugach, M. Aragon, and Y. Li for critically
reading the article; and S. Labadessa for technical
assistance. Support was provided by Grant No.
DE011089 from the NIDCR, NIH.
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