Japanese Dental Science Review (2014) 50, 47—54

Available online at www.sciencedirect.com

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Review Article

New insights into the functions of enamel

matrices in calcified tissues

a, a a

Satoshi Fukumoto *, Takashi Nakamura , Aya Yamada ,

a a b a

Makiko Arakaki , Kan Saito , Juan Xu , Emiko Fukumoto , b

Yoshihiko Yamada

a

Division of Pediatric Dentistry, Department of Oral Health and Development Sciences, Tohoku University

Graduate School of Dentistry, Sendai 980-8575, Japan

b

Laboratory of Cell and Developmental Biology, NIDCR, National Institutes of Health, Bethesda, MD 20892, USA

Received 5 July 2013; received in revised form 27 December 2013; accepted 8 January 2014

KEYWORDS Summary secrete enamel matrix , including , ameloblastin,

, amelotin, and Apin/odontogenic -associated (Apin/ODAM). Ame-

Enamel matrix;

Amelogenin; logenin is the major protein component of the enamel matrix. Amelogenin, ameloblastin, and

Ameloblastin; enamelin are expressed during the secretory stage of ameloblast, while amelotin and Apin/ODAM

Enamelin; are expressed during the maturation. Amelogenin and ameloblastin are also expressed in

Amelotin; osteoblasts, and they regulate bone formation. In addition, recent studies show the importance

Odontogenic of protein—protein interactions between enamel matrix components for enamel formation. In a

ameloblast-associated mouse model mimicking a mutation of the amelogenin in imperfect (AI) in

protein; humans, the mutated amelogenin forms a complex with ameloblastin, which accumulates in the

Apin endoplasmic reticulum/Golgi apparatus and causes ameloblast dysfunction resulting in AI

phenotypes. Ameloblastin is a cell adhesion molecule that regulates cell proliferation. It inhibits

odontogenic tumor formation and regulates osteoblast differentiation through binding to CD63.

Amelotin interacts with Apin/ODAM, but not ameloblastin, while Apin/ODAM binds to amelo-

blastin. These interactions may be important for enamel mineralization during amelogenesis. The

enamel matrix are clustered on human 4 except for the amelogenin genes

located on the sex . Genes for these enamel matrix proteins evolved from a common

ancestral gene encoding secretory calcium-binding phosphoprotein.

# 2014 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 48

2. Amelogenin (AMEL) ...... 49

3. Ameloblastin (AMBN) ...... 50

* Corresponding author. Tel.: +81 22 717 8380; fax: +81 22 717 8386.

E-mail address: [email protected] (S. Fukumoto).

1882-7616/$ — see front matter # 2014 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdsr.2014.01.001

48 S. Fukumoto et al.

4. Enamelin (ENAM) ...... 51

5. Amelotin (AMTN) and Apin/ODAM ...... 52

6. Evolution of enamel matrix genes ...... 52

7. Conclusion ...... 53

Acknowledgements...... 53

References ...... 53

completely degraded by specific proteases such as MMP-

1. Introduction

20, mainly produced during the secretory/transition stage,

Dental enamel, known as the hardest tissue in vertebrates, is and KLK4, mainly produced during the transition/maturation

formed by ameloblasts derived from the oral epithelium. In stage, resulting in a highly ordered and purposefully designed

the initial stage of the ’s development, the oral meshwork of carbonated hydroxyapatite crystals with aston-

epithelium invades the dental mesenchyme, followed by ishing mechanical properties [1].

differentiation into four types of epithelial cells, including Cell adhesion to the extracellular matrix is of fundamental

the , the , the importance for a wide range of cellular functions, including

, and the . Among cell differentiation, proliferation, and survival [2]. Inner and

these cell types, the inner enamel epithelium differentiates outer enamel epithelial cells interact with the basement

into enamel matrix-secreting ameloblasts. The formation of membrane, of which major constituents are type IV collagen,

dental enamel is a prototype of functional organ develop- laminin, and heparan-sulfate proteoglycan perlecan. For

ment through a matrix mineralization process (Fig. 1). Unlike example, laminin a5 (Lama5)-deficient mice show small

dentin and bone matrices, in which collagen I is the major germs without a cusp [3]. In addition, proliferation and polar-

matrix protein, enamel matrices consist of a distinct set of ization of the dental epithelium are inhibited in these mice,

matrix molecules with amelogenin being the major compo- indicating that interactions between the dental epithelium

nent. The different matrix components in enamel contribute and the basement membrane are important to determine

to its larger and more rigid hydroxyapatite crystal structures tooth sizes and dental epithelial cell differentiation.

than dentin and bone. Enamel matrix proteins are produced Ameloblasts are polarized in the secretory stage and

at their highest levels by ameloblasts during the secretory secrete enamel matrix components, including amelogenin

and transition stages of amelogenesis and collectively (AMEL), ameloblastin (AMBN), and enamelin (ENAM).

orchestrate the proper assembly and growth of crystals Furthermore, amelotin (AMTN) and Apin/odontogenic ame-

within mineralized enamel. These proteins are nearly loblast-associated (ODAM) are secreted from those in the

Figure 1 Oral epithelium differentiates into four types of dental epithelium: inner and outer enamel epithelium, stratum

intermedium, and stellate reticulum. Inner enamel epithelium differentiates into pre-ameloblast and ameloblast. Inner enamel

epithelium and pre-ameloblast secrete and bind to basement membrane. Secretory-stage ameloblasts secrete enamel matrices AMEL,

AMBN, and ENAM. Maturation-stage ameloblasts secrete AMTN and Apin/ODAM.

New insights into the functions of enamel matrices 49

Table 1 Summary of enamel matrix.

Matrix name Expression Human cytogenetic Mouse KO phenotype Reference

(abbreviation) localization

Amelogenin (AMEL) Ameloblast (secretory), Xp22.2 (AMELX), [9]

, osteoblast, Yp11.2 (AMELY) tooth root resorption

cementoblast, epithelial reduced bone formation

rests of Malassez

Ameloblastin (AMBN) Ameloblast (secretory), 4q13.3 Amelogenesis imperfecta [18]

odontoblast, osteoblast, reduced bone formation

cementoblast, epithelial tumor formation

rests of Malassez

Enamelin (ENAM) Ameloblast (secretory) 4q13.3 Amelogenesis imperfecta [33]

(ENU induced)

Amelotin (AMTN) Ameloblast (maturation) 4q13.3

Apin/odontogenic, Ameloblast (maturation) 4q13

ameloblast-associated (ODAM)

maturation stage. These enamel matrix components are ancestor of extant mammals, whereas the 3-prime portion

considered important for enamel crystal formation. In fact, evolved independently within different mammal species.

AMEL and ENAM mutations are identified in patients with The boundary is marked by a transposon insertion in intron

amelogenesis imperfecta (AI), a common group of inherited 2, which is shared by all of the examined species [9].

defects of dental enamel formation that exhibit marked Mutations in the AMEL gene in humans and AMEL deficiency

genetic and clinical heterogeneity, with at least 14 different in mice cause defective enamel [10] and result in the diverse

sub-types recognized on the basis of their clinical appearance group of genetically altered conditions collectively known as AI

and mode of inheritance [4—6]. However, a recent study that in humans. AMEL null mice showed remarkable resorption of

used a gene-targeting method and in vitro analysis showed and root dentin via the receptor activator of

that these matrices also have a large number of biological nuclear factor-kappa B ligand-mediated osteoclastogenesis

functions in addition to enamel formation, including bone [11], suggesting that AMEL is negative regulator of osteoclast

formation, tumorigenesis, and the regulation of stem cell genesis [12]. In fact, in vivo application of AMEL suppresses

function. Mineralized tissue formation and maintenance are root resorption [13]. The enamel layer in AMEL null mice is

controlled by matrix proteins such as several secretory cal- hypoplastic and missing a characteristic prism pattern, while it

cium-binding phospho-proteins (SCPPs), which are encoded has an elemental composition consistent with a hydroxyapa-

by SCPP genes clustered on human [7]. Half of tite-like mineral [10], suggesting that AMEL is necessary for

these proteins, including bone sialoprotein, osteopontin, enamel organization, but not mineral crystal initiation. A

dentin sialophosphoprotein, and dentin matrix protein, are tyr64-to-his missense mutation in the tri-tyrosyl domain of

associated with bone and dentin formation, while the other the enamel extracellular matrix protein in mice (AMELX)

half of consists of enamel-associated proteins, including results in severe defects of enamel biomineralization asso-

AMBN, ENAM, AMTN, and Apin/ODAM (Table 1). They promote ciated with an absence of the full-length AMEL protein in the

mineral nucleation through calcium ion sequestration and enamel matrix; this mutation also increases ameloblast apop-

control crystal growth by adsorption onto growing crystal tosis [14]. In that study, affected ameloblasts express, but fail

surfaces [8]. Here, we present the functions of enamel matrix to secrete full-length AMEL, leading to engorgement of the

molecules in enamel formations and review recently endoplasmic reticulum/Golgi apparatus, and both AMEL and

reported new functions of these molecules in other tissues AMBN accumulated in the affected cells of mice. Furthermore,

and diseases. the co-transfection of AMBN and mutant AMELX in cells reveals

intracellular abnormalities and increases cytotoxicity com-

2. Amelogenin (AMEL) pared with cells singly transfected with wild-type AMELX,

mutant AMELX, or AMBN, or co-transfected with both wild-

Tooth enamel is a highly organized hierarchical nano-compo- type AMELX and AMBN, indicating that intracellular protein—

site material consisting of parallel arrays of elongated apa- protein interactions mediated via the amelogenin tri-tyrosyl

titic crystallites that form an intricate three-dimensional motif may be a key mechanistic factor underpinning the

microstructure. Amelogenin (AMEL) is a highly conserved molecular pathogenesis in this example of AI.

protein secreted by ameloblasts that constitutes 90% of Abundant AMEL proteins are responsible for generating

the enamel matrix. It is also the most abundant protein in proper enamel thickness and structure, and most of those

the organic enamel matrix and has structural characteristics proteins include a conserved hydrophilic C-terminus. Mice

that are indispensable for enamel formation. Genomic that express AMEL lacking the C-terminal show deficient

sequence analysis of the AMEL gene in five primates and enamel in the molar teeth, suggesting that AMEL, especially

three other mammals and FISH analysis on the comparison of the C-terminus, is essential for proper enamel density,

humans with seven other mammals revealed that the 5-prime volume, and organization [15]. AMEL regulates the formation

portion of the AMEL loci began to evolve from the common of these crystalline arrays via cooperative interactions with

50 S. Fukumoto et al.

the forming mineral phase. Cryoelectron microscopy analysis amorphous mineral particles along the fibril axes, triggering

has shown that AMEL undergoes stepwise hierarchical self- the mineralization of the bulk of collagen fibrils, indicating

assembly. Furthermore, interactions between AMEL hydro- that interactions between collagen and AMEL play an impor-

philic C-terminal telopeptides are essential for oligomer tant role in forming the dentin—enamel boundary that pro-

formation and subsequent steps of hierarchical self-assem- vides structural continuity between dentin and enamel [20].

bly. The pre-nucleation clusters subsequently fuse together Further, posttranslational modification like glycosylation,

to form needle-shaped mineral particles, leading to the phosphorylation and sulfation is important for protein func-

formation of bundles of crystallites, the hallmark structural tion. Tyrosyl motif in amelogenin binds N-acetyl-D-glucosa-

organization of enamel forming at a nanoscale size [16]. mine and N-acetyl-D-glucosamine-mimicking peptide motif of

AMEL is soluble at a low pH and self-assembles to form cytokeratin [21]. In enamel formation, amelogenin interacts

higher order structures at physiological pH. In FTIR spectro- with sytokeratin 14 [22] and binds to ameloblastin via ame-

scopy (FTIRS) studies of the pH-triggered assembly of recom- logenin tyrosyl motif [23]. That domain has an affinity for

binant porcine AMEL performed to elucidate the mechanisms binding N-acetyl-D-glucosamine of cleavage products of

of its assembly and interactions with calcium phosphate enamelin, suggesting amelogenin—enamelin interaction

mineral, AMEL at a pH of 3.0 is found to exist in an unfolded [24].

disordered state, while increases in pH lead to structural

ordering, manifested by increases in intra- and intermole-

cular beta-sheet structures and decreases in random coil and 3. Ameloblastin (AMBN)

beta-turns. AMEL assembled at a pH of 7.2 is also found to

contain large portions of extended intramolecular beta-sheet Ameloblastin (AMBN) is a non-AMEL protein that is highly

structures, and interactions with minerals lead to a reduction expressed by ameloblasts in the secretory stage. AMBN-null

in protein structural organization. These findings indicate mice develop severe enamel hypoplasia (Fig. 2) [25]. Further-

that AMEL has an intrinsic structural flexibility to accommo- more, though the dental epithelium in these mice differenti-

date interactions with both forming and mature calcium ates into ameloblasts, the cells become detached from the

phosphate mineral phases and provide new insights into matrix and subsequently lose cell polarity; thus, proliferation

the potential importance of AMEL—mineral interactions in is resumed. In addition, recombinant AMBN binds to amelo-

enamel biomineralization [17]. blasts and inhibits cell proliferation, indicating that AMBN is

Recent studies have also revealed that AMEL has cell an essential cell adhesion molecule for amelogenesis and

signaling properties. Although the AMEL protein has been plays a role in maintaining secretory-stage ameloblasts by

described as a specific product of ameloblasts, recent find- binding to ameloblasts and inhibiting proliferation [25].

ings have shown that AMEL is expressed in bone marrow AMBN expression is regulated by BMP2 and neurotrophic

stromal cells. Researchers have also demonstrated that factor NT-4, while NT-4 and its receptor TrkB regulate the

the full-length AMEL protein interacts with LAMP1, an AMEL differentiation of ameloblasts in tooth development [26].

receptor, and increases the proliferation of mesenchymal Recent studies have shown that NT-4-induced AMBN expres-

stem cells through the MAPK-ERK signaling pathway [18]. sion is regulated by glycosphingolipids. The exogenous glyco-

One of the AMEL splicing isoforms, leucine-rich amelogenin sphingolipids GM3 and LacCer in dental epithelial cells induce

peptide (LRAP), induces osteogenesis in osteoprogenitor AMBN expression. It is also interesting to note that GM3

cells. LPAP activates the canonical Wnt signaling pathway synergistically enhances NT-4-mediated AMBN expression.

to induce the osteogenic differentiation of mouse ES cells Furthermore, depleting the glycosphingolipids from dental

through the concerted regulation of Wnt agonists and antago- epithelial cells using D-PDMP inhibits AMBN expression as well

nists. There is also a reciprocal relationship between osteo- as ERK1/2 phosphorylation. In contrast, adding exogenous

genic and adipogenic differentiation in bone marrow GM3 or LacCer rescues the phosphorylation of ERK1/2

mesenchymal stem cells; Wnt10b-mediated activation of repressed by pre-treatment with D-PDMP, suggesting that

canonical Wnt signaling has also been shown to regulate GM3 and LacCer are essential for the NT-4-mediated induc-

mesenchymal stem cell fate. Using the bone marrow stromal tion of AMBN expression and contribute to dental epithelial

cell line ST2, LRAP activates the canonical Wnt/b-catenin cells’ differentiation into ameloblasts [27].

signaling pathway. LRAP treatment also elevates the Wnt10b Although first discovered in ameloblasts [28], AMBN is also

expression level, whereas Wnt10b knockdown by siRNA abro- expressed by osteoblasts [29], cementoblasts [30], and

gates its effects, indicating that LRAP promotes mesenchy- epithelial rests of Malassez in the periodontal ligament

mal stem cells’ osteogenesis at the expense of adipogenesis [31]. The AMBN protein sequence features a fibronectin

by up-regulating Wnt10b expression to activate Wnt signaling interaction site [32] and heparin-binding domains [33]. In

[19]. rodents, there is a potential a2b1 integrin-binding domain

Collagen I and AMEL are major extracellular organic and a thrombospondin cell adhesion motif in the AMBN

matrix proteins of dentin and enamel, respectively, which protein [34]. The effect of AMBN on cell adhesion involves

represent two mineralized tissues that comprise the tooth RhoA signaling and cell cycle progression through p27 [35].

crown. Both are present at the dentin—enamel boundary, a AMBN is expressed in mouse calvarial bone and adjacent

remarkably robust interface that holds dentin and enamel condensed mesenchyme, and an AMBN transgenic mouse

together. Collagen fibrils guide the assembly of AMEL into an model shows delayed posterior frontal suture fusion and

elongated chain or filament-like structures oriented along incomplete suture closure. Furthermore, AMBN-overexpres-

the long axes of the fibrils. The interactions between col- sing mice show reduced cell proliferation in suture blastemas

lagen fibrils and amelogenin—calcium phosphate mineral and mesenchymal cells from posterior frontal sutures. In

complexes lead to the oriented deposition of elongated these mice, the expression of Msx2 in calvaria and suture

New insights into the functions of enamel matrices 51

Figure 2 X-ray analysis of an AMBN knockout mouse shows severe enamel hypoplasia. Tooth cusps were flat because of attrition. The

entire craniofacial bone size was not different than that of a wild-type mouse. Similar tooth phenotypes were observed in AMEL and

ENAM mutant mice.

mesenchymal cells is decreased. Finally, AMBN overexpres-

4. Enamelin (ENAM)

sion significantly reduces the expression of Msx2 downstream

target molecules, including osteogenic transcription factors Enamelin (ENAM) is a secreted glycoprotein known to be

Runx2 and Osterix. Together, these results suggest that AMBN critical for dental enamel formation. The analysis of a

plays a crucial role in the regulation of cranial bone growth three-generation family with an autosomal-dominant smooth

and suture closure via Msx2 suppression and proliferation hypoplastic form of AI showed a mutation in the splice donor

inhibition [36]. site of intron 7 of the ENAM gene in affected family members

Tooth root formation begins after the completion of crown [39]. Using N-ethyl-N-nitrosourea (ENU), an ENAM mutation

morphogenesis. At the edge of the tooth crown end, inner mouse model [40] showed AI-like phenotypes in the incisors

and outer enamel epithelia form the Hertwig’s epithelial root and molars. Ameloblasts in ENAM null mice develop atypical

sheath (HERS), which extends along with dental follicular features that include the absence of a Tomes’ process, an

tissue for root formation. AMBN is a matrix protein secreted expanded endoplasmic reticulum, an apparent loss of polar-

by ameloblasts and HERS-derived cells. In one study, AMBN ity, and a pooling of extracellular matrix in all directions,

siRNA treatment of 10-day-postnatal molars was found to including between ameloblasts and the stratum interme-

inhibit root formation. Furthermore, HERS in these mice dium. Ameloblast apoptosis has been observed in these mice

revealed a multilayered appearance, and BrdU-positive cells starting in the secretory stage and with no apparent altera-

were increased in the outer layers, indicating that AMBN tion in cell proliferation. In the absence of ENAM, ameloblasts

regulates HERS cell proliferation and functions as a trigger for undergo pathological changes early in the secretory stage,

normal root formation [37]. tooth surface detachment, apoptosis, and ectopic calcifica-

Dental epithelial tumor formation has been observed in tion formation, both outside and inside the dystrophic

AMBN knockout mice [25], while AMBN has also been shown to enamel organ [41]. Furthermore, ENAM null mice have alveo-

be expressed in osteosarcoma cells. AMBN binds to CD63, a lar bone height reduction [42], indicating that ENAM may be

member of the transmembrane-4 glycoprotein superfamily, important for calcification and bone formation.

and promotes CD63 binding to integrin b1. Furthermore, the b-Catenin/LEF1 is a key transcriptional complex involved

0

interaction between CD63 and integrin b1 induces Src kinase in tooth development and bone formation via Wnt. The 5 -

inactivation via the binding of CD63 to Src, indicating that flanking region of the mouse ENAM gene contains two con-

AMBN is expressed in osteoblasts and functions as a promoting served LEF1 responsive elements that may augment its

factor for osteogenic differentiation via a novel pathway transcriptional activity. Further, Wnt/b-Catenin signaling

through an interaction between CD63 and integrin b1 [38]. might function in ENAM expression by direct interactions

52 S. Fukumoto et al.

through these two elements of the ENAM gene in ameloblast- suggests that it is involved in adhesive mechanisms active at

like cells [43]. these sites, while its presence in other epithelial tissues

indicates that it likely possesses broader physiological roles

[49].

5. Amelotin (AMTN) and Apin/ODAM

The expression of Apin/ODAM is increased in the secre-

tory-stage ameloblasts of AMTN transgenic animals, indicat-

Amelotin (AMTN) is an enamel matrix protein originally

ing that AMTN may regulate its expression [46]. Both Apin/

identified by the differential display PCR assays of various

ODAM and AMTN are localized in the basal lamina of matura-

micro-dissected dental organ cell types obtained from incisor

tion-stage ameloblasts. At the beginning of maturation, a

teeth. The predicted translated protein sequence is unique

concentration of Apin/ODAM exists on the cell side of the

and shows a significant homology only with its human ortho-

basal lamina, while AMTN appears to be more concentrated

log. The AMTN gene in mice and humans displays a similar

on the enamel side. In the late maturation stage, such

exon—intron structure and is located in loci on chromosomes

differential distribution is no longer apparent [50]. Using

5 and 4, respectively. AMTN mutations are associated with

two-yeast hybrid screening systems, AMTN interacts with

various forms of AI. AMTN mRNA expression is restricted to

itself and ODAM, but not with AMEL, AMBN, or ENAM. Using

the maturation stage of ameloblasts in developing murine

ODAM as bait, the interaction with AMTN is confirmed. ODAM

molars and incisors. In addition, the AMTN protein is effi-

binds to itself and AMBN, as well as weakly to AMEL, but not to

ciently secreted from cultured transfected cells, indicating

ENAM. The distinct expressions of AMTN and ODAM and their

that AMTN is an extracellular matrix molecule produced by

interaction are involved in defining the enamel microstruc-

ameloblasts [44]. In addition, the AMTN gene is conserved in

ture on the enamel’s surface [51].

mammals, while it is absent in fish, birds, and amphibians

[45].

AMTN expression is detected in a transient fashion during 6. Evolution of enamel matrix genes

ameloblast differentiation in molars. The expression level

declines at the time of . Prominent AMTN Most ECM proteins contain domain structures such as EGF-

expression in the maturation stage of ameloblasts in con- like, fibronectin type III (FN3), laminin G (LamG), Ig-like,

tinuously erupting incisors persists into adulthood, whereas thrombospondin (TSPN), von Willebrand factor A (VWA), and

AMEL, AMBN, and ENAM are predominantly found during the collagenous domains. Variations in their arrangements give

early secretory stage. Furthermore, odontogenic ameloblast- rise to the characteristic features of ECM proteins. Approxi-

associated amyloid in Pindborg tumors and kallikrein 4 mately 300 genes are identified that are involved in coding

expression in ameloblasts’ maturation stage were found to ECM proteins in mammals by searching domains character-

parallel that of AMTN. Secreted AMTN is also detected at the istic to ECM molecules through the whole genome sequence

interface between ameloblasts and mineralized enamel. [52]. The total number of ECM molecules corresponds to

AMTN transgenic mice under the control of the amelogenin about 1.5% of proteasomes, a list of all proteins expressed

gene promoter show a histological appearance similar to that by the genome. ECM proteins typically consist of repeated

of ameloblasts or supporting cellular structures, while domains, which are coded in the genome as separate exonic

expressions of the enamel proteins AMEL and AMBN are not units and evolve through multiple processes such as exon

altered [46]. These results suggest that AMTN plays a primary duplication and shuffling, and gene duplication and conver-

role in the late stages of enamel mineralization [47]. sion [53,54]. Analyses of the genomes in different organisms

Apin/ODAM is identified in calcified epithelial odontogenic suggest that some ECM molecules in mammals retain ancient

tumor (CEOT) specimens. Its subcellular localization varies forms, which are found in all metazoans, while some are

during ameloblast differentiation, though it is stage-specific. derived from newer proteins.

Apin/ODAM mRNA is not expressed in pre-ameloblasts and Mineralization is one of the hallmark processes that has

only weakly expressed in secretory ameloblasts, whereas taken place during vertebrate evolution. Three major miner-

expression is strong in maturation-stage ameloblasts as well alized tissues, enamel, dentin, and bone, have evolved over

as the junctional epithelium attached to the enamel of 500 million years likely from a common ancestral process

erupted molars. In maturation-stage ameloblasts, Apin/ [55]. The genes for enamel matrix proteins expressed by

ODAM protein is conspicuous in the supranuclear area (Golgi ameloblasts, AMEL, ENAM, AMBN, AMTN, and Apin/ODAM,

complex) of smooth-ended ameloblasts as well as in both the have evolved from a common ancestral gene encoding an

supranuclear area and the ruffle end of ruffle-ended amelo- SCPP [56,57]. Enamel matrix genes are clustered on human

blasts. Furthermore, its overexpression and inactivation chromosome 4q13. However, AMEL is located on sex chromo-

cause an increase in matrix metalloproteinase-20 (MMP-20) somes. AMELX and AMELY are located on the X and Y chromo-

expression and a decrease in expression, indicating somes, respectively. About 90% of the AMEL transcripts are

that Apin/ODAM plays a functional role in enamel minerali- expressed on the X locus. SCPP family genes are also mapped

zation and maturation that is mediated by the expression of on human chromosome 4. Analysis of the enamel matrix

MMP-20 and tuftelin [48]. genes in different species has shown that ENEM is the most

The Apin/ODAM gene is highly conserved in mammals, ancient gene and the other four enamel genes are derived

while it is absent in fish, birds, and amphibians, as seen with from ENEM. AMEL is derived from AMBN and was later trans-

AMTN [49]. The Apin/ODAM protein is modified in a post- located to sex chromosomes [57]. The chromosomal synteny

translational manner, which is consistent with the presence of these genes is conserved in primates and rodents. In birds,

of predicted sites for phosphorylation and O-linked glycosy- which lost their teeth about 100 million years ago, the

lation. The presence of Apin/ODAM at cell-tooth interfaces enamel matrix gene cluster is lacking [58].

New insights into the functions of enamel matrices 53

[13] Yagi Y, Suda N, Yamakoshi Y, Baba O, Moriyama K. In vivo

7. Conclusion

application of amelogenin suppresses root resorption. J Dent

Res 2009;88(2):176—81.

Initially, enamel matrices were identified as enamel-specific

[14] Barron MJ, Brookes SJ, Kirkham J, Shore RC, Hunt C, Mironov A,

molecules. However, the expression of enamel matrix pro-

et al. A mutation in the mouse Amelx tri-tyrosyl domain results

teins has been shown in other tissues outside of the tooth in impaired secretion of amelogenin and phenocopies human X-

germ as a result of improved sensitivity in detection analyses. linked amelogenesis imperfecta. Hum Mol Genet 2010;19(7):

Furthermore, the overexpression or knockdown of these 1230—47.

[15] Pugach MK, Li Y, Suggs C, Wright JT, Aragon MA, Yuan ZA, et al.

matrices not only causes enamel defects, but also aberrant

The amelogenin C-terminus is required for enamel develop-

tooth roots, bone formation, and tumor phenotypes. The

ment. J Dent Res 2010;89(2):165—9.

enamel matrix proteins are evolutionally related, but have

[16] Fang PA, Conway JF, Margolis HC, Simmer JP, Beniash E. Hier-

distinct functions. The newly identified activities of enamel

archical self-assembly of amelogenin and the regulation of

matrix proteins will provide a better understanding of the

biomineralization at the nanoscale. Proc Natl Acad Sci USA

mechanisms of craniofacial development, including enamel, 2011;108(34):14097—102.

dentin, and craniofacial bone formation.

[17] Beniash E, Simmer JP, Margolis HC. Structural changes in ame-

logenin upon self-assembly and mineral interactions. J Dent Res

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[18] Huang YC, Tanimoto K, Tanne Y, Kamiya T, Kunimatsu R, Michida

M, et al. Effects of human full-length amelogenin on the pro-

This work was supported by grants-in-aid (20679006 to S.F.,

liferation of human mesenchymal stem cells derived from bone

21792054 to A.Y., 21792154 to E.F.) from the Ministry of marrow. Cell Tissue Res 2010;342(2):205—12.

Education, Science, and Culture of Japan, the NEXT program [19] Wen X, Cawthorn WP, MacDougald OA, Stupp SI, Snead ML, Zhou

(LS010 to S.F.), and by grants from the Intramural Program of Y. The influence of leucine-rich amelogenin peptide on MSC fate

by inducing Wnt10b expression. Biomaterials 2011;32(27):

National Institute of Dental and Craniofacial Research,

6478—86.

National Institutes of Health to Y. Y.

[20] Deshpande AS, Fang PA, Simmer JP, Margolis HC, Beniash E.

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