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Hindawi Publishing CorporationISRN DentistryVolume 2013 Article ID 684607 24 pageshttpdxdoiorg1011552013684607

Review ArticleDental Enamel Development Proteinases and Their EnamelMatrix Substrates

John D Bartlett

Harvard School of Dental Medicine amp Chair Department of Mineralized Tissue Biology The Forsyth Institute245 First Street Cambridge MA 02142 USA

Correspondence should be addressed to John D Bartlett jbartlettforsythorg

Received 27 June 2013 Accepted 15 July 2013

Academic Editors A Jager and J H Jeng

Copyright copy 2013 John D Bartlett This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogeninameloblastin and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secretedinto the enamel matrix After an overview of enamel development this review focuses on these enamel proteins by describingtheir nomenclature tissue expression functions proteinase activation and proteinase substrate specificity These proteins andtheir respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromicenamelmalformations termed amelogenesis imperfecta Pertinent controversies are addressed For example do any of these proteinshave a critical function in addition to their role in enamel development Does amelogenin initiate crystallite growth does itinhibit crystallite growth in width and thickness or does it do neither Detailed examination of the null mouse literature providesunmistakable clues andor answers to these questions and this data is thoroughly analyzed Striking conclusions from this analysisreveal that widely held paradigms of enamel formation are inadequate The final section of this review weaves the recent data intoa plausible new mechanism by which these enamel matrix proteins support and promote enamel development

1 Introduction

Tooth development is a highly orchestrated process thatbegins with the defined placement of individual teeth ofspecific shapes and sizes within the jaw Precise signalingpathways to and from epithelial and mesenchymal cells arerequired for each tooth to initiate and continue along itsdevelopmental path [1 2] The complexity of these pathwaysis reflected by their high rate of incompletion Deficiencyof third molars second premolars and lateral incisors iscommon The reported incidence of selective agenesis variesfrom 16 to 96 for all but third molars Agenesis ofthird molars occurs in approximately 20 of the Worldrsquospopulation [3]Therefore the study of tooth development hastaught us how genes and tissues interact to form complexdental structures that each occupies a prespecified placewithin the jaw and has taught us about what can go wrongwith the intricate developmental signaling pathways

Teeth are composed of three differentmineralized tissuescementum dentin and enamel Cementum is found along

the tooth root and primarily serves to hold the tooth in placeby binding collagen fibers (Sharpeyrsquos fibers) that are contin-uous with the principal fibers of the periodontal ligamentThese fibers are orientatedmore or less perpendicularly to thecementum surface and play a major role in tooth anchorage[4] Dentin is a bone-like matrix that forms the bulk of thetooth It is characterized by closely packed dentinal tubulesand is slightly harder than bone but softer than enamelDentin has an elastic quality that provides flexibility thatprevents fracture of the overlying brittle enamel Dentin andenamel are firmly bound at the dentin-enamel junction (DEJ)[4] The enamel layer covers the crown of the tooth and isunique because it is the only epithelial derived calcified tissuein vertebrates and it is the hardest substance in the bodyIts hardness is between that of iron and carbon steel buthas a higher elasticity [5] Enamel hardness is a function ofits high mineral content Unlike bone and dentin (20ndash30organic material by weight) fully formed enamel containsvery little protein (less than 1 organic material) [6 7]Therefore within the body teeth are the most resistant to

2 ISRN Dentistry

deterioration and have given us important anthropologicalclues as to how humans evolved Although DNA analysishas taught us much about the migration patterns of ourancient ancestors teeth have perhaps been more importantin identifying our ancestorrsquos food preferences and lifestylesTheir shape wear patterns and carbon isotope compositionsare unique indicators of our past behaviors [8]

2 Overview of Enamel Development

This review focuses on enamel proteinases their substratesand on recent discoveries that help us to better understandthe process of enamel development For outstanding reviewarticles about enamel development prior to 1999 pleasesee the following reviews [9ndash11] The review by Simmerand Fincham [9] provides an excellent historical perspectiveabout enamel development the review by Smith [11] providesa detailed examination on how ions are controlled duringenamel formation and the review byBartlett and Simmer [10]focuses on what was known about the enamel proteins andproteinases prior to 1999

Enamel development (amelogenesis) can be broken downinto four defined stages presecretory secretory transitionand maturation The stages are defined by the morphologyand function of the ameloblasts (Figure 1) The ameloblastsare a single cell layer that covers the developing enamel andis responsible for enamel composition Ameloblasts are partof the enamel organ that is composed of an outer epitheliallayer the stellate reticulum the stratum intermedium andthe inner enamel epithelium (ameloblast layer) The basal(proximal) end of the preameloblast is attached by desmo-somes to the stratum intermedium and the apical (distal) endis attached by hemidesmosomes to a basement membrane(basal lamina) located at the future site of the DEJ

21 Organization of the Enamel Organ The outer enamelepithelium is a single layer of cells that covers the enamelorgan and is contiguous at the cervical loop with theameloblasts (inner enamel epithelium) located initially atthe future DEJ The cells of the stellate reticulum are sand-wiched between the outer dental epithelium and stratumintermedium and secrete hydrophilic glycosaminoglycansinto the extracellular compartment This causes water todiffuse into the enamel organ which in turn forces these cellsapart Since these cells are all interconnected by desmosomesthey are stretched into a star shape and are therefore termedthe stellate reticulum [4] The stratum intermedium formsa boundary between the stellate reticulum and the innerenamel epithelium and may be important for shuttling ionsto and from the ameloblasts [19]The ameloblasts are respon-sible for secreting enamel matrix proteins and proteinasesinducing mineral ribbons to form and organizing them intorod and interrod patterns typical for each vertebrate species

22 Presecretory Stage One of the earliest events occurringduring the presecretory stage just prior tomineral formationis the deposition of predentin by odontoblasts at the futureDEJ [20] This occurs first at the cusp tips and progresses

to the cervical regions of a tooth Predentin is composedprimarily of collagen but also contains noncollagenous pro-teins Predentin is the first to mineralize [21] starting slightlyunder what will become the DEJThe dentin becomes thickeras the wave of mineralization moves latterly and away fromthe DEJ towards the future pulp chamber This decreases thesize of the chamber as mineralization proceeds Howeveralmost immediately after initial dentin mineralization nearthe DEJ differentiating preameloblasts extend cytoplasmicprojections through the basement membrane that removeand destroy it and then the ameloblasts begin secretingenamel matrix proteins which rapidly initiate mineralization[4]

23 Secretory Stage Thepreameloblasts transform into secre-tory stage ameloblasts by elongating into tall columnar cellsand by forming Tomesrsquo processes at their apical ends nearestthe forming enamelThe Tomesrsquo process is a conical structurethat points toward the forming enamelmatrix Enamelmatrixproteins are primarily secreted from one side of the Tomesrsquoprocess (secretory face) and all ameloblasts within a rowsecrete protein from the same side of their Tomesrsquo processesThe first enamel crystals (ribbons) formed grow betweenthe dentin crystals perhaps by mineralizing around dentinproteins such as collagen These crystal ribbons elongate atthe mineralization front where enamel proteins are secreted[22] Secretory stage enamel is protein rich and has a softcheese-like consistency

The ameloblasts start secreting large amounts of enamelmatrix proteins as they move away from the dentin surfaceso that the nascent enamel layer can thicken In associationwith newly secreted proteins long thin mineral ribbons formrapidly normal to the secretory surface of the ameloblastsThe parallel crystallite ribbons approximately 10000 to40000 [23] will eventually form into a rod (prism) and eachameloblast is responsible for creating one enamel prism allof which collectively form a highly ordered 3D structureSoon after the initial formation of crystallite ribbons theameloblasts develop their apical Tomesrsquo processesThis estab-lishes a two-compartmental system where proteins destinedto form interrod enamel tend to exit near the ldquobaserdquo of theprocess whereas those involved in rod formation tend toexit from the ldquotiprdquo (secretory face) of the process Duringthis time protein cleavage products are either reabsorbedby the ameloblasts or may accumulate between the rod andinterrod enamel Mineral crystallites forming within the rodwill growprogressively in 119888-axis length parallel to one anotheras ameloblasts move away from the dentin surface Mineralcrystallites developing between the rods (interrod) may havemore limited lengths but they are always positioned spatiallyto be at angles relative to rod crystallites [4]

During the secretory stage ameloblasts not only moveaway from the dentin as the enamel thickens but they alsomove in groups that slide by one another and this movementculminates in the characteristic decussating enamel prismpattern observed in rodent incisors [24] or the entwinedgnarled prism pattern seen in human molars [25] As thisoccurs ameloblasts secrete four different proteins into the

ISRN Dentistry 3

Basementmembrane

12

3

4 5 6 7

Enamel

Dentin

Figure 1 Ameloblast changes during enamel formationThe epithelial cells of the inner enamel epithelium (1) rest on a basement membranecontaining laminin These cells increase in length and become differentiating ameloblasts above the predentin matrix (2) Presecretoryameloblasts send processes through the degenerating basement membrane as they initiate the secretion of enamel proteins on the villoussurface ofmineralizing dentin (3) After establishing the dentin-enamel junction andmineralizing a thin layer of aprismatic enamel secretoryameloblasts develop a secretory specialization or Tomesrsquo process Along the secretory face of the Tomesrsquo process in place of the absentbasement membrane secretory ameloblasts secrete proteins at a mineralization front where the enamel crystals grow in length (4) Eachenamel rod follows a retreating Tomesrsquo process from a single ameloblast At the end of the secretory stage ameloblasts lose their Tomesrsquoprocess and produce a thin layer of aprismatic enamel (5) At this point the enamel has achieved its final thickness During the transitionstage the ameloblasts undergo a major restructuring that diminishes their secretory activity and changes the types of proteins secreted (6)KLK4 is secreted which degrades the accumulated protein matrix During the maturation stage ameloblasts modulate between ruffled andsmooth-ended phases (7) Their activities harden the enamel layer The histology of the developing tooth was adapted from Uchida et alArch Histol Cytol 54527-538 1991 and the schematic plus tooth was published in Hu et al Cells Tissues Organs 18678-85 2007 DOI101159000102683

enamel matrix Three are presumed structural proteins andone is a proteinase The structural proteins are amelogenin(AMELX) ameloblastin (AMBN) and enamelin (ENAM)and the proteinase is matrix metalloproteinase-20 (MMP20enamelysin) Amelogenin comprises approximately 80ndash90of the organic matter within the secretory stage enamelmatrix and ameloblastin and enamelin comprise roughly5 and 3ndash5 respectively [13] MMP20 is present in traceamounts The precise function of these proteins remainsunclear However human mutations in AMELX ENAM andMMP20 genes and mouse knock-out models have providedstriking clues that have dramatically changed the way weenvision amelogenesis and this has occurred within a spanof less than two decades For example as will be describedin detail below enamel crystals will form in the absenceof amelogenin but will not form if either ameloblastin orenamelin is absent By the end of the secretory stage theenamel layer has achieved its full thickness It is not untilthe end of the maturation stage when the proteins arealmost completely removed that the enamel achieves its finalhardened form

24 Transition and Maturation Stages The beginning ofthe transition stage is dependent on the species and onthe specific developing tooth However prior to when theenamel layer reaches its full thickness the ameloblasts nolonger move relative to each other They retract their Tomesrsquoprocesses smooth off the enamel surface with a final coatingof aprismatic enamel transition (transition stage) into shorterand fatter maturation stage cells and reapply a new basallamina and the ameloblasts start modulating between ruffleand smooth-ended cells at the enamel surface [11] It is

during the maturation stage that ameloblasts actively secretekallikrein-related peptidase-4 (KLK4) to help remove themass of previously secreted and partially hydrolyzed matrixproteins from the enamel layer so that the rod and interrodcrystallites can expand in volume to occupy as much space aspossible within the enamel layer

Enamelmineral is very similar to hydroxyapatite [Ca5OH

(PO4)3] but also contains low percentages of carbonate

sodium and magnesiumThe initial enamel ribbons are onlya few apatitic unit cells in thickness (about 10 nm) with awidth of approximately 30 nm [26 27] and a length that mayextend through the entire thickness of the enamel layer Afterthe enamel rods have formed an area exists in some speciesbetween the rod and interrod enamel that contains a thinorganic matrix with no crystals [28ndash30] This structure isoften called the rod sheath or sheath space and is prominentin humans [31]

25 Summary Enamel development can be broken downinto four defined stages During the presecretory stage theameloblasts poke through and remove the basal laminaand start secreting enamel matrix proteins at the formingDEJ Soon after as the ameloblasts enter the secretorystage they elongate develop Tomesrsquo processes and secretelarge amounts of proteins into the enamel matrix whichare necessary for the enamel crystallite ribbons to formand lengthen Once the enamel reaches full thickness theameloblasts transition into shorter protein reabsorbing cellsthat define the maturation stage of development and at theend of this stage the enamel will achieve its final hardenedform These general features of amelogenesis are remarkablyconstant in different species [32]

4 ISRN Dentistry

3 Enamel Proteinases

Approximately 50 years ago it was demonstrated that devel-oping enamel had a high protein content whereas matureenamel did not [33 34] It was discovered that the enamelmatrix proteins were removed during the maturation stageof amelogenesis [35] Studies on developing bovine enameldemonstrated that the percentage of protein by weightdropped from 30 during the secretory stage to 2 duringthe early maturation stage [36] In the rat incisor a similardecline was associated with a significant change in the aminoacid composition of total enamelmatrix proteins [37]Thus arole for proteinases in the degradation and export of enamelproteins was advanced Approximately 25 years ago severalinvestigations suggested that as the forming enamel passesthrough the secretory stage and into the maturation stageof development the enamel proteinases undergo a changein profile This change was first identified by Overall andLimeback [38] who used enzyme inhibitors to demonstratethat metalloproteinase(s) were present during early enameldevelopment and that serine proteinase(s) were presentduring the later stages Although there was some overlapthis change in enzyme profile was confirmed by several otherinvestigators [39ndash43]Therefore prior to identification of anyspecific protease within developing dental enamel evidencesuggested that at least two classes of enzymes were presentA proteinase of the metalloproteinase class was present earlyduring the secretory stage and a proteinase of the serineclass was present late during the maturation stage of enameldevelopment

The proteinase expressed during the secretory throughearly maturation stage is MMP20 [44] and the proteinaseexpressed from the transition through maturation stages isKLK4 [45] To date these are the only two proteinases knownto be secreted into the enamel matrix Both proteinases arepresent in trace amounts during enamel development andeach proteinase was separately cloned by performing PCR-based homology cloning [46 47] KLK4was originally namedenamel matrix serine proteinase-1 (EMSP1) but its name waschanged to kallikrein-4-related peptidase-4 because the geneencoding KLK4 locates in the kallikrein gene cluster

4 Matrix Metalloproteinase-20Enamelysin

41 MMP20 Nomenclature MMP20 was originally clonedfrom a porcine enamel organ cDNA library [46] Althoughit has since been shown to be expressed in odontoblasts ofthe pulp organ [48] originally its expression was thoughtto be confined to the enamel organ This novel MMP wastherefore named ldquoenamelysinrdquo At the 1997 Gordon ResearchConference on Matrix Metalloproteinases a group headedby Dr J Fredrick Woessner designated this novel MMP asldquoMMP20rdquo and this designation was first published with thecloning of the humanMMP20mRNA [49]

42MMP20 Localization MMP20was cloned by PCR-basedhomology cloning from pig enamel organ and supportingnorthern blots demonstrated its tissue restricted pattern ofexpressionTherefore it remained uncertain as to which cells

of the enamel organ expressedMMP20 andwhetherMMP20was secreted into the enamel matrix These issues wereresolved by a report that used immunogold labeling analysisto identify MMP20 location and used a unique zymographytechnique that started with a gel overloaded with porcineenamel matrix proteins The zymogram was incubated for ashort time to demonstrate proteolysis of the copolymerizedsubstrate and was then subjected to western blotting todemonstrate that those zones of proteolysis were attributed toMMP20 [48] Another paper showed in situ hybridization todemonstrate that both ameloblasts and odontoblasts expressMmp20 transcripts [44] Thus MMP20 became the firstproteinase to be definitively identified as expressed by theameloblasts of the enamel organ and was identified by nameas the first proteinase secreted into the developing enamelmatrix

43 MMP20 Tissue Expression MMP20 has a highlyrestricted pattern of expression Very few tissues or cell linesexpress MMP20 Reverse-transcription-PCR was used todetect various MMP expression in 51 different cell linesHowever none of the cell lines tested positive for MMP20[50] In contrast MMP20 was expressed in a few pathologictissues such as in ghost cells of calcifying odontogenic cysts[51] odontogenic tumors [52] human tongue carcinomacells [53] and in bradykinin treated granulosa cells isolatedfrom the follicles of porcine ovaries [54] No recent reportshave observed MMP20 expression in any tested cell line(reviewed in [55])

Our laboratory assessed Mmp20 expression byquantitative-real time PCR (qPCR) of mRNA isolatedfrom various mouse tissues We found that except fordeveloping teeth Mmp20 was only expressed at very lowlevels in the large intestine MMP20 expression in theintestine was too low to be detectable by northern blottingand was approximately 5000 times lower than the levelsobserved in 4-day-old tooth budsMmp20 was not expressedin small intestine brain heart kidney liver lung pancreasspleen or stomach [56] Incomprehensively in 2009 onegroup published a report stating that an SNP withinMMP20was associated with kidney aging Two years later the samegroup published a review article reiterating their findingAn MMP20 association with kidney aging has not beenconfirmed by any other group and no other papers havebeen published on this subject The finding was problematicbecause MMP20 is not expressed in the kidney An NCBIsearch for ldquoMMP20rdquo in the UniGene search engine revealsthat not a single MMP20 EST has been recovered from thekidney Of even greater consequence it was discovered thatin Baleen whales Mmp20 is a pseudogene Baleen whaleslack teeth and since nonfunctional Mmp20 genes are foundonly in mammals lacking enamel the authors postulated thatthe only nonoverlapping essential function of MMP20 is indental enamel formation [57] Therefore to date MMP20 isconsidered a tooth-specific MMP

44 MMP20 Activation How MMP20 becomes activatedremains an enigma Two MMP20 bands of approximately 46

ISRN Dentistry 5

and 41 kDA are observed on immunoblots and zymograms[48] Normally one would consider the upper band to bepro-MMP20 and the lower band to be active MMP20 withits propeptide cleaved However The identity of the 46and 41 kDa forms of purified porcine MMP20 was assessedby performing immunoblots zymography reverse-phaseHPLC and protein sequencing after exposure to oxidizingand reducing conditions [58] The oxidizing and reducingconditions were performed to leave intact (oxidizing) orrelease (reducing) the disulfide bond that connects the firstand last amino acids of the C-terminal hemopexin domainWhen the disulfide bond was left intact both the 46 and41 kDa forms of MMP20 were observed When the disulfidebond was released the 41 kDa band was replaced by acatalytically active 27 kDa band Edman sequencing of thethree bands showed they all contained the catalytic domainat their N-termini (YRLFPGEPK) proving that none of thebands corresponded to the MMP20 zymogen In additionunder reducing conditions that release the disulfide bonda 17 kDa protein band stained positive for MMP20 on theimmunoblot and its N-terminal sequence started with Ile336of the hemopexin domain In aggregate these observationsdemonstrate that one of the 46 or 41 kDaMMP20 bands is theactive intact protease while the other band is active proteasethat has been cleaved in the hemopexin domain after Thr335[58] This C-terminal peptide is covalently attached by thedisulfide bridge and when that bridge is released a portionof the cleaved hemopexin domain falls away to generate the27 kDa catalytic domain and the 17 kDa hemopexin domainThe MMP20 zymogen is not often observed within thesecretory stage enamel matrix presumably due to its efficientactivation in vivo [58] andor because the demineralizationprocedures necessary to extractMMP20 from thematrixmayactivate the zymogen

TheMMP20 propeptide does not contain an RXXR furinconsensus sequence that allows activation in the trans-Golginetwork However recombinant MMP20 autoactivates [49]and appears to readily remove its hemopexin-like domain toform a catalytically active species of approximately 22ndash27 kDa[59ndash61] Therefore MMP20 was postulated to autoactivatein vivo Additionally membrane-type MMP1 (MT1-MMPMMP14) has a transmembrane domain and binds to cellmembranes with its catalytic domain located extracellularlyMMP14 was identified on the cell surface of ameloblasts andodontoblasts of the developing tooth [62] and MMP14 doesactivate the MMP20 zymogen [63] So this is also a possiblemeans of activation in vivo To date no strong evidence existson howMMP20 becomes activatedwithin the enamelmatrix

45 MMP20 Substrate Specificity Soon after its discoveryMMP20 was shown to cleave the most abundant enamelmatrix protein amelogenin [49] Since that time MMP20substrate specificity was characterized by use of an iterativemixture-based random dodecamer peptide library screenwith Edman sequencing of MMP20 cleavage productsMMP20 was found to have a broad substrate specificity witha deep and wide catalytic pocket that can accommodatesubstrates with large aromatic residues in the P11015840 position

As is typical for MMPs MMP20 is highly selective forhydrophobic residues at the P11015840 position and its preferredamino acid at this position is leucine with methionine andtyrosine also being strongly selected MMP20 was relativelynonselective at the P21015840 position and had a slight preferencefor smaller residues at the P31015840 position which had beenpreviously observed in a few other MMPs Alanine andproline are preferred amino acids at the P3 position Thisstudy suggested that MMP20 expression may be restrictedto tooth tissues because of its broad substrate specificitythat might otherwise cause tissue destruction if expressedelsewhere [56] This conclusion is reinforced by the fact thatthe MMP20 zymogen is rarely observed in vivo

MMP20 has been well characterized for its ability tocleave themost abundant enamelmatrix protein amelogenin[48 49 60 61 64ndash66] Two different studies identifiedthe exact MMP20 cleavage sites in amelogenin The firstused recombinant MMP20 and amelogenin [61] and for thesecond study the authors used their considerable proteinpurification expertise to purify native amelogenin and nativeMMP20 from developing pig teeth and also used quenchedfluorescent peptides to confirm their cleavage site results[66] The precise cleavage sites were identified by variousmeans including mass spectrometry and protein sequencingThese cleavage sites were then compared to previously iden-tified amelogenin cleavage products isolated from extractedporcine enamel All of the MMP20 amelogenin cleavage sitesgenerated in vitro were also identified from amelogeninsextracted from normal porcine enamel Therefore sinceno other amelogenin cleavage products were identified insecretory stage porcine enamel other than those generatedby MMP20 it was concluded that MMP20 is likely the onlyproteinase present in the enamel matrix during the secretorystage of enamel development [61 66]

The same group assessed the other structural enamelmatrix proteins (ameloblastin enamelin) to determine if theywere MMP20 substrates Similar exacting protocols used toidentify amelogenin cleavage products were also used toidentify ameloblastin and enamelin cleavage products Aswasobserved for amelogenin all of the ameloblastin MMP20cleavage sites identified in vitro were also identified fromameloblastin extracted from porcine enamel in vivo [67 68]It is difficult to identify enamelin cleavage sites because enam-elin is so quickly cleaved within the enamel matrix Enamelinhas a 186 kDa apparent molecular weight and is highly glyco-sylated However only a 32 kDa enamelin cleavage productaccumulates within the maturing subsurface enamel layer[69] MMP20 does not cleave the glycosylated 32 kDa enam-elin [70] Therefore based in part on the amelogenin andameloblastin MMP20 cleavage results it was concluded thatMMP20 is likely responsible for generating the 32 kDa enam-elin cleavage product in vivo MMP20 will also cleave theKLK4propeptide to produce catalytically activeKLK4 [71] InadditionMMP20 is expressed in the odontoblasts of the pulporgan as is MMP2 and both MMP20 and MMP2 were eachdemonstrated to cleave dentin sialophosphoprotein whichis the major noncollagen secretory product of odontoblastsresponsible for dentin formation [72] Therefore the firstevidence suggesting that MMP20 plays an critical role in

6 ISRN Dentistry

enamel development was with the first report demonstratingthat all amelogenin cleavage products observed in vivo areexpected MMP20 cleavage products [61]

In addition to enamel and dentin proteins MMP20 wasalso demonstrated to cleave E-cadherin [73] casein andorgelatin [48 53 59] aggrecan and cartilage oligomeric matrixprotein [74] type V collagen [56] type XVIII collagen [75]fibronectin type IV collagen tenascin-C and laminin-1 and -5 but not type I or type II collagen [53]These reports confirmthe broad substrate specificity of MMP20 and lend credenceto the theory that MMP20 has a highly restricted patternof expression because its expression elsewhere could causetissue damage (reviewed in [55])

46 The Mmp20 Null Mouse The MMP20 preproenzyme iscomposed of 483 amino acids while the proenzyme has 461residues and the active form has 376 amino acids [64] ThemouseMmp20 gene consists of 10 exons (all coding) spanningapproximately 65 kb within the MMP gene cluster at the cen-tromeric end of chromosome 9 [76] TheMmp20 null mousewas engineered by deleting the majority of exon 4 and exon5 [77] Exon 5 encodes the highly conserved zinc binding site(HEXGHXXGXXH) present in the catalytic domain ofMMPfamilymembersThis deletion renderedMMP20 catalyticallyinactive The Mmp20 null mouse was demonstrated to notprocess amelogenin properly had an altered enamel proteinand enamel rod pattern had hypoplastic (thin) enamel(Figure 2) had enamel that broke off from the dentin andhad a deteriorating enamel and enamel organ morphologyas enamel development progressed [77] A subsequent studyshowed that the weight percent ofMmp20 null mousematureenamel was 7ndash16 less than that of wild-type controls andthat the overall enamel mineral was reduced by 50 andenamel hardness was decreased by 37 Remarkably thebiggest difference in mineral content between the Mmp20null and controls occurred in the nearly mature enamelwhen Mmp20 was normally no longer expressed [78] Thissuggested that MMP20 acts directly or indirectly to facilitatethe removal of maturation stage enamel matrix proteins

Recent reports examining the Mmp20 ablated mice havesuggested that MMP20 does something else besides cleavingenamel matrix proteins For example Tomesrsquo processes arenormally formed after ameloblasts have formed an initialthin layer of mineralized aprismatic enamel at the DEJTheseTomesrsquo processes are later retracted permanently just prior towhen the ameloblasts produce the final thin layer of miner-alized aprismatic enamel at the outer enamel surface This iswhen ameloblasts start their transition into the maturationstage HoweverMmp20 null ameloblasts abnormally extendretract and later reextend their Tomesrsquo processes duringenamel development [73] This suggests that the signalingmechanism responsible for developmental progression tothe maturation stage is deficient in the null mice So howcould MMP20 play a role in ameloblast cell signaling Itwas proposed that MMP20 does this by cleaving the extra-cellular domains of cadherins that are part of the adherensjunction (AJ) complex responsible for ameloblast cell-celladhesion [19 73] Cadherins are transmembrane proteins

where the extracellular domains connect through homotypictranspairing between cadherins on adjacent cells and theintracellular domains are linked to the actin cytoskeletonby catenins (reviewed in [79]) Ameloblasts express E-N- and P-cadherins 120573-catenin and p120-catenin duringdental enamel development [80ndash86] A major pathway forsignal transduction by AJs involves regulation of 120573-cateninand p120-catenin which can act as either structural pro-teins at cell-cell junctions or as transcription factors inthe cell nucleus (reviewed in [87]) When MMPs cleave acadherinrsquos extracellular domain 120573-catenin and p120-cateninare removed from their position near the cell membraneand under certain circumstances will translocate to thecell nucleus thereby promoting cell migration cell invasionandor cell proliferation [88ndash92] E-cadherin is among thecadherins expressed by ameloblasts [19] and MMP20 wasshown to cleave the E-cadherin extracellular domain [73]Therefore a possible way that MMP20 could play a role inameloblast cell signaling is by cleaving cadherin extracellulardomains on ameloblasts that in turn release 120573-catenin andp120-catenin from their disassembled intracellular domainsThese released catenins would then be transported to theameloblast nucleus where they would participate in cell sig-naling It was definitively demonstrated that 120573-catenin p120-catenin and cadherins are essential for tooth and enameldevelopment [80 93] and thatMMP20 cleaves the E-cadherinextracellular domain in vitro [73] However it remains to bedetermined if MMP20 actually cleaves cadherins in vivo toinitiate ameloblast cell movement andor a signaling cascade

The enamel from Mmp20 ablated mice has strikingfeatures Normally a thin highly mineralized initial layerof enamel begins forming during the secretory stage at thedentin-enamel junction However this does not occur inMmp20 null mice and this may be a primary reason why theenamel from these mice sheers off the dentin In additionthe fully developed null mouse enamel appears histologicallyas two distinct layers and the enamel surface is marred bycalcified nodules that vary greatly in their dimensions [1494 95] Neither layer resembles wild-type enamel nor doeseither layer have the characteristic rodinterrod organizationThe inner layer closest to the dentin appears homogenous isnot well mineralized and does not vary greatly in thicknessHowever the outer layer closest to the ameloblasts showslarge variations in thickness and large nodules can beobserved protruding from this layer It is not known whythe Mmp20 null mouse enamel forms in this manner Itwas proposed that during the secretory stage a 25ndash30120583mdefective mineral layer is deposited on top of the dental-enamel junction that contains abundant uncleaved enamelproteins and that during the maturation stage ions thatnormally contribute to the maturation of the crystallites can-not penetrate the inner enamel layer and instead precipitateas a second layer on top of the first [95] The reasons forwhy the calcified nodules form are equally perplexing TheMmp20 null mouse ameloblasts do cover the nodules butit remains uncertain as to whether the ameloblasts ldquoball uprdquoprior to nodule formation or if they acquire their dysplasticshape because the nodules form first KLK4 is expressedand is active in Mmp20 ablated mice However unabsorbed

ISRN Dentistry 7

(a) (b)

(c) (d)

Figure 2 Enamel rod patterns of mandibular incisors from wild-type and Mmp20 null mice The wild-type enamel had crisscrossing(decussating) rows of enamel rods (a) The Mmp20 null enamel may have a poorly organized rod pattern (b) no rod pattern with a poorlyorganized rod layer beneath (c) or virtually no rod pattern whatsoever (d) 1 and 2 designate the two different enamel layers All magnificationbars are 10120583m in length This figure was originally published in Bartlett et al Eur J Oral Sci 119 (Suppl 1) 199-205 2011 D01101111j1600-0722201100864x

protein can be observed in the maturation stage at theameloblast-enamel interface It was therefore proposed thatthese proteins may promote ectopic nodule calcification [14]This proposal supports the notion that the nodules form firstand disrupt the normally smooth ameloblast layer Takentogether theMmp20 ablatedmice have taught usmuch aboutthe function of MMP20 and enamel formation but we stillhave much more to learn

47 Human MMP20 Mutations Human MMP20 isexpressed from a gene on chromosome 11q22-q23 thathas 10 exons (all coding) The MMP20 protein has 483amino acids and its domain structure includes a signalpeptide necessary for MMP20 secretion a propeptide thatmaintains enzyme latency a catalytic domain with a zincbinding site and a hinge domain that links the catalyticdomain to the C-terminal hemopexin-like domain [49]Its only posttranslational modification is a disulfide bridgeconnecting the first and last amino acids of the hemopexindomain [58] Although MMP20 is not glycosylated as aresome other MMPs MMP20 does share the characteristicdomain structure found in most other MMP familymembers

Inherited enamel defects that occur in the absence of ageneralized syndrome are collectively designated as amelo-genesis imperfecta (AI) AI can be inherited by autosomaldominant (ADAI) autosomal recessive (ARAI) and X-linked modes of transmission Classification of AI can be

divided into fourteen distinct subtypes based on clinicalphenotype and mode of inheritance [96] However this canbe narrowed down to threemain typesThese are hypoplastichypomaturation and hypocalcified AI Hypoplastic enamelis thin and is associated with defective matrix synthesis thatoccurs as the enamel increases in thickness Hypomaturationenamel is soft and typically stained but is of normal thicknessand is associated with a failure to remove enamel matrixproteinsHypocalcified enamel is themost severe and appearsto represent a more fundamental disturbance that affectsboth early and late stage enamel development Hypocalcifiedenamel is typically soft rough and is rapidly lost by attrition(reviewed in [97])

Seven different human MMP20 mutations are known tocause autosomal recessive hypomaturation or hypoplastic-hypomaturation amelogenesis imperfecta Five of these muta-tions cause pigmented hypomaturation AI [98ndash101] and tworesulted in hypoplastic-hypomaturation AI [102 103] In allseven cases the teeth are normal in size but the enamel layerdoes not contrast well with dentin on radiographs and theenamel tends to chip away from the underlying dentinOne ofthe hypoplastic-hypomaturation phenotypes has enamel withsurface roughness and a yellowish-brown pigmentation thatis present during tooth eruption suggesting that the stainingis intrinsic and not acquired [102] Six of the seven MMP20mutations known to causeAI are homozygousmutations andone is a compound heterozygous mutationThe homozygousMMP20 mutations include in order of publication date

8 ISRN Dentistry

mutations in the intron 6 spice acceptor (IVS6-2A-T) thatlikely causes themRNA to be degraded by nonsensemediateddecay a missense mutation in the conserved active siteresidue (pHis226Gln) of the catalytic domain that eliminatesenzyme activity a premature stop codon in the propeptide(pTrp34X) a mutation in a highly conserved residue presentin the hemopexin domain (pAla304Thr) that likely causesmisfolding with resulting endoplasmic reticulum-associateddegradation a missense mutation in the conserved activesite residue (pHis204Arg) of the catalytic domain thatcoordinates the structural zinc ion and a missense mutationin an invariant residue (pThr130Ile) present in the catalyticdomainThe compound heterozygousmutation has one allelewith the just described pThr130Ile mutation and the otherallele has a nucleotide deletion leading to a premature stopcodon (pAsn120fzlowast2) Other than defective tooth enamelno other phenotype is observed in these patients Thereforegenetic mutations in both mice and humans and the lack offunctional MMP20 in mammalian species without enamel(baleen whales) demonstrate that MMP20 is essential forenamel formation but is not essential for any other biologicalfunction

5 Kallikrein-Related Peptidase-4

51 KLK4Nomenclature KLK4was originally named enamelmatrix serine proteinase-1 (EMSP1) [47] Later it was foundin normal and neoplastic prostate epithelial tissues and wasnamed ldquoprostaserdquo [104] Another group named it kallikrein-like proteinase-1 (KLK-L1) [105] Finally the Human GeneNomenclature Committee (London UK) adopted the tissueneutral term ldquoserine proteinase 17rdquo (PRSS17) However thisdesignation was later deemed unsatisfactory and the officialdesignation was changed to kallikrein-4 (KLK4) It was sonamed because KLK4 is the fourth member of a cluster of15 serine protease genes that comprise the human kallikreinlocus near the telomere on the long arm of chromosome19 However even this name required further refinement bythe Nomenclature Committee Now the KLK4 designationrefers to ldquokallikrein-related peptidase 4rdquo Unfortunately aPubMed search using the search term ldquoKLK4rdquo will notretrieve the original publication describing the first cloning ofKLK4EMSP1 [47] or other papers using early designations

52 KLK4 Localization In 1977 a protease was purified frompig enamel [106] that was later demonstrated to be inhibitedby the serine proteinase inhibitors phenylmethylsulfonyl flu-oride (PMSF) and diisopropylfluorophosphate (DIFP) [107]This protease was expressed during the early maturationstage when the enamel proteins are reabsorbed from thehardening enamel [38] Like MMP20 KLK4 was clonedby PCR-based homology cloning from porcine cDNA withsubsequent screening of a porcine cDNA library This wasaccomplished by one team of investigators However unlikeMMP20 it was already known that KLK4 was secreted intothe enamel matrix because another team of investigatorshad been purifying KLK4 protein from 2000 uneruptedpig incisors for protein sequencing and eventual cloning

At first neither team knew of each otherrsquos KLK4 researchHowever the epiphany occurred at a Gordon ResearchConference when the principle investigator (Bartlett) fromone team displayed a KLK4 poster that was directly adjacentto the KLK4 poster from one of the principle investigators(Simmer) from the other team After careful considerationof the avenues to pursue we decided to collaborate [47] andour collaborative research has continued ever since ThusKLK4EMSP1 became the second proteinase identified byname that is secreted into the developing enamel matrix

53 KLK4 Tissue Expression KLK4 is a glycosylatedchymotrypsin-like serine protease that is expressed andsecreted by transition to maturation stage ameloblasts[45 108 109] KLK4 protein has not been isolated from anytissue other than from developing teeth [66 71] Howeverseveral studies have performed immunoassays or qPCRtechniques to identify KLK4 in various tissues and manyof these studies conflict with one another as to exactlywhere KLK4 is expressed (reviewed in [110]) All prior KLKexpression studies in nondental tissues (excluding cancers)were performed in adult mice Clarity from these convoluteddata was made possible by the development of a gene-targeted mouse strain that has a LacZ reporter gene with amouse nuclear localization signal (NLS-120573gal) inserted at thenatural Klk4 translation initiation site which can be used toassay Klk4 expression using 120573-galactosidase histochemistry[15] So the Klk4 knock-outLacZ knock-in mice were usedto identify the tissues expressing Klk4 The tissues testedwere teeth adult prostrate liver kidneys submandibularsalivary glands ovaries testes vas deferens and epididymisThe results demonstrated that the expression of KLK4 bymaturation stage ameloblasts was far stronger than thatof any soft tissue tested In the adult organs surveyed thestriated ducts of the submandibular salivary gland and smallpatches of prostate epithelia were the only sites that showedunambiguous KLK4 expression Furthermore no obviousmorphological abnormalities were observed in any of thenondental tissues examined suggesting that their normaldevelopment is not Klk4 dependent [110] As is true forMMP20 it appears that the only essential nonoverlappingfunction of KLK4 is in enamel development

54 KLK4Activation It remains uncertain howKLK4 is acti-vated in vivo Active KLK4 has a predicted molecular weightof 24 kDa but this value does not take into account posttrans-lational modifications Mouse and pig KLK4 each have threeAsn residues in the appropriate context for glycosylationwhile human KLK4 has just one [111] The KLK4 zymogenhas not been observed in the enamel matrix (J P Simmerpersonal communication) So as for MMP20 it is likely thatmostly active KLK4 resides in the maturing enamel Removalof the KLK4 propeptide is essential for activation because itallows a salt linkage to form between the new N-terminusand the side chain of Asp194 and this is essential for enzymeactivity [112] Unlike the other kallikrein-related peptidasesKLK4 has a Gln as the last residue of its propeptide and notan Arg or Lys which means that KLK4 cannot be activated

ISRN Dentistry 9

by trypsin-like enzymes [113] KLK4 cannot activate itself butcan be activated by MMP20 and thermolysin in vitro [71]However KLK4 is active in Mmp20 ablated mice [114] soMMP20 cannot be the sole KLK4 activator Although it hasnot been directly demonstrated perhaps the best candidatefor the activation of KLK4 in vivo is dipeptidyl peptidase I(Cathepsin C CTSC) CTSC activates KLK4 in vitro and isalmost ubiquitously expressed In the enamel organ CTSCis expressed at progressively increasing levels as developmentprogresses to the earlymaturation stagewhenKLK4begins itsexpression Furthermore this same study demonstrated thatenamel from CTSC null mice was significantly softer thanenamel from wild-type controls [115] Therefore it remainsa possibility that this cysteine aminopeptidase is the primaryenzyme that activates KLK4

55 KLK4 Substrate Specificity The first report demon-strating that KLK4 cleaves amelogenin used native porcineKLK4 incubated with recombinant pig amelogenin andthis resulted in the generation of twelve cleavage prod-ucts which were characterized by N-terminal sequencing[71] It was subsequently demonstrated that the primaryMMP20 N-terminal cleavage product tyrosine-rich amel-ogenin polypeptide (TRAP) was further cleaved by KLK4which was consistent with the notion that KLK4 cleavesenamel matrix proteins into small peptides to facilitatetheir export out of the enamel as the enamel hardens [66]Porcine ameloblastin was stably expressed and secreted fromHEK293-N cells and was purified for digestion by KLK4Thecleavage productswere characterized byN-terminal sequenc-ing and KLK4 was shown to cleave ameloblastin at ninedifferent sites [68] The 32 kDa enamelin is presumed to beanMMP20 cleavage product and it is the only domain of theparent protein that accumulates in the deeper more matureenamel layer Native porcine KLK4 was incubated withnative porcine 32 kDa enamelin and the digestion productswere fractionated by reverse-phase high-performance liquidchromatography (RP-HPLC) and characterized by Edmansequencing amino acid analysis and mass spectrometryKLK4 digestion of the 32-kDa enamelin generated ninemajor cleavage products [70] Therefore KLK4 cleaves allthe structural enamel matrix proteins that are known to besecreted into the enamelmatrix and recent evidence suggeststhat KLK4 may also hydrolyze MMP20 This is because inKlk4 ablated mice MMP20 is active well into the maturationstage when MMP20 activity has normally ceased [114]

KLK4 was assessed for its substrate specificity by usingrecombinant KLK4 to screen tetrapeptide positional scan-ning synthetic combinatorial libraries (PS-SCL) The identi-fied preferred P1-P4 positions were P1-Arg P2-GlnLeuValP3-GlnSerVal and P4-IleVal Based on these results adatabase search for substrates was performed and it wasdemonstrated that KLK4 activates pro-KLK3 and cleavesmembers of the insulin-like growth factor binding proteinfamily (IGFBP-3 -4 -5 and -6) [116] KLK4 also acti-vates pro-prostate specific antigen and degrades prostaticacid phosphatase [117] It will activate meprin 120573 [118] andurokinase-type plasminogen activator (uPA) [117] and also

cleaves its receptor (uPAR) [119] Recombinant human KLK4mediates limited cleavage of types I and IV collagen effi-ciently degrades the 120572-chain of fibrinogen [120] and was alsoshown to activate all pro-KLKs except for itself and KLK-7-8 and -10 [121] Additionally KLK4 was proposed to havea signaling function via protease activated receptors (PARs)of the family of G protein coupled receptors particularlyPAR1and PAR

2[122ndash125] However because during normal

development (cancer excluded) the only apparent essentialnonoverlapping function of KLK4 is in enamel formationthe substrate specificity of KLK4 is of little consequenceunless those substrates are present during the transitionto maturation stage of enamel development when KLK4 isnormally expressed

56 The Klk4 Knock-outLacZ Knock-in Mouse The KLK4preproenzyme is composed of 254 amino acids while theproenzyme has 230 residues and the active form has 224amino acids [47]TheKLK4 genes of both mouse and humanhave 6 exons the first of which is noncodingThemouse Klk4gene is approximately 10 kb in size and locates in cytogenicregion B2 on mouse chromosome 7 [109] Gene targetingwas used to generate a mouse strain carrying a null allele ofKlk4 that has a nuclear LacZ reporter gene inserted directlyinto the Klk4 translation initiation site Therefore the LacZcode was positioned in the same genomic context as wild-type Klk4 and so provided a sensitive tissue reporter fornative Klk4 expression [15] Other than a tooth phenotypethe Klk4 ablated mice were normal The teeth were normalthe enamel attained normal thickness and no abnormalitieswere observed until the enamel reached the transition toearly maturation stage of development At this point thenormal export of enamel matrix proteins from the matrixback to the ameloblasts destined for lysosomal degradationwas impeded The enamel retained proteins that should havebeen removed and the soft protein-rich enamel abraded fromthe mouse teeth (Figures 3(a)ndash3(c)) This strongly supportsthe supposition that KLK4 functions to cleave enamel matrixproteins to facilitate their export out of the hardening enamel[15] However the Klk4 ablated mice did reveal unexpectedsurprisesThe rod enamel sometimes pulled away from inter-rod enamel as if the rod enamel were pegs on a cribbage boardand the interrod enamel was defining the peg holes (Figures3(d) and 3(e)) During the secretory stage the ribbon-likeenamel crystallites are surrounded by protein but the approx-imate 10000 to 40000 crystallites that will interlock to forman enamel rod [126] are themselves surrounded by a tube-likeprotein layer (Felicitas Bidlack personal communication)So if this protein layer was not substantially removed it canbe envisioned that the rod and interrod enamel would notproperly interlock with one another which would allow theldquopegs to fall out of the holesrdquo Another surprise was that theindividual crystallites themselves were prone to fall out of therods This was described as much like a circular bunch ofldquouncooked angel hair spaghettirdquo from which the individualstrands fell Although the normal rod pattern was presentin the Klk4 ablated enamel the 10000 to 40000 crystallitesthat the rod is composed of failed to interlock properly and

10 ISRN Dentistry

(d)

(c)

(a)

(b)

(e)

Figure 3 Scanning electronmicroscopy of themandibularmolars (a and b) andmandibular incisor (cndashe) of aKlk4 null mouse at 7 weeksTheenamel of all molars showed a significant loss of enamel from all working surfaces (buccal cusps occlusal surface andmarginal ridges) (a andb) Similarly the enamel layer was abraded at the working (buccal) surface of the mandibular incisor at its tip (c) Higher magnification of thechipped area near the tip of the incisor showed that the break was in the enamel layer close to but not at the DEJThe broken surface appearsto be composed of interrod (ir) enamel with holes where enamel rods (r) had pulled out and separated (d) from the initial deposit of interrodenamel near the DEJ The holes are too numerous to be made by odontoblastic processes penetrating the enamel (enamel spindles) Theorientation of the crystallites on the walls of the holes is parallel to the direction of the tubular holes and to the crystallites between the holes(e) This figure was originally published in Simmer et al J Biol Chem 284 (28)19110-19121 2009 The American Society for Biochemistryand Molecular Biology DOI 101074jbc M109013623

the crystallites fell from the rods [15] Strikingly the fact thatthe crystallites grew until they were expected to interlockwith one another flies in the face of conventional theoriesof enamel formation Conventional theories postulate thatamelogenins inhibit growth inwidth and thickness of crystal-lites and that this growth will not occur until amelogenins areremovedduring thematuration stage of enamel developmentIn the Klk4 ablated mice the amelogenins were not properlyremoved from the maturation stage enamel Despite this thecrystallite ribbons grew into ldquospaghetti strandsrdquo thick enoughto define an enamel rod and were almost ready to interlockwith adjacent ldquospaghetti strandsrdquo Therefore amelogeninsdid not inhibit crystallite growth in width and thicknessby selectively binding to specific sides of the crystallitesand consequently our ldquoconventional theoryrdquo requires seriousreexamination

More recent examination of the Klk4 null mouse demon-strates that the outer enamel layer (most recently formed) is

muchharder than the inner enamel layers and that the enamelshows progressively less mineralization with depth [94 95]The reasons for this are unclear Recall that odontoblastsdo not express KLK4 so odontoblasts do not contribute toenamel matrix removal in the deep enamel layers during thematuration stage of development Also recall that MMP20activity is observed in maturation stage enamel from Klk4ablated mice It was postulated that the continued MMP20activity and endocytosis by ameloblasts combine to removeproteins from the surface enamel but that KLK4 may benecessary to break up aggregates of accumulated enamelprotein cleavage products in the deeper regions of the enamellayer so that they can return to the ameloblast for endocytosis[95] All-in-all the Klk4 knock-inknock-out mouse hasrevealed surprises about enamel formation and has forced usto reexamine some of our more firmly held beliefs about howcrystallites grow in width and thickness to form an enamelrod

ISRN Dentistry 11

57 Human KLK4 Mutations The human KLK4 gene islocated near the telomere of chromosome 19 (19q133-19q134)in a cluster of genes including the KLK family of ser-ine proteases Its gene exonintron structure and proteindomain structure are identical to those of the mouse [127]A difference between the human KLK4 and KLK4 frommouse and pig is that human KLK4 has only one potentialglycosylation site (Asn139) while the pig and mouse eachhave three potential glycosylation sites (pig Asn104 Asn139and Asn184 mouse Asn93 Asn139 and 184) The reason forKLK4 glycosylation is incompletely known but glycosyla-tions can affect protein conformation stability and solubilitycan protect against proteolysis and can affect protein-proteinand protein-mineral interactions Native human KLK4 hasnever been isolated but it was demonstrated that both pigand mouse KLK4 are variably glycosylated Commerciallyavailable recombinant human KLK4 is not glycosylated andwas shown to lose activity rapidly when compared to nativepig and mouse KLK4 However native pig and mouse KLK4did lose activity when they were deglycosylated So it wasdeemed likely that glycosylation is important for KLK4 stabil-ity presumably by protecting it from proteolytic degradation[128]

Two different humanKLK4mutations are known to causeautosomal recessive hypomaturation AI The first discoveredis a nonsense mutation occurring upstream of the KLK4catalytic domain (pTrp153X) This tryptophan residue iscompletely conserved inmouse and pig KLK4 and expressionof this mutated gene would result in a truncated proteinlacking the final 101 amino acids which includes the catalytictriad (His71 Asp116 and Ser207)This homozygous mutationoccurred in two female siblings and both their primary andpermanent dentitions were similarly affected The siblingrsquosteeth were yellow-brown in color and were excessively sen-sitive to hot and cold The enamel was normal in thicknessbut radiographically showed only a slight increase in opacityover that of the underling dentin indicating a decreasedenamel mineral content This soft enamel fractured fromthe occlusal surfaces of the primary molars [129] No otherphenotype resulted from this nonsense mutation in KLK4The second human KLK4 mutation was recently discoveredby use of whole exome sequencing which identified a singlenucleotide deletion (pGly82Alafslowast87) in both alleles of anine-year-old female The frameshift was in the third of fivecoding exons so the mutant KLK4 transcripts may havebeen degraded by nonsense-mediated decay If translatedthe mutant protein would lack the same catalytic triad thatwas also lacking in the first discovered KLK4 mutation Asfor the previously discovered KLK4 mutation the enamelcovering this probandrsquos teeth appeared normal in size shapebut was discolored yellow-brown and chipped on multipleteeth This proband was also secondarily affected with dentalcaries [103] No other phenotype was observed due to thenucleotide deletion in KLK4 Therefore both humans andmice have shown us that KLK4 is essential for enamel toachieve its final hardened form and that just as for MMP20the only nonoverlapping function of KLK4 is in dentalenamel development

6 Other Enamel Matrix Proteinases

Human and mouse mutations in both MMP20 and KLK4demonstrate that no other proteinase has an extensive over-lapping function with either of these proteinases If this wasthe case no severe enamel phenotype would likely occur ifthe activity of MMP20 or KLK4 was compromised Howeverin the past prior to our knowledge of MMP20 and KLK4mutations we and others have proposed that various MMPsare present within the enamel matrix [62 130ndash132] Althoughsome of the data are compelling it is nonetheless difficultto reconcile that hydrolysis by MMP20 accounts for allthe isolated amelogenin and ameloblastin cleavage productsextracted from normal secretory stage porcine enamel [6166] If another active MMP was in the enamel matrix wewould expect that amelogenin would be cleaved as has beendemonstrated in vitro for at leastMMP2 [132] Also no enamelphenotype was demonstrated when these other MMPs wereablated from mice Recently MMP9 was proposed to beinvolved in controlling amelogenin processing and enamelformation [133] These authors had Mmp9 knockout micebut failed to show an enamel phenotype for these miceChymotrypsin C (caldecrin CTRC) was also recently shownas expressed in the enamel organ and was upregulated duringthe maturation stage of enamel development The authorssuggested that cathepsin C (CTSC) activates KLK4 whichin turn activates CTRC [134] However although loss ofCTRC function is a risk factor for pancreatitis an associatedenamel phenotype has not been described Surprisingly inmice lacking MMP20 a 40 kDa proteinase was observed byzymography of enamel extracts that were purified by reverse-phase high-performance liquid chromatography Its presencewas suggested to be a response by ameloblasts to the faultyarray of inputs they receive from the defective extracellularmatrix [114]

In all likelihood most if not all of these proteinases areexpressed in the enamel organ of forming teeth The enamelorgan is a dynamic structure that moves back to accom-modate appositional mineral growth and it also flattens asenamel matures This moving and changing morphologicalstructure would almost certainly require the enlistmentof several active proteolytic enzymes The difficult part isdetermining if these proteinases are secreted into the enamelmatrix and are important for enamel formation If anyproteinase other thanMMP20 and KLK4 function within theenamel matrix it is likely they have overlapping functionswith at least one other proteinase that precludes an enamelphenotype with loss of function

7 Enamel Matrix Proteins

71 Location and Function The three major ldquostructuralrdquoproteins in the enamel matrix of developing teeth are amel-ogenin enamelin and ameloblastin [135] These proteinsare derived from an ancestral gene belonging to the secre-tory calcium-binding phosphoprotein (SCCP) family [136]Enamelin and ameloblastin map to a small area on theq arm of human chromosome 4 (4q13) [137] The humanamelogenin gene is on the X and Y chromosomes but it

12 ISRN Dentistry

was proposed that the amelogenin gene translocated to thesex chromosomeswhile enamelin and ameloblastin remainedon their original chromosome [138ndash140] Mutations in theX-chromosome amelogenin gene (AMELX) and mutationsin the enamelin gene (ENAM) cause nonsyndromic enamelmalformation (AI) Although disease causingmutations havenot yet been observed in the human ameloblastin gene(AMBN) homozygous deletion of exons 5 and 6 in themouseAmbn gene results in almost no enamel formation [17 141]

Two important points are worth highlighting about theseenamel matrix proteins First they are necessary for properenamel formation However they are later reabsorbed by theameloblasts that originally secreted them into the matrixSo these proteins are necessary for enamel formation butthey are not part of the final mature product Only traceamounts of protein remain in mature enamel Second genesencoding these enamelmatrix proteins have degenerated intopseudogenes in multiple toothless or enamelless species thatdescended from ancestors with teeth covered by enamelBoth birds and toothless baleen whales have degeneratedamelogenin ameloblastin and enamelin genes [57 142 143]and a functional enamelin gene is absent in four differentorders of placenta mammals that are toothless andor enam-elless [144] This suggests that amelogenin ameloblastin andenamelin are only essential for dental enamel developmentand that there is no selective advantage in having thesegenes functionally maintained in species without enamelSeveral groups have postulated that in addition to their rolein enamel formation specific enamel matrix proteins areimportant signaling molecules If this was true one wouldexpect that these genes would remain functional in toothlessand enamelless mammals due to the selection pressure ofkeeping the signaling pathways active However this is notthe case

72 Amelogenin Amelogenin is the most abundant enamelmatrix protein and it is essential for enamel formation [145ndash147] Humans and pigs have two amelogenin genes each onthe X-and Y-chromosomes while mice have just one on theirX-chromosome [148] The amelogenin amino acid sequenceat its N- and C-termini is highly conserved amongmammalsand the C-terminal region (13ndash15 residues) is highly charged(pI 42) whereas the entire protein has a pI of 80 [135] Inhuman males approximately 90 of amelogenin mRNA isexpressed from the X-chromosome [149] Mice with Amelxablated from their genome have defective enamel that ishypoplastic and disorganized and that when fractured lacks adiscernable enamel rod pattern (Figure 4) [16] Amelogeninhas only one posttranslational modification whereby Ser16is phosphorylated [150 151] but its transcripts undergoextensive alternative splicing [149 152 153] to generate at least16 X-chromosomal murine amelogenin mRNAs [154ndash156]The function of amelogenin alternative splicing is unclearHowever a recent study used transgenes to express the twomost abundant Amelx transcripts in the amelogenin nullmouse These transcripts separately encoded the mouse 180amino acid amelogenin protein (M180) and the mouse 59amino acid protein (M59) termed leucine-rich amelogenin

protein (LRAP) The amelogenin null mouse has enamelthat is 10ndash20 of the usual thickness of wild-type enamelThe M180 but not the M59 transgene increased the enamelthickness and improved the rod pattern However whenboth transgenes were expressed in the same mouse animprovement in enamel thickness and rod structure occurredover that of the M180 transgene alone This enamel was a bitmore than half the thickness of the wild-type molar enameland was only about one third the thickness of wild-typeincisor enamel [157] Therefore although the reasons for thealternatively spliced amelogenin transcripts remain unclearthey do appear to contribute to the formation of fully thickenamel with a proper decussating enamel rod pattern

Interestingly numerous in vitro studies have addressedthe question of how amelogenin promotes enamel formationSince amelogenin is the most abundant enamel matrixprotein these studies made sense when little was knownabout the other matrix proteins that are much less abundantAmelogenin is also relatively easy to express and purifyand unlike ameloblastin which is glycosylated and enamelinwhich is highly glycosylated amelogenin has only one post-translational modification consisting of a single phosphateTherefore amelogenin can be purified in bacterial expressionsystems or due to its abundance purified from immaturepig teeth However neither of these purification schemesis feasible for the mass purification of ameloblastin andenamelin necessary for the large posttranslationally modifiedquantities required to perform in vitro crystal growth exper-iments Currently we know from mouse knock-outknock-in studies that amelogenin by itself cannot initiate crystalgrowth or promote enamel formation When the enamelin[18] or ameloblastin [17] gene is ablated from mice thesemice have no true enamel and no crystal structure Strikinglythe amelogenin null mice have measurable crystals with awell defined organization The crystals are much smallerand less well organized than in wild-type mice but they arenonetheless present [158] It is unfortunate that ameloblastinand enamelin are so difficult to isolate and purify because wenow know from knock-outknock-in mice just how essentialthey are for enamel development

To date eighteen AMELX mutations including two com-plete gene deletions [159] were shown to cause human X-linked AI X-linked AI accounts for about 5 of all AI cases[160] The enamel phenotype varies with the location of themutation but a unique aspect of certain AMELX mutationsis that affected women may have vertically ridged teethwith alternating bands of normal and hypoplastic enamelThis is presumably due to ameloblasts that have randomlyinactivated either the normal or defective X-chromosomesduring development [161] Affected males have a more severephenotype No AI cases have been reported from mutationsin AMELY and two individuals with AMELY deletions hadnormal teeth [162] Although AMELX and AMELY eachcontain seven exons these genes have diverged and do notundergo homologous recombination [163] This is why theyare used in forensics for sex determination Furthermoresince theAMELY gene does not function in enamel formationor have any other known function no positive selection exitsfor its maintenance and forensic analyses have demonstrated

ISRN Dentistry 13

(a) (b) (c)

Figure 4 Scanning electron microscopy of fractured incisors from 16-week-old wild type and amelogenin null mice The enamel (E) andjunction with dentin (D) are shown (a) wild type mouse (b) the enamel from the Amelx null mouse does not have a normal prismaticstructure and is markedly reduced in thickness compared with that of the wild type mouse shown at the same magnification as (a) (c) highermagnification of the enamel layer from the null mouse Arrowheads indicate enamel thickness Bars in (a) and (b) = 10 120583m bar in (c) = 1 120583mThis figure was originally published in Gibson et al J Biol Chem 267 (34)31871-31875 2001 The American Society for Biochemistry andMolecular Biology DOI 101074jbcM104624200

that AMELY is frequently deleted from the Y-chromosome[164] It is worth noting that like amelogenin ablated micedeletion of the amelogenin gene (AMELX) in humans doesresult in a thin enamel layer This layer is the thickest onthe cusp tips and marginal ridges relative to the lateral toothsurfaces [159] It appears that amelogenin is not necessaryfor the nucleation of enamel crystallites but is necessary forthe crystallites to continue to grow in length in an organizedmanner

73 Ameloblastin Ameloblastin is the secondmost abundantenamel matrix protein The AMBN gene locates to chro-mosome 4q21 and has 13 exons [165 166] Ameloblastinwas first described as a nonamelogenin protein extractedfrom pig that migrated between 13 and 17 kDa on SDS-PAGE [167] Immunohistochemical experiments showed thatthis protein locates between the enamel rods in an areatermed the sheath space for which the proteins were thereforenamed sheath proteins [12] The proteins locating to thesheath space are ameloblastin cleavage products In contrastintact ameloblastin in the outermost newly formed enamelaccumulates on the enamel rods and not in the sheath spaceas do the cleavage products [168 169] This was the firstdata suggesting that full-length ameloblastin performs onefunction at the mineralizing front and that it performs adifferent function once it is cleaved and accumulates in thesheath space [10]

At approximately the same time three groups indepen-dently cloned ameloblastin cDNAs from rat (two groups) andfrom pig Therefore three different names were proposedThe rat protein was named ldquoameloblastinrdquo [165] and ldquoamelinrdquo[170] and the pig protein was named ldquosheathlinrdquo [171] Thegene name has been designated ldquoameloblastinrdquo (Ambn) Inpig the intact secreted ameloblastin protein is composed of395 amino acids but migrated on SDS-PAGE at a higher than

expected molecular mass of approximately 62 kDa Subse-quently It was discovered that ameloblastin is O-glycosylatedat Ser86 [172] and Thr348 [173] hydroxylated at Pro11 [171]and Pro324 [173] and has four serines (Ser15 Ser17 Ser209and Ser210) in the required context for phosphorylationby Golgi casein kinase [174] The ameloblastin mRNA isalternatively spliced and splicing determines the glycosyla-tion state of the ameloblastin protein [172] Excluding thesignal peptide the translation products are 380 and 395amino acids and the smaller ameloblastin isoform differsfrom the larger by the absence of 15 amino acids encodedat the 51015840 end of exon 5 Although its function remainsunknown this N-terminal region is highly conserved amongspecies and contains the Ser86 O-glycosylation site [172]Therefore ameloblastin undergoes several posttranslationalmodifications and is alternatively spliced and translation ofthe smaller splice product results in the loss of a highlyconserved domain that contains one of only two ameloblastinO-linked phosphorylation sites

Intact ameloblastin is a trace component of develop-ing enamel and has never been isolated in vivo [68] Asmentioned previously cleavage of ameloblastin by MMP20accounts for all the known ameloblastin cleavage productsin the porcine enamel matrix The initial cleavages releasethree products from the N-terminal region which include the17 and 13 kDa products originally observed on SDS-PAGEgels [167] and also include a 15 kDa cleavage product [175] Asubsequent comprehensive analysis of MMP20 ameloblastincleavage site preferences suggested that MMP20 initiallycleaves the large ameloblastin splice product (395 residues)at one of three sites near the N-terminus (after Gln130Arg170 or Ala222) to generate cleavage products Theseinitial products are then cleaved a second or third timeat these same sites as well as at specific secondary sitesthat are located mostly near the C-terminus [68] The N-terminal cleavage products accumulate in the sheath space

14 ISRN Dentistry

throughout the enamel layer while the calcium binding C-terminal cleavage products are on the rods and are notdetectable beyond a depth of 50 120583m from the surface ofthe newly formed enamel [168] This is consistent with theproposed 3D human ameloblastin model demonstrating thatameloblastin has N- and C-terminal domains connected byan unstructured linker that is susceptible to degradation[176]Therefore this reinforces previous data by showing thatameloblastin N- and C-terminal cleavage products locate todifferent areas of the forming enamel and likely play differentroles in enamel development

Ameloblastin is a pseudogene in certain species ofbaleen whales [57] and although published reports sug-gest critical functions in other tissues deletion of Ambnfrom mice appears to only affect enamel development Arecent publication suggested that ameloblastin is importantin root development However the report of the origi-nal ameloblastin mutated mouse showing a severe enamelphenotype stated that ldquo root formation in the mutantmice was not different from wild-type and 119860119898119887119899+minus micerdquo[17] Perhaps the most published alternative function forameloblastin is in bone remodeling and repair Howeverif the NCBI UniGene database is accessed and AMBN isused as a search term the resulting EST Profile demonstratesthat not a single AMBN transcript has been found in eitherhuman or mouse bone Conversely the mouse but not thehuman EST Profile contains an EST analysis from molarand Ambn transcripts were found in mouse molar tissuesThe evidence for ameloblastin expression in bone may beconclusively confirmed or denied by the soon to be publishedcharacterization of the newly engineeredAmbn-LacZ-knock-in mouse that substitutes LacZ into and Ambn out of thenatural Ambn translational start site (JP Simmer personalcommunication)

Although humanmutations inAMBN that cause AI havenot yet been discovered a mouse model exists where exons5 and 6 are deleted from Ambn Originally this mouse wasdeemed as a true Ambn knockout [17] but subsequently itwas discovered that in these same mice an Ambn mRNAlacking exons 5 and 6 was expressed and that this truncatedmRNA was also translated in cells from mouse enamelorgan [141] However as mentioned previously the mutatedprotein causes a severe enamel phenotype where a very thinlayer of dysplastic mineralized material is deposited on thedentin and this material has no rods no crystals and doesnot resemble enamel (Figure 5) [17 141] Interestingly duringthe presecretory stage when the ameloblasts are attachedto a basement membrane adjacent to where the enamel willform the Ambn mutant ameloblasts are normally arrangedand start their characteristic process of elongation intosecretory ameloblasts However after the early secretorystage when the basement membrane is destroyed thesesame ameloblasts abnormally detach from the matrix layerlose their cell polarity and appeared to fold over one anotherto form multilayered cell structures [17] This was attributedto ameloblastin playing a role in cell adhesion Howeverablation of the enamelin gene results in a near identicalprogressive dysplastic ameloblast layer morphology as is

observed in the Ambn mutated mouse [177] An alternativeexplanation for the dysplastic ameloblast layer morphologymay be that since no enamel layer forms in theAmbnmutatedand Enam ablated mice ameloblasts could fail to adhereto the unnatural surface even if their attachment apparatusremained intact Once the basement membrane is degradedameloblasts no longer have a tight binding site adjacent tothe mineral surface and as they progress into the secretorystage the ameloblasts normally adhere weakly to the enamelsurface Furthermore wild-type ameloblasts move back asthe enamel layer rapidly thickens and cell proliferation at thecervical loop compensates for this movement of ameloblastsaway from the dentin So when enamel thickening isabsent as in the case with Ambn and Enam mutations theameloblasts occupy a smaller surface than normal that couldcontribute to their dysplastic morphology [177] Althoughthe Ambnmutant mice have an ameloblast layer morphologythat is disrupted after the early secretory stage this may bea secondary effect from the almost complete absence of therapidly thickening enamel layer Ameloblastin and enamelinlikely play a more central role in crystallite nucleation andsubsequent crystallite elongation

74 Enamelin The human enamelin gene (ENAM) spans18 kb on chromosome 4q11-21 and its transcript has nineexons with a single noncoding exon 1 [178] The humanenamelin mRNA encodes a preprotein of 1142 amino acidsand no alternatively spliced enamelin mRNAs have ever beenidentified [69] The enamelin protein is secreted as a 186 kDaprecursor phosphorylated glycoprotein which quickly under-goes a series of proteolytic cleavages [127 179 180] Thesecreted 186 kDa (amino acids 1ndash1104) porcine parent proteincan only be foundwithin 1120583mof the enamel surface [70]Thisprotein is rapidly processed from its C-terminus to generate155 145 and 89 kDa N-terminal cleavage products that areshort lived and are only found near the enamel surface The32 kDa enamelin cleavage product (amino acids 136ndash241) isthe only stable domain that accumulates in the deeper enamel[70] This 32 kDa enamelin proteolytic fragment is highlyconserved among species The three N-linked glycosylationsites originally described in the porcine 32 kDa enamelinwereunchanged during mammalian evolution [178]This suggeststhat the 32 kDa enamelin cleavage product plays an importantfunctional role in enamel formation Furthermore amongmammals sites of enamelin posttranslational modificationsare highly conserved This includes several potential andknown phosphorylation andN-linked glycosylation sites andincludes six cysteines that are thought to form disulphidebridges [142] Therefore enamelin is a large extensivelypost-translationally modified protein that is highly con-served amongmammals andundergoes proteolysis soon afterits secretion Like amelogenin ameloblastin and MMP20enamelin function is only required in mammals that haveenamel on their teeth In mammals without enamel orwithout teeth enamelin may become a pseudogene [57]

Mutations in the mouse enamelin gene were origi-nally induced with the mutagen N-ethyl-N-nitrosourea andfour separate point mutations were identified These were

ISRN Dentistry 15

(a) (b)

Figure 5 Defects in enamel formation of ameloblastin null mice SEM analysis of 8-wk-old incisor cross sections from heterozygote andhomozygote mice Note the lack of a true enamel layer on the homozygous mutated Ambn incisor E enamel D dentin P pulp (D) SEManalysis of incisor E enamel D dentin P pulp dE defective enamelThis figure was originally published in Fukumoto et al J Cell Biol 167(5)973-983 2004 D01101083jcb200409077

pSer55Ile pGlu57Gly a T to A substitution at the splicedonor site in exon 4 and pGln176XThe heterozygousmousephenotypes included a rough and pitted enamel surface andthe homozygous mutant mice had enamel agenesis [181 182]Since then as was performed for KLK4 (see Section 56) anEnam knock-outLacZ knock-in mouse line was developedGene targeting was used to generate a knock-in mousecarrying a null allele of Enam that has a LacZ reportergene replacing the Enam translation initiation site and genesequences through exon 7 [18] Thus the LacZ reporterconstruct was in the precise genomic location that Enamhad been knocked out of and was therefore transcriptionallyregulated in precisely the same way as the Enam gene LacZexpression provided a sensitive reporter for native Enamexpression and the knock-in mice showed LacZ expressiononly in the ameloblasts of developing teeth In additionteeth from 119864119899119886119898minusminus mice lacked enamel and had a whiteopaque appearance with severe dentin abrasion along thelabial and lingual sides of the teeth The molars from thesemice had pronounced occlusal wear so that the cusp tipswere flattened and rounded The erupted portions of theincisors had very thin enamel that was coated with a layer ofsmall calcifiedmaterial that ldquofelt gritty and sandpaper-like inconsistencyrdquo (Figure 6) 120583CT reconstructions confirmed thelack of mineralized enamel in the molar crown and incisorcrown from Enamminusminus mice Strikingly the secretory stageenamel layer of 119864119899119886119898minusminus mice was von Kossa negative exceptfor small mineralized nodules that were more abundant nearthe dentin-enamel junction The mandibular incisors from119864119899119886119898

+minus mice were consistently chalky white whereas themaxillary incisors were variable ranging from near normalto chalky white The mandibular incisors were also subject toocclusal wear and the functional incisal edge of the incisor

was always missing Also the enamel from mandibularincisors of 119864119899119886119898+minus mice had a mineral to protein ratiothat was normal during the secretory stage but in the earlymaturation to nearly mature stages this ratio was half thatof wild-type mice [18 183] A thick layer of enamel proteinaccumulates in the extracellular space beneath the secretoryameloblasts in the 119864119899119886119898minusminus mice but no mineralizationoccurs at the mineralization front Therefore enamelin isthought to be a critical component of themineralization frontthat promotes or catalyzes the extension of enamel crystallites[18] Both enamelin and ameloblastin appear to have similarfunctions with regard to crystallite initiation and elongationwhereas amelogenin appears to create a framework thatallows the continued elongation of the already initiatedcrystallites

Mutations in ENAM cause AI The first reported AI-causing mutation in ENAM was a heterozygous G to Atransition in the first nucleotide of intron 8 that was predictedto cause a deletion of exon 8 (pA158-Q178del) during mRNAprocessing [184] The tooth crowns were small thin andyellow with little or no enamel When a single Enam alleleis defective the phenotype can be nonpenetrant [185] or bemanifested as enamel pits [186] horizontal groves [187] orgeneralized thin enamel [184] When both ENAM alleles aredefective the enamel is extremely thin or nonexistnt [186]In most cases however ENAM mutations cause autosomaldominant AI To date twelve novel ENAM disease-associatedmutations have been characterized two of which are causedby two different mutations in each ENAM allele (compoundheterozygosity) The compound herterozygous mutationswere discovered in a family where the proband and his fatherhad an AG insertion (p422FsX448) in ENAM previouslyidentified in AI kindreds from Slovenia and Turkey whereas

16 ISRN Dentistry

Enam++

(a)

Enam+minus

(b)

Enamminusminus

(c)

Enamminusminus

(d)

(e) (f) (g) (h)

Figure 6 Scanning electron microscopy of wild-type heterozygous and enamelin null mouse enamel from incisors and molars at 7 weeksSEM was used to examine fractured sections of mouse incisors (andashd) and molars (endashh) The enamel of wild type (a and e) and heterozygous(b and f) both showed a thick enamel layer with well-defined rod (prism) structures In contrast the enamel of Enam null mice (c d g andh) was extremely thin and irregular with a rough surface In some places (g) the enamel did not even form sufficiently to complete the DEJArrowheads delineate the DEJ This figure was originally published in Hu et al J Biol Chem 283 (16)10858-10871 2008 by The AmericanSociety for Biochemistry and Molecular Biology DOI 101074jbcM710565200

the mother and proband had a novel missense mutation thatsubstitutes leucine for a phosphorylated serine (pSer216Leu)in the 32 kDa enamelin cleavage product [188] The probandhad discolored small teeth consistent with hypoplastic AIBoth the father and themother had teeth that appeared highlypolished with localized pitting defects The most recentlydiscovered ENAM mutation identified a novel heterozy-gous frameshift mutation in exon 4 (pAsn36Ilefs56) Thisframeshift occurs in the coding region for the signal peptidewhich is predicted to preclude synthesis of the entire secretedprotein Both the proband and his father had thin softenamel and the incisal edges of their primary anterior teethwere chipped [189] Therefore enamelin is the largest leastabundant and most highly post-translationally modifiedenamel matrix protein It is essential for enamel developmentbut may become a pseudogene in enamelless or toothlessmammals [144]

8 Summary and Perspective

This review on ldquoDental Enamel Development Proteinasesand their Enamel Matrix Substratesrdquo has thus far provided amostly fact-based update on the proteins known to be presentwithin the enamel matrix In this section these facts will bewoven into a plausible mechanism by which these enamel

matrix proteins support and promote enamel developmentThe new facts support a somewhat different interpretation ofenamel development than what was previously accepted Itmust be stated that although the proteins of the enamelmatrix(amelogenin ameloblastin enamelin and MMP20 KLK4)are essential for enamel formation because the malfunctionof any one of them causes AI they are not the only proteinsthat are essential Mutations in FAM83H [190] and LAMB3[191] cause autosomal dominantAI andmutations inWRD72[192] C4orf26 [193] and SCLA24A4 [194] cause autosomalrecessive AI Several other gene mutations cause defectiveenamel as part of a syndrome [103] However the proposedmechanism of enamel development focuses on just what mayoccur within the enamel matrix itself so that the enamel candevelop and ultimately achieve its final hardened form

Recall that each enamel rod is composed of approximately10000 to 40000 crystallites and that each crystallite starts inthe shape of a ribbon (about 26 nmby 68 nm in cross-section)[126] Previously it was widely accepted that amelogeninserves to inhibit crystallite growth in width and thickness asthe crystallites extend from the dentin-enamel junction tothe surface of the tooth MMP20 was and still is proposed tocleave enamel matrix proteins so that the cleavage productswill locate to different areas of the forming enamel to supportthe elongation of the enamel crystallites KLK4 was proposedto cleave the enamel matrix proteins principally amelogenin

ISRN Dentistry 17

to facilitate protein removal and to allow the crystallites togrow in width and thickness However from the results ofthe Klk4 ablated mice [15] we now know that amelogenindoes not completely inhibit crystallite growth in width andthickness Ablation of Klk4 in mice resulted in enamel witha higher protein content [94] because the proteins werenot efficiently removed from the enamel matrix Howeverdespite this the enamel crystallites grew substantially inwidth and thickness The crystallites did not interlock andactually spilled out of the rods as what appeared to be ldquoangelhair spaghettirdquo It is possible that the crystallites grew to thepoint where they could interlock but the excessive proteinpresent may have prevented this Regardless the presence ofamelogenin still allowed the crystallites to grow substantiallyin width and thickness to the point where they could almostinterlock

Conversely as described in Section 72 amelogenin byitself cannot initiate crystal growth or promote enamelformationWhen the enamelin [18] or ameloblastin [17] geneis ablated from mice these mice have no true enamel andno crystal structure However the amelogenin null micehave measurable crystals with a well-defined organizationThe crystals are much smaller and less well organized thanin wild-type mice but they are nonetheless present [158]Therefore amelogenin does not inhibit crystallite growth inwidth and thickness by binding selectively to specific sides ofthe crystallites and it does not initiate crystallite growth Sowhat is the most abundant enamel matrix protein doing topromote enamel development A significant clue comes fromthe finding that newly formed enamel ribbons start as amor-phous calcium phosphate (ACP) that then transform intohydroxyapatite (HAP) [195 196] This means that the enamelribbons are established prior to their crystallization The sizeshape and spatial organization of the enamel crystallites mustbe set when the ACP is first formed so that it can crystallizeinto the proper ribbon shape Therefore amelogenin isproposed to form a mold that sets the boundaries for thecrystallite ribbons Much as cement is poured into a moldfor a house foundation ACP would be poured in or establishitself within the confines of the amelogenin ribbon moldAlthough the 32 kDa enamelin cleavage product that is highlyconserved among mammals and that persists in the enamelmatrix has not been shown to bind with amelogenin in vivoit was shown to colocalize with amelogenin [197] Thus itis likely that the 32 kDa enamelin cleavage product is also astructural component of the postulated amelogenin ribbonmolds Along this same line of reasoning it would also makesense that the ameloblastin MMP20 cleavage products thataccumulate in the enamel sheath space may also provide astructural component for the mold Certainly the mold mustbe adequately supportive so that the long thin ribbons do notbreak as they grow in length

So it is possible that full-length enamelin andameloblastin andor their C-terminal cleavage productspromote the growth of the future crystallites in lengthThis makes sense because the full-length proteins andortheir C-terminal cleavage products are only present at themineralization front and are not present in the older deeperenamel layers Subsequently and almost immediately these

proteins and amelogenin are cleaved by MMP20 and someof these N-terminal cleavage products accumulate within theenamel to form a mold that establishes the shape size andorientation of the forming crystallites Once the end of thesecretory stage is attained and the ribbons stop growing inlength KLK4 is secreted to cleave the enamel matrix proteinmold so that it can be exported from the hardening enamelso that the maturing crystallites can interlock and form acohesive rod structure

This new theory [126] represents a departure fromprevious beliefs that amelogenin by itself initiates enamelformation and from the thought that amelogenin inhibitscrystallite growth in width and thickness Results fromgenetically altered mice clearly demonstrate that neitherof these widely held beliefs is true The newly proposedtheory incorporates the results obtained from geneticallyaltered mice and is therefore based on what occurs in vivoduring enamel development Theories are only the closestapproximate of the truth that we have at this point in timeFuture theories if they are to be taken seriously will have tobe consistent with results generated from studies performedin vivo After all it is nature that guides us to its mysteries

Acknowledgments

The author would like to sincerely thank The Forsyth Insti-tute and the National Institute of Dental and CraniofacialResearch for their support of his research endeavors over theyears Special thanks go to Charles E Smith and James PSimmer for their critical review of this manuscript and fortheir generosity in helping the author to better understandenamel development over our many years of productiveinteraction

References

[1] M Mina and E J Kollar ldquoThe induction of odontogene-sis in non-dental mesenchyme combined with early murinemandibular arch epitheliumrdquo Archives of Oral Biology vol 32no 2 pp 123ndash127 1987

[2] IThesleff A Vaahtokari P Kettunen and T Aberg ldquoEpithelial-mesenchymal signaling during tooth developmentrdquo ConnectiveTissue Research vol 32 no 1ndash4 pp 9ndash15 1995

[3] A Tucker and P Sharpe ldquoThe cutting-edge of mammaliandevelopment how the embryo makes teethrdquo Nature ReviewsGenetics vol 5 no 7 pp 499ndash508 2004

[4] A Nanci Ten Catersquos Oral Histology Development Structure andFunction Mosby St Louis Mo USA 2003

[5] A S Cole and J E Eastoe Eds Biochemistry and Oral BiologyButterworth London UK 1988

[6] M Deakins and J F Volker ldquoAmount of organic matter inenamel from several types of human teethrdquo Journal of DentalResearch vol 20 no 2 pp 117ndash121 1941

[7] M L LeFevre andR SManly ldquoMoisture inorganic and organiccontents of enamel and dentin from carious teethrdquo Journal of theAmerican Dental Association vol 24 pp 233ndash242 1938

[8] M Sponheimer Z Alemseged T E Cerling F E Grine WH Kimbel et al ldquoIsotopic evidence of early hominin dietsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 26 pp 10513ndash10518 2013

18 ISRN Dentistry

[9] J P Simmer and A G Fincham ldquoMolecular mechanisms ofdental enamel formationrdquo Critical Reviews in Oral Biology andMedicine vol 6 no 2 pp 84ndash108 1995

[10] J D Bortlett and J P Simmer ldquoProteinases in developing dentalenamelrdquo Critical Reviews in Oral Biology and Medicine vol 10no 4 pp 425ndash441 1999

[11] C E Smith ldquoCellular and chemical events during enamelmaturationrdquo Critical Reviews in Oral Biology and Medicine vol9 no 2 pp 128ndash161 1998

[12] T Uchida T Tanabe M Fukae and M Shimizu ldquoImmuno-cytochemical and immunochemical detection of a 32 kDanonamelogenin and related proteins in porcine tooth germsrdquoArchives of Histology and Cytology vol 54 pp 527ndash538 1991

[13] J C-C Hu Y-H P Chun T Al Hazzazzi and J P SimmerldquoEnamel formation and amelogenesis imperfectardquo Cells TissuesOrgans vol 186 no 1 pp 78ndash85 2007

[14] J D Bartlett Z Skobe A Nanci and C E Smith ldquoMatrixmetalloproteinase 20 promotes a smooth enamel surface astrong dentino-enamel junction and a decussating enamel rodpatternrdquo European Journal of Oral Sciences vol 119 supplement1 pp 199ndash205 2011

[15] J P Simmer Y Hu R Lertlam Y Yamakoshi and J C-CHu ldquoHypomaturation enamel defects in Klk4 knockoutLacZknock in micerdquo Journal of Biological Chemistry vol 284 no 28pp 19110ndash19121 2009

[16] C W Gibson Z-A Yuan B Hall et al ldquoAmelogenin-deficientmice display an amelogenesis imperfecta phenotyperdquo Journal ofBiological Chemistry vol 276 no 34 pp 31871ndash31875 2001

[17] S Fukumoto T Kiba B Hall et al ldquoAmeloblastin is a celladhesion molecule required for maintaining the differentiationstate of ameloblastsrdquo Journal of Cell Biology vol 167 no 5 pp973ndash983 2004

[18] J C-C Hu Y Hu C E Smith et al ldquoEnamel defects andameloblast-specific expression in Enam knock-outlacZ knock-in micerdquo Journal of Biological Chemistry vol 283 no 16 pp10858ndash10871 2008

[19] J D Bartlett andC E Smith ldquoModulation of cell-cell junctionalcomplexes by matrix metalloproteinasesrdquo Journal of DentalResearch vol 92 no 1 pp 10ndash17 2013

[20] H C Slavkin Developmental Craniofacial Biology Lea andFebiger Philadelphia Pa USA 1979

[21] I A Mjor and F Ole Human Oral Embryology and HistologyMunksgaard Copenhagen Denmark 1986

[22] E Ronnholm ldquoAn electron microscopic study of the amelogen-esis in human teeth I The fine structure of the ameloblastsrdquoJournal of Ultrasructure Research vol 6 no 2 pp 229ndash2481962

[23] G Daculsi J Menanteau L M Kerebel and D Mitre ldquoLengthand shape of enamel crystalsrdquoCalcified Tissue International vol36 no 5 pp 550ndash555 1984

[24] E J Reith and M H Ross ldquoMorphological evidence for thepresence of contractile elements in secretory ameloblasts of theratrdquo Archives of Oral Biology vol 18 no 3 pp 445ndash448 1973

[25] A Boyde ldquoEnamelrdquo in Handbook of Microscopic Anatomy AOksche and L Vollrath Eds pp 309ndash473 Springer BerlinGermany 1989

[26] G Daculsi and B Kerebel ldquoHigh-resolution electron micro-scope study of human enamel crystallites size shape andgrowthrdquo Journal of Ultrastructure Research vol 65 no 2 pp163ndash172 1978

[27] F J G Cuisinier P Steuer B Senger J C Voegel and RM Frank ldquoHuman amelogenesis I high resolution electronmicroscopy study of ribbon- like crystalsrdquo Calcified TissueInternational vol 51 no 4 pp 259ndash268 1992

[28] E Kallenbach ldquoFine structure of secretory ameloblasts in thekittenrdquo American Journal of Anatomy vol 148 no 4 pp 479ndash511 1977

[29] D F Travis andM J Glimcher ldquoThe structure and organizationof and the relationship between the organic matrix and theinorganic crystals of embryonic bovine enamelrdquoThe Journal ofCell Biology vol 23 pp 447ndash497 1964

[30] M Wakita H Tsuchiya T Gunji and S Kobayashi ldquoThree-dimensional structure of Tomesrsquo processes and enamel prismformation in the kittenrdquoArchivumHistologicum Japonicum vol44 no 3 pp 285ndash297 1981

[31] H J Orams ldquoAn examination of the prism core prism sheathand interprismatic substance using the electon microscoperdquoAustralian Dental Journal vol 11 no 2 pp 93ndash104 1966

[32] C Robinson J Kirkham J A Weatherell A Richards KJosephsen and O Fejerskov ldquoMineral and protein concentra-tions in enamel of the developing permanent porcine dentitionrdquoCaries Research vol 22 no 6 pp 321ndash326 1988

[33] J E Eastoe ldquoOrganic matrix of tooth enamelrdquo Nature vol 187no 4735 pp 411ndash412 1960

[34] J E Eastoe ldquoThe amino acid composition of proteins from theoral tissues-II The matrix proteins in dentine and enamel fromdeveloping human deciduous teethrdquo Archives of Oral Biologyvol 8 no 5 pp 633ndash652 1963

[35] R C Burgess and C M Maclaren ldquoProteins in developingbovine enamelrdquo in Tooth Enamel I M V Stack andW FR Edspp 74ndash82 John Wright amp Sons LTD Bristol England 1965

[36] M Fukae and M Shimizu ldquoStudies on the proteins of develop-ing bovine enamelrdquo Archives of Oral Biology vol 19 no 5 pp381ndash386 1974

[37] C Robinson N R Lowe and J A Weatherell ldquoChanges inamino acid composition of developing rat incisor enamelrdquoCalcified Tissue International vol 23 no 1 pp 19ndash31 1977

[38] C M Overall and H Limeback ldquoIdentification and characteri-zation of enamel proteinases isolated from developing enamelAmelogeninolytic serine proteinases are associatedwith enamelmaturation in pigrdquo Biochemical Journal vol 256 no 3 pp 965ndash972 1988

[39] P K DenBesten and L M Heffernan ldquoSeparation by poly-acrylamide gel electrophoresis of multiple proteases in rat andbovine enamelrdquo Archives of Oral Biology vol 34 no 6 pp 399ndash404 1989

[40] C E Smith S Borenstein A Fazel and A Nanci ldquoIn vitrostidies of the proteases which degrade amelogenenins in devel-oping rat incisor enamelrdquo in Tooth Enamel V R W F Ed pp286ndash293 Florence Publishers Yokohama Japan 1989

[41] C E Smith M Issid H C Margolis and E C MorenoldquoDevelopmental changes in the pHof enamel fluid and its effectson matrix-resident proteinasesrdquo Advances in Dental Researchvol 10 no 2 pp 159ndash169 1996

[42] T Tanabe M Fukae T Uchida and M Shimizu ldquoThe localiza-tion and characterization of proteinases for the initial cleavageof porcine amelogeninrdquo Calcified Tissue International vol 51no 3 pp 213ndash217 1992

[43] T Tanabe M Fukae and M Shimizu ldquoDegradation of enam-elins by proteinases found in porcine secretory enamel in vitrordquoArchives of Oral Biology vol 39 no 4 pp 277ndash281 1994

ISRN Dentistry 19

[44] C Begue-Kirn P H Krebsbach J D Bartlett and W T ButlerldquoDentin sialoprotein dentin phosphoprotein enamelysin andameloblastin tooth-specific molecules that are distinctivelyexpressed duringmurine dental differentiationrdquo European Jour-nal of Oral Sciences vol 106 no 5 pp 963ndash970 1998

[45] J C C Hu X Sun C Zhang S Liu J D Bartlett and JP Simmer ldquoEnamelysin and kallikrein-4 mRNA expression indeveloping mouse molarsrdquo European Journal of Oral Sciencesvol 110 no 4 pp 307ndash315 2002

[46] J D Bartlett J P Simmer J Xue H C Margolis and E CMoreno ldquoMolecular cloning and mRNA tissue distribution ofa novel matrix metalloproteinase isolated from porcine enamelorganrdquo Gene vol 183 no 1-2 pp 123ndash128 1996

[47] J P Simmer M Fukae T Tanabe et al ldquoPurification charac-terization and cloning of enamel matrix serine proteinase 1rdquoJournal of Dental Research vol 77 no 2 pp 377ndash386 1998

[48] M Fukae T Tanabe T Uchida et al ldquoEnamelysin (matrixmetalloproteinase-20) localization in the developing tooth andeffects of pH and calcium on amelogenin hydrolysisrdquo Journal ofDental Research vol 77 no 8 pp 1580ndash1588 1998

[49] E Llano A M Pendas V Knauper et al ldquoIdentification andstructural and functional characterization of human enam-elysin (MMP-20)rdquo Biochemistry vol 36 no 49 pp 15101ndash151081997

[50] G M Grant T A Giambernardi A M Grant and R J KlebeldquoOverview of expression of matrix metalloproteinases (MMP-17 MMP-18 and MMP-20) in cultured human cellsrdquo MatrixBiology vol 18 no 2 pp 145ndash148 1999

[51] T Takata M Zhao H Nikai T Uchida and T Wang ldquoGhostcells in calcifying odontogenic cyst express enamel-relatedproteinsrdquo Histochemical Journal vol 32 no 4 pp 223ndash2292000

[52] T Takata M Zhao T Uchida et al ldquoImmunohistochemicaldetection and distribution of enamelysin (MMP-20) in humanodontogenic tumorsrdquo Journal of Dental Research vol 79 no 8pp 1608ndash1613 2000

[53] A Vaananen R Srinivas M Parikka et al ldquoExpression andregulation of MMP-20 in human tongue carcinoma cellsrdquoJournal of Dental Research vol 80 no 10 pp 1884ndash1889 2001

[54] A Kimura T Kihara R Ohkura K Ogiwara and T Taka-hashi ldquoLocalization of bradykinin B2 receptor in the fol-licles of porcine ovary and increased expression of matrixmetalloproteinase-3 and -20 in cultured granulosa cells bybradykinin treatmentrdquo Biology of Reproduction vol 65 no 5pp 1462ndash1470 2001

[55] J D Bartlett ldquoMatrix metalloproteinase-20enamelysinrdquo inHandbook of Proteolytic Enzymes N D Rawlings and G SSalvesen Eds pp 835ndash840 Academic Press Oxford UK 2013

[56] B E Turk D H Lee Y Yamakoshi et al ldquoMMP-20 ispredominately a tooth-specific enzyme with a deep catalyticpocket that hydrolyzes type V collagenrdquo Biochemistry vol 45no 12 pp 3863ndash3874 2006

[57] R W Meredith J Gatesy J Cheng and M S SpringerldquoPseudogenization of the tooth gene enamelysin (MMP20) inthe common ancestor of extant baleen whalesrdquo Proceedings ofThe Royal Society vol 278 no 1708 pp 993ndash1002 2011

[58] Y Yamada Y Yamakoshi R F Gerlach C CHu KMatsumotoet al ldquoPurification and characterization ofenamelysin fromsecretory stage pig enamelrdquo Archives of Comparative Biology ofTooth Enamel vol 8 pp 21ndash25 2003

[59] P K den Besten J S Punzi and W Li ldquoPurification andsequencing of a 21 kDa and 25 kDa bovine enamel metallopro-teinaserdquo European Journal of Oral Sciences vol 106 no 1 pp345ndash349 1998

[60] W Li D Machule C Gao and P K DenBesten ldquoActivationof recombinant bovine matrix metalloproteinase-20 and itshydrolysis of two amelogenin oligopeptidesrdquo European Journalof Oral Sciences vol 107 no 5 pp 352ndash359 1999

[61] O H Ryu A G Fincham C-C Hu et al ldquoCharacterization ofrecombinant pig enamelysin activity and cleavage of recombi-nant pig and mouse amelogeninsrdquo Journal of Dental Researchvol 78 no 3 pp 743ndash750 1999

[62] C Caron J Xue and J D Bartlett ldquoExpression and localizationof membrane type 1 matrix metalloproteinase in tooth tissuesrdquoMatrix Biology vol 17 no 7 pp 501ndash511 1998

[63] H Palosaari Y Ding M Larmas et al ldquoRegulation andinteractions ofMT1-MMPandMMP-20 in humanodontoblastsand pulp tissue in vitrordquo Journal of Dental Research vol 81 no5 pp 354ndash359 2002

[64] J D Bartlett O H Ryu J Xue J P Simmer and H C Mar-golis ldquoEnamelysin mRNA displays a developmentally definedpattern of expression and encodes a protein which degradesamelogeninrdquo Connective Tissue Research vol 39 no 1ndash3 pp101ndash109 1998

[65] J Moradian-Oldak I Jimenez D Maltby and A G FinchamldquoControlled proteolysis of amelogenins reveals exposure of bothcarboxy- and amino-terminal regionsrdquo Biopolymers vol 58 no7 pp 606ndash616 2001

[66] T Nagano A Kakegawa Y Yamakoshi et al ldquoMmp-20 andKlk4 cleavage site preferences for amelogenin sequencesrdquo Jour-nal of Dental Research vol 88 no 9 pp 823ndash828 2009

[67] T Iwata Y Yamakoshi J C-C Hu et al ldquoProcessing ofameloblastin by MMP-20rdquo Journal of Dental Research vol 86no 2 pp 153ndash157 2007

[68] Y-H P Chun Y Yamakoshi F Yamakoshi et al ldquoCleavage sitespecificity ofMMP-20 for secretory-stage ameloblastinrdquo Journalof Dental Research vol 89 no 8 pp 785ndash790 2010

[69] J C-C Hu and Y Yamakoshi ldquoEnamelin and autosomal-dominant amelogenesis imperfectardquo Critical Reviews in OralBiology and Medicine vol 14 no 6 pp 387ndash398 2003

[70] Y Yamakoshi J C-C Hu M Fukae F Yamakoshi and J PSimmer ldquoHow do enamelysin and kallikrein 4 process the 32-kDa enamelinrdquo European Journal of Oral Sciences vol 114 no1 pp 45ndash51 2006

[71] O Ryu J C C Hu Y Yamakoshi et al ldquoPorcine kallikrein-4activation glycosylation activity and expression in prokaryoticand eukaryotic hostsrdquo European Journal of Oral Sciences vol110 no 5 pp 358ndash365 2002

[72] Y Yamakoshi J C-C Hu T Iwata K Kobayashi M Fukaeand J P Simmer ldquoDentin sialophosphoprotein is processed byMMP-2 and MMP-20 in vitro and in vivordquo Journal of BiologicalChemistry vol 281 no 50 pp 38235ndash38243 2006

[73] J D Bartlett Y Yamakoshi J P Simmer A Nanci and C ESmith ldquoMMP20 cleaves E-cadherin and influences ameloblastdevelopmentrdquo Cells Tissues Organs vol 194 no 2ndash4 pp 222ndash226 2011

[74] J O Stracke A J Fosang K Last et al ldquoMatrix metallopro-teinases 19 and 20 cleave aggrecan and cartilage oligomericmatrix protein (COMP)rdquo FEBS Letters vol 478 no 1-2 pp 52ndash56 2000

20 ISRN Dentistry

[75] AVaananen L Tjaderhane L Eklund et al ldquoExpression of col-lagen XVIII and MMP-20 in developing teeth and odontogenictumorsrdquoMatrix Biology vol 23 no 3 pp 153ndash161 2004

[76] J Caterina J Shi S Krakora et al ldquoIsolation characterizationand chromosomal location of the mouse enamelysin generdquoGenomics vol 62 no 2 pp 308ndash311 1999

[77] J J Caterina Z Skobe J Shi et al ldquoEnamelysin (matrixmetalloproteinase 20)-deficient mice display an amelogenesisimperfecta phenotyperdquo Journal of Biological Chemistry vol 277no 51 pp 49598ndash49604 2002

[78] J D Bartlett E Beniash D H Lee and C E Smith ldquoDecreasedmineral content in MMP-20 null mouse enamel is prominentduring the maturation stagerdquo Journal of Dental Research vol83 no 12 pp 909ndash913 2004

[79] A Hartsock and W J Nelson ldquoAdherens and tight junctionsstructure function and connections to the actin cytoskeletonrdquoBiochimica et Biophysica Acta vol 1778 no 3 pp 660ndash6692008

[80] J D Bartlett J M Dobeck C E Tye et al ldquoTargeted p120-catenin ablation disrupts dental enamel developmentrdquo PLoSOne vol 5 no 9 Article ID e12703 2010

[81] J-L Fausser O Schlepp D Aberdam GMeneguzzi J V RuchandH Lesot ldquoLocalization of antigens associatedwith adherensjunctions desmosomes and hemidesmosomes during murinemolar morphogenesisrdquo Differentiation vol 63 no 1 pp 1ndash111998

[82] R Heymann I About U Lendahl J-C Franquin B ObrinkandTAMitsiadis ldquoE- andN-cadherin distribution in develop-ing and functional human teeth under normal and pathologicalconditionsrdquo American Journal of Pathology vol 160 no 6 pp2123ndash2133 2002

[83] N Obara Y Suzuki Y Nagai andM Takeda ldquoExpression of E-and P-cadherin during tooth morphogenesis and cytodifferen-tiation of ameloblastsrdquoAnatomy and Embryology vol 197 no 6pp 469ndash475 1998

[84] J Palacios N Benito R Berraquero A Pizarro A Cano andC Gamallo ldquoDifferential spatiotemporal expression of E- andP-cadherin during mouse tooth developmentrdquo InternationalJournal of Developmental Biology vol 39 no 4 pp 663ndash6661995

[85] B C Sorkin M Y Wang J M Dobeck K L Albergo and ZSkobe ldquoThe cadherin-catenin complex is expressed alternatelywith the adenomatous polyposis coli protein during rat incisoramelogenesisrdquo Journal of Histochemistry andCytochemistry vol48 no 3 pp 397ndash406 2000

[86] C Terling R Heymann B Rozell B Obrink and J Wrob-lewski ldquoDynamic expression of E-cadherin in ameloblasts andcementoblasts in micerdquo European Journal of Oral Sciences vol106 no 1 pp 137ndash142 1998

[87] H GMunshi andM S Stack ldquoReciprocal interactions betweenadhesion receptor signaling and MMP regulationrdquo Cancer andMetastasis Reviews vol 25 no 1 pp 45ndash56 2006

[88] J K McGuire Q Li and W C Parks ldquoMatrilysin (matrixmetalloproteinase-7) mediates E-cadherin ectodomain shed-ding in injured lung epitheliumrdquoAmerican Journal of Pathologyvol 162 no 6 pp 1831ndash1843 2003

[89] A Lochter S Galosy J Muschler N Freedman Z Werb andM J Bissell ldquoMatrix metalloproteinase stromelysin-1 triggers acascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype inmammary epithelial cellsrdquo Journal of Cell Biology vol 139 no 7pp 1861ndash1872 1997

[90] B Fingleton C C Lynch T Vargo-Gogola and LMMatrisianldquoCleavage of E-cadherin by matrix metalloproteinase-7 pro-motes cellular proliferation in nontransformed cell lines viaactivation of RhoArdquo Journal of Oncology vol 2010 Article ID530745 11 pages 2010

[91] J Sanceau S Truchet and B Bauvois ldquoMatrixmetalloproteinase-9 silencing by RNA interference triggers themigratory-adhesive switch in Ewingrsquos sarcoma cellsrdquo Journal ofBiological Chemistry vol 278 no 38 pp 36537ndash36546 2003

[92] K D C Dahl J Symowicz Y Ning et al ldquoMatrix metallopro-teinase 9 is a mediator of epidermal growth factor-dependentE-cadherin loss in ovarian carcinoma cellsrdquo Cancer Researchvol 68 no 12 pp 4606ndash4613 2008

[93] J Chen Y Lan J-A Baek Y Gao and R Jiang ldquoWntbeta-catenin signaling plays an essential role in activation of odonto-genic mesenchyme during early tooth developmentrdquo Develop-mental Biology vol 334 no 1 pp 174ndash185 2009

[94] C E Smith A S Richardson Y Hu J D Bartlett J C-C Hu and J P Simmer ldquoEffect of kallikrein 4 loss onenamel mineralization comparison with mice lacking matrixmetalloproteinase 20rdquo Journal of Biological Chemistry vol 286no 20 pp 18149ndash18160 2011

[95] Y Hu J C-C Hu C E Smith J D Bartlett and J P SimmerldquoKallikrein-related peptidase 4 matrix metalloproteinase 20and the maturation of murine and porcine enamelrdquo EuropeanJournal of Oral Sciences vol 119 no 1 pp 217ndash225 2011

[96] C J Witkop Jr ldquoAmelogenesis imperfecta dentinogenesisimperfecta and dentin dysplasia revisited problems in classifi-cationrdquo Journal of Oral Pathology vol 17 no 9-10 pp 547ndash5531988

[97] S Becerik D Cogulu G Emingil T Han P S Hart and T CHart ldquoExclusion of candidate genes in seven Turkish familieswith autosomal recessive amelogenesis imperfectardquo AmericanJournal ofMedical Genetics A vol 149 no 7 pp 1392ndash1398 2009

[98] S-K Lee F Seymen H-Y Kang et al ldquoMMP20 hemopexindomainmutation in amelogenesis imperfectardquo Journal ofDentalResearch vol 89 no 1 pp 46ndash50 2010

[99] B Gasse E Karayigit E Mathieu S Jung A Garret et alldquoHomozygous and compound heterozygousMMP20mutationsin amelogenesis imperfectardquo Journal of Dental Research vol 92no 7 pp 598ndash603 2013

[100] J-W Kim J P Simmer T C Hart et al ldquoMMP-20 mutation inautosomal recessive pigmented hypomaturation amelogenesisimperfectardquo Journal of Medical Genetics vol 42 no 3 pp 271ndash275 2005

[101] D Ozdemir P S Hart O H Ryu et al ldquoMMP20 active-sitemutation in hypomaturation amelogenesis imperfectardquo Journalof Dental Research vol 84 no 11 pp 1031ndash1035 2005

[102] P Papagerakis H-K Lin K Y Lee et al ldquoPremature stop codonin MMP20 causing amelogenesis imperfectardquo Journal of DentalResearch vol 87 no 1 pp 56ndash59 2008

[103] S K Wang Y Hu J P Simmer F Seymen N M Estrella etal ldquoNovel KLK4 and MMP20 mutations discovered by whole-exome sequencingrdquo Journal of Dental Research vol 92 no 3pp 266ndash271 2013

[104] P S Nelson L Gan C Ferguson et al ldquoMolecular cloningand characterization of prostase an androgen-regulated serineprotease with prostate-restricted expressionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 96 no 6 pp 3114ndash3119 1999

[105] G M Yousef C V Obiezu L-Y Luo M H Black and E PDiamandis ldquoProstaseKLK-L1 is a new member of the human

ISRN Dentistry 21

kallikrein gene family is expressed in prostate and breast tissuesand is hormonally regulatedrdquoCancer Research vol 59 no 17 pp4252ndash4256 1999

[106] M Fukae T Tanabe and M Shimizu ldquoProteolytic enzymeactivity in porcine immature enamelrdquo Tsurumi Shigaku vol 3no 1 pp 15ndash17 1977

[107] M Shimizu T Tanabe and M Fukae ldquoProteolytic enzyme inporcine immature enamelrdquo Journal of Dental Research vol 58pp 782ndash789 1979

[108] J C-C Hu O H Ryu J J Chen et al ldquoLocalization ofEMSP1 expression during tooth formation and cloning ofmouse cDNArdquo Journal of Dental Research vol 79 no 1 pp 70ndash76 2000

[109] J C-C Hu C Zhang X Sun et al ldquoCharacterization of themouse and human PRSS17 genes their relationship to otherserine proteases and the expression of PRSS17 in developingmouse incisorsrdquo Gene vol 251 no 1 pp 1ndash8 2000

[110] J P Simmer A S Richardson C E Smith Y Hu and J C-CHu ldquoExpression of kallikrein-related peptidase 4 in dental andnon-dental tissuesrdquo European Journal of Oral Sciences vol 119no 1 pp 226ndash233 2011

[111] J P Simmer ldquoKallekrein-related peptidase-4rdquo in Handbook ofProteolytic Enzymes N D Rawlings and G S Salvesen Edspp 2768ndash2772 Academic Press Oxford UK 2013

[112] M Debela V Magdolen V Grimminger et al ldquoCrystal struc-tures of human tissue kallikrein 4 activity modulation by aspecific zinc binding siterdquo Journal of Molecular Biology vol 362no 5 pp 1094ndash1107 2006

[113] A Lundwall and M Brattsand ldquoKallikrein-related peptidasesrdquoCellular and Molecular Life Sciences vol 65 no 13 pp 2019ndash2038 2008

[114] Y Yamakoshi A S Richardson S M Nunez et al ldquoEnamelproteins and proteases in Mmp20 and Klk4 null and double-null micerdquo European Journal of Oral Sciences vol 119 no 1 pp206ndash216 2011

[115] C E Tye C T Pham J P Simmer and J D Bartlett ldquoDPPImay activate KLK4 during enamel formationrdquo Journal of DentalResearch vol 88 no 4 pp 323ndash327 2009

[116] M Matsumura A S Bhatt D Andress et al ldquoSubstrates ofthe prostate-specific serine protease prostaseKLK4 defined bypositional-scanning peptide librariesrdquoProstate vol 62 no 1 pp1ndash13 2005

[117] T K Takayama B A McMullen P S Nelson M Mat-sumura and K Fujikawa ldquoCharacterization of hK4 (prostase)a prostate-specific serine protease activation of the precursor ofprostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acidphosphataserdquo Biochemistry vol 40 no 50 pp 15341ndash153482001

[118] C Becker-Pauly M Howel T Walker et al ldquoThe 120572 and 120573subunits of the metalloprotease meprin are expressed in sep-arate layers of human epidermis revealing different functionsin keratinocyte proliferation and differentiationrdquo Journal ofInvestigative Dermatology vol 127 no 5 pp 1115ndash1125 2007

[119] N Beaufort M Debela S Creutzburg et al ldquoInterplay ofhuman tissue kallikrein 4 (hK4) with the plasminogen acti-vation system hK4 regulates the structure and functions ofthe urokinase-type plasminogen activator receptor (uPAR)rdquoBiological Chemistry vol 387 no 2 pp 217ndash222 2006

[120] C VObiezu I PMichaelM A Levesque and E P DiamandisldquoHuman kallikrein 4 enzymatic activity inhibition and degra-dation of extracellular matrix proteinsrdquo Biological Chemistryvol 387 no 6 pp 749ndash759 2006

[121] H Yoon G Laxmikanthan J Lee et al ldquoActivation profiles andregulatory cascades of the human kallikrein-related peptidasesrdquoJournal of Biological Chemistry vol 282 no 44 pp 31852ndash31864 2007

[122] A J Ramsay Y Dong M L Hunt et al ldquoKallikrein-relatedpeptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs) KLK4 and PAR-2 are co-expressedduring prostate cancer progressionrdquo Journal of Biological Chem-istry vol 283 no 18 pp 12293ndash12304 2008

[123] G J Mize W Wang and T K Takayama ldquoProstate-specifickallikreins-2 and -4 enhance the proliferation of DU-145prostate cancer cells through protease-activated receptors-1 and-2rdquoMolecular Cancer Research vol 6 no 6 pp 1043ndash1051 2008

[124] V Gratio N Beaufort L Seiz et al ldquoKallikrein-related pepti-dase 4 a new activator of the aberrantly expressed protease-activated receptor 1 in colon cancer cellsrdquo American Journal ofPathology vol 176 no 3 pp 1452ndash1461 2010

[125] WWang G JMize X Zhang and T K Takayama ldquoKallikrein-related peptidase-4 initiates tumor-stroma interactions inprostate cancer through protease-activated receptor-1rdquo Interna-tional Journal of Cancer vol 126 no 3 pp 599ndash610 2010

[126] J P Simmer A S Richardson Y Y Hu C E Smith and JChing-Chun Hu ldquoA post-classical theory of enamel biominer-alization and why we need onerdquo International Journal of OralScience vol 4 no 3 pp 129ndash134 2012

[127] C-CHu T CHart B R Dupont et al ldquoCloning human enam-elin cDNA chromosomal localization and analysis of expres-sion during tooth developmentrdquo Journal of Dental Research vol79 no 4 pp 912ndash919 2000

[128] Y Yamakoshi F Yamakoshi J C-C Hu and J P SimmerldquoCharacterization of kallikrein-related peptidase 4 glycosyla-tionsrdquo European Journal of Oral Sciences vol 119 supplement1 pp 234ndash240 2011

[129] P S Hart T C Hart M D Michalec et al ldquoMutation inkallikrein 4 causes autosomal recessive hypomaturation amel-ogenesis imperfectardquo Journal of Medical Genetics vol 41 no 7pp 545ndash549 2004

[130] M Goldberg D Septier K Bourd et al ldquoImmunohistochemi-cal localization ofMMP-2 MMP-9 TIMP-1 and TIMP-2 in theforming rat incisorrdquoConnective Tissue Research vol 44 no 3-4pp 143ndash153 2003

[131] R Hall D Septier G Embery and M Goldberg ldquoStromelysin-1 (MMP-3) in forming enamel and predentine in rat incisormdashcoordinated distribution with proteoglycans suggests a func-tional rolerdquo Histochemical Journal vol 31 no 12 pp 761ndash7701999

[132] C Caron J Xue X Sun J P Simmer and J D Bartlett ldquoGelati-nase A (MMP-2) in developing tooth tissues and amelogeninhydrolysisrdquo Journal of Dental Research vol 80 no 7 pp 1660ndash1664 2001

[133] J Feng J S McDaniel H H Chuang O Huang A Rakian etal ldquoBinding of amelogenin to MMP-9 and their co-expressionin developing mouse teethrdquo Journal of Molecular Histology vol43 no 5 pp 473ndash485 2012

[134] R S Lacruz C E Smith S M Smith et al ldquoChymotrypsin C(caldecrin) is associated with enamel developmentrdquo Journal ofDental Research vol 90 no 10 pp 1228ndash1233 2011

22 ISRN Dentistry

[135] A G Fincham J Moradian-Oldak and J P Simmer ldquoThestructural biology of the developing dental enamel matrixrdquoJournal of Structural Biology vol 126 no 3 pp 270ndash299 1999

[136] K Kawasaki T Suzuki and K M Weiss ldquoGenetic basis for theevolution of vertebrate mineralized tissuerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 no 31 pp 11356ndash11361 2004

[137] J D Bartlett B Ganss M Goldberg et al ldquoProtein-proteininteractions of the developing enamel matrixrdquo Current Topicsin Developmental Biology vol 74 pp 57ndash115 2006

[138] M Iwase Y Satta Y Hirai H Hirai H Imai and N TakahataldquoThe amelogenin loci span an ancient pseudoautosomal bound-ary in diverse mammalian speciesrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no9 pp 5258ndash5263 2003

[139] K Kawasaki and K M Weiss ldquoMineralized tissue and verte-brate evolution the secretory calcium-binding phosphoproteingene clusterrdquo Proceedings of the National Academy of Sciences oftheUnited States of America vol 100 no 7 pp 4060ndash4065 2003

[140] J-Y Sire S Delgado D Fromentin and M Girondot ldquoAmelo-genin lessons from evolutionrdquo Archives of Oral Biology vol 50no 2 pp 205ndash212 2005

[141] R M Wazen P Moffatt S F Zalzal Y Yamada and A NancildquoA mouse model expressing a truncated form of ameloblastinexhibits dental and junctional epithelium defectsrdquoMatrix Biol-ogy vol 28 no 5 pp 292ndash303 2009

[142] N Al-Hashimi A-G Lafont S Delgado K Kawasaki and J-YSire ldquoThe enamelin genes in lizard crocodile and frog and thepseudogene in the chicken provide new insights on enamelinevolution in tetrapodsrdquoMolecular Biology and Evolution vol 27no 9 pp 2078ndash2094 2010

[143] J-Y Sire S C Delgado and M Girondot ldquoHenrsquos teeth withenamel cap from dream to impossibilityrdquo BMC EvolutionaryBiology vol 8 no 1 article 246 2008

[144] R W Meredith J Gatesy W J Murphy O A Ryder and M SSpringer ldquoMolecular decay of the tooth gene enamelin (ENAM)mirrors the loss of enamel in the fossil record of placentalmammalsrdquo PLoS Genetics vol 5 no 9 Article ID e10006342009

[145] P S Hart T C Hart J P Simmer and J T Wright ldquoAnomenclature for X-linked amelogenesis imperfectardquo Archivesof Oral Biology vol 47 no 4 pp 255ndash260 2002

[146] T J Wright P S Hart M J Aldred et al ldquoRelationship ofphenotype and genotype in X-linked amelogenesis imperfectardquoConnective Tissue Research vol 44 no 1 pp 72ndash78 2003

[147] J-W Kim J P Simmer Y Y Hu et al ldquoAmelogenin pM1T andpW4S mutations underlying hypoplastic X-linked amelogen-esis imperfectardquo Journal of Dental Research vol 83 no 5 pp378ndash383 2004

[148] Y Yamakoshi ldquoPorcine amelogenin alternative splicing pro-teolytic processing protein-protein interactions and possiblefunctionsrdquo Journal of Oral Biosciences vol 53 no 3 pp 275ndash283 2011

[149] EC Salido PHYenKKoprivnikar L-C Yu andL J ShapiroldquoThe human enamel protein gene amelogenin is expressed fromboth the X and the Y chromosomesrdquo American Journal ofHuman Genetics vol 50 no 2 pp 303ndash316 1992

[150] A G Fincham and J Moradian-Oldak ldquoRecent advances inamelogenin biochemistryrdquo Connective Tissue Research vol 32no 1ndash4 pp 119ndash124 1995

[151] T Takagi M Suzuki T Baba K Minegishi and S SasakildquoComplete amino acid sequence of amelogenin in developingbovine enamelrdquo Biochemical and Biophysical Research Commu-nications vol 121 no 2 pp 592ndash597 1984

[152] C W Gibson E Golub W Ding et al ldquoIdentification ofthe leucine-rich amelogenin peptide (LRAP) as the translationproduct of an alternatively spliced transcriptrdquo Biochemical andBiophysical Research Communications vol 174 no 3 pp 1306ndash1312 1991

[153] E C Lau J P Simmer P Bringas Jr et al ldquoAlternative splicingof the mouse amelogenin primary RNA transcript contributesto amelogenin heterogeneityrdquo Biochemical and BiophysicalResearch Communications vol 188 no 3 pp 1253ndash1260 1992

[154] Y Li Z-A Yuan M A Aragon A B Kulkarni and C WGibson ldquoComparison of body weight and gene expression inamelogenin null and wild-type micerdquo European Journal of OralSciences vol 114 supplement 1 pp 190ndash193 2006

[155] J D Bartlett R L Ball T Kawai C E TyeM Tsuchiya and J PSimmer ldquoOrigin splicing and expression of rodent amelogeninexon 8rdquo Journal of Dental Research vol 85 no 10 pp 894ndash8992006

[156] O BabaN Takahashi T TerashimaW Li P KDenBesten andY Takano ldquoExpression of alternatively spliced RNA transcriptsof amelogenin gene exons 8 and 9 and its end products in therat incisorrdquo Journal of Histochemistry andCytochemistry vol 50no 9 pp 1229ndash1236 2002

[157] C W Gibson Y Li C Suggs et al ldquoRescue of the murineamelogenin null phenotype with two amelogenin transgenesrdquoEuropean Journal of Oral Sciences vol 119 no 1 pp 70ndash74 2011

[158] J T Wright Y Li C Suggs M A Kuehl A B Kulkarni and CW Gibson ldquoThe role of amelogenin during enamel-crystallitegrowth and organization in vivordquo European Journal of OralSciences vol 119 supplement 1 pp 65ndash69 2011

[159] J C Hu H C Chan S G Simmer F Seymen A S Richardsonet al ldquoAmelogenesis imperfecta in two families with definedAMELX deletions in ARHGAP6rdquo PLoS One vol 7 Article IDe52052 2012

[160] B Backman and G Holmgren ldquoAmelogenesis imperfecta agenetic studyrdquoHumanHeredity vol 38 no 4 pp 189ndash206 1988

[161] M D Berkman and A Singer ldquoDemonstration of the lyonhypothesis in X-linked dominant hypoplastic amelogenesisimperfectardquo Birth Defects Original Article Series vol 7 no 7 pp204ndash209 1971

[162] W Lattanzi M C di Giacomo G M Lenato et al ldquoA largeinterstitial deletion encompassing the amelogenin gene on theshort arm of the Y chromosomerdquo Human Genetics vol 116 no5 pp 395ndash401 2005

[163] A Akane ldquoSex determination by PCR analysis of the X-Yamelogenin generdquo Methods in Molecular Biology vol 98 pp245ndash249 1998

[164] M A Jobling I C C Lo D J Turner et al ldquoStructuralvariation on the short arm of the human Y chromosomerecurrent multigene deletions encompassing Amelogenin YrdquoHuman Molecular Genetics vol 16 no 3 pp 307ndash316 2007

[165] P H Krebsbach S K Lee Y Matsuki C A Kozak K MYamada and Y Yamada ldquoFull-length sequence localizationand chromosomal mapping of ameloblastin a novel tooth-specific generdquo Journal of Biological Chemistry vol 271 no 8 pp4431ndash4435 1996

[166] C K Mardh B Backman D Simmons et al ldquoHumanameloblastin gene genomic organization andmutation analysis

ISRN Dentistry 23

in amelogenesis imperfecta patientsrdquo European Journal of OralSciences vol 109 no 1 pp 8ndash13 2001

[167] M Fukae and T Tanabe ldquoNonamelogenin components ofporcine enamel in the protein fraction free from the enamelcrystalsrdquo Calcified Tissue International vol 40 no 5 pp 286ndash293 1987

[168] C Murakami N Dohi M Fukae et al ldquoImmunochemical andimmunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine toothgermrdquo Histochemistry and Cell Biology vol 107 no 6 pp 485ndash494 1997

[169] T Uchida C Murakami N Dohi K Wakida T Satoda andO Takahashi ldquoSynthesis secretion degradation and fate ofameloblastin during the matrix formation stage of the ratincisor as shown by immunocytochemistry and immunochem-istry using region-specific antibodiesrdquo Journal of Histochemistryand Cytochemistry vol 45 no 10 pp 1329ndash1340 1997

[170] R Cerny I Slaby LHammarstrom andTWurtz ldquoAnovel geneexpressed in rat ameloblasts codes for proteins with cell bindingdomainsrdquo Journal of Bone andMineral Research vol 11 no 7 pp883ndash891 1996

[171] C-C Hu M Fukae T Uchida et al ldquoSheathlin cloningcDNApolypeptide sequences and immunolocalization ofporcine enamel sheath proteinsrdquo Journal of Dental Research vol76 no 2 pp 648ndash657 1997

[172] K Kobayashi Y Yamakoshi J C-C Hu et al ldquoSplicingdetermines the glycosylation state of ameloblastinrdquo Journal ofDental Research vol 86 no 10 pp 962ndash967 2007

[173] Y Yamakoshi T Tanabe S Oida C-C Hu J P Simmerand M Fukae ldquoCalcium binding of enamel proteins and theirderivatives with emphasis on the calcium-binding domain ofporcine sheathlinrdquo Archives of Oral Biology vol 46 no 11 pp1005ndash1014 2001

[174] A M Brunati O Marin A Bisinella A Salviati and L APinna ldquoNovel consensus sequence for the Golgi apparatuscasein kinase revealed using proline-rich protein-1 (PRP1)-derived peptide substratesrdquo Biochemical Journal vol 351 part3 pp 765ndash768 2000

[175] M Fukae M Kanazashi T Nagano T Tanabe S Oida andK Gomi ldquoPorcine sheath proteins show periodontal ligamentregeneration activityrdquoEuropean Journal ofOral Sciences vol 114supplement 1 pp 212ndash218 2006

[176] J Vymetal I Slaby A Spahr J Vondrasek and S P Lyn-gstadaas ldquoBioinformatic analysis and molecular modellingof human ameloblastin suggest a two-domain intrinsicallyunstructured calcium-binding proteinrdquo European Journal ofOral Sciences vol 116 no 2 pp 124ndash134 2008

[177] J C-C Hu R Lertlam A S Richardson C E Smith M DMcKee and J P Simmer ldquoCell proliferation and apoptosis inenamelin null micerdquo European Journal of Oral Sciences vol 119supplement 1 pp 329ndash337 2011

[178] NAl-Hashimi J-Y Sire and SDelgado ldquoEvolutionary analysisof mammalian enamelin the largest enamel protein supportsa crucial role for the 32-kDa peptide and reveals selectiveadaptation in rodents and primatesrdquo Journal of MolecularEvolution vol 69 no 6 pp 635ndash656 2009

[179] C-C HuM Fukae T Uchida et al ldquoCloning and characteriza-tion of porcine enamelin mRNAsrdquo Journal of Dental Researchvol 76 no 11 pp 1720ndash1729 1997

[180] M Fukae T Tanabe C Murakami N Dohi T Uchida and MShimizu ldquoPrimary structure of the porcine 89-kDa enamelinrdquoAdvances in Dental Research vol 10 no 2 pp 111ndash118 1996

[181] H Seedorf M Klaften F Eke H Fuchs U Seedorf and MHrabe DeAngelis ldquoAmutation in the enamelin gene in amousemodelrdquo Journal of Dental Research vol 86 no 8 pp 764ndash7682007

[182] H Masuya K Shimizu H Sezutsu et al ldquoEnamelin (Enam)is essential for amelogenesis ENU-induced mouse mutants asmodels for different clinical subtypes of human amelogenesisimperfecta (AI)rdquo Human Molecular Genetics vol 14 no 5 pp575ndash583 2005

[183] C E Smith R Wazen Y Hu et al ldquoConsequences for enameldevelopment andmineralization resulting from loss of functionof ameloblastin or enamelinrdquo European Journal of Oral Sciencesvol 117 no 5 pp 485ndash497 2009

[184] M H Rajpar K Harley C Laing R M Davies and M JDixon ldquoMutation of the gene encoding the enamel-specificprotein enamelin causes autosomal-dominant amelogenesisimperfectardquoHumanMolecular Genetics vol 10 no 16 pp 1673ndash1677 2001

[185] H-Y Kang F Seymen S-K Lee et al ldquoCandidate gene strategyreveals ENAM mutationsrdquo Journal of Dental Research vol 88no 3 pp 266ndash269 2009

[186] T C Hart P S HartM C Gorry et al ldquoNovel ENAMmutationresponsible for autosomal recessive amelogenesis imperfectaand localised enamel defectsrdquo Journal of Medical Genetics vol40 no 12 pp 900ndash906 2003

[187] C K Mardh B Backman G Holmgren J C-C Hu J PSimmer and K Forsman-Semb ldquoA nonsense mutation in theenamelin gene causes local hypoplastic autosomal dominantamelogenesis imperfecta (AIH2)rdquo Human Molecular Geneticsvol 11 no 9 pp 1069ndash1074 2002

[188] H-C Chan L Mai A Oikonomopoulou et al ldquoAltered enam-elin phosphorylation site causes amelogenesis imperfectardquo Jour-nal of Dental Research vol 89 no 7 pp 695ndash699 2010

[189] S G Simmer N M Estrella R N Milkovich and J C HuldquoAutosomal dominant amelogenesis imperfecta associated withENAM frameshift mutation pAsn36Ilefs56rdquo Clinical Geneticsvol 83 no 2 pp 195ndash197 2013

[190] J-W Kim S-K Lee Z H Lee et al ldquoFAM83H mutations infamilies with autosomal-dominant hypocalcified AmelogenesisImperfectardquo American Journal of Human Genetics vol 82 no2 pp 489ndash494 2008

[191] J A Poulter W El-Sayed R C Shore J Kirkham C FInglehearn et al ldquoWhole-exome sequencing without priorlinkage identifies a mutation in LAMB3 as a cause of domi-nant hypoplastic amelogenesis imperfectardquo European Journal ofHuman Genetics 2013

[192] W El-Sayed D A Parry R C Shore et al ldquoMutations in thebeta propeller WDR72 cause autosomal-recessive hypomatu-ration amelogenesis imperfectardquo American Journal of HumanGenetics vol 85 no 5 pp 699ndash705 2009

[193] D A Parry S J Brookes C V Logan J A Poulter W El-Sayed et al ldquoMutations in C4orf26 encoding a peptide within vitro hydroxyapatite crystal nucleation and growth activitycause amelogenesis imperfectardquo American Journal of HumanGenetics vol 91 no 3 pp 565ndash571 2012

[194] D A Parry J A Poulter C V Logan S J Brookes H Jafriet al ldquoIdentification of mutations in SLC24A4 encoding apotassium-dependent sodiumcalcium exchanger as a cause ofamelogenesis imperfectardquoAmerican Journal ofHumanGeneticsvol 92 no 2 pp 307ndash312 2013

[195] P Bodier-Houlle P Steuer J M Meyer L Bigeard and F J GCuisinier ldquoHigh-resolution electron-microscopic study of the

24 ISRN Dentistry

relationship between human enamel and dentin crystals at thedentinoenamel junctionrdquo Cell and Tissue Research vol 301 no3 pp 389ndash395 2000

[196] E Beniash R A Metzler R S K Lam and P U P A GilbertldquoTransient amorphous calcium phosphate in forming enamelrdquoJournal of Structural Biology vol 166 no 2 pp 133ndash143 2009

[197] V Gallon L Chen X Yang and J Moradian-Oldak ldquoLocaliza-tion and quantitative co-localization of enamelin with amelo-geninrdquo Journal of Structural Biology 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oral OncologyJournal of

DentistryInternational Journal of



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Biomaterials


BioMed Research International


Case Reports in Dentistry


Oral ImplantsJournal of


Anesthesiology Research and Practice


Radiology Research and Practice

Environmental and Public Health

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


Dental SurgeryJournal of

Drug DeliveryJournal of



Oral DiseasesJournal of


Computational and Mathematical Methods in Medicine

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PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Preventive MedicineAdvances in


EndocrinologyInternational Journal of



OrthopedicsAdvances in

2 ISRN Dentistry

deterioration and have given us important anthropologicalclues as to how humans evolved Although DNA analysishas taught us much about the migration patterns of ourancient ancestors teeth have perhaps been more importantin identifying our ancestorrsquos food preferences and lifestylesTheir shape wear patterns and carbon isotope compositionsare unique indicators of our past behaviors [8]

2 Overview of Enamel Development

This review focuses on enamel proteinases their substratesand on recent discoveries that help us to better understandthe process of enamel development For outstanding reviewarticles about enamel development prior to 1999 pleasesee the following reviews [9ndash11] The review by Simmerand Fincham [9] provides an excellent historical perspectiveabout enamel development the review by Smith [11] providesa detailed examination on how ions are controlled duringenamel formation and the review byBartlett and Simmer [10]focuses on what was known about the enamel proteins andproteinases prior to 1999

Enamel development (amelogenesis) can be broken downinto four defined stages presecretory secretory transitionand maturation The stages are defined by the morphologyand function of the ameloblasts (Figure 1) The ameloblastsare a single cell layer that covers the developing enamel andis responsible for enamel composition Ameloblasts are partof the enamel organ that is composed of an outer epitheliallayer the stellate reticulum the stratum intermedium andthe inner enamel epithelium (ameloblast layer) The basal(proximal) end of the preameloblast is attached by desmo-somes to the stratum intermedium and the apical (distal) endis attached by hemidesmosomes to a basement membrane(basal lamina) located at the future site of the DEJ

21 Organization of the Enamel Organ The outer enamelepithelium is a single layer of cells that covers the enamelorgan and is contiguous at the cervical loop with theameloblasts (inner enamel epithelium) located initially atthe future DEJ The cells of the stellate reticulum are sand-wiched between the outer dental epithelium and stratumintermedium and secrete hydrophilic glycosaminoglycansinto the extracellular compartment This causes water todiffuse into the enamel organ which in turn forces these cellsapart Since these cells are all interconnected by desmosomesthey are stretched into a star shape and are therefore termedthe stellate reticulum [4] The stratum intermedium formsa boundary between the stellate reticulum and the innerenamel epithelium and may be important for shuttling ionsto and from the ameloblasts [19]The ameloblasts are respon-sible for secreting enamel matrix proteins and proteinasesinducing mineral ribbons to form and organizing them intorod and interrod patterns typical for each vertebrate species

22 Presecretory Stage One of the earliest events occurringduring the presecretory stage just prior tomineral formationis the deposition of predentin by odontoblasts at the futureDEJ [20] This occurs first at the cusp tips and progresses

to the cervical regions of a tooth Predentin is composedprimarily of collagen but also contains noncollagenous pro-teins Predentin is the first to mineralize [21] starting slightlyunder what will become the DEJThe dentin becomes thickeras the wave of mineralization moves latterly and away fromthe DEJ towards the future pulp chamber This decreases thesize of the chamber as mineralization proceeds Howeveralmost immediately after initial dentin mineralization nearthe DEJ differentiating preameloblasts extend cytoplasmicprojections through the basement membrane that removeand destroy it and then the ameloblasts begin secretingenamel matrix proteins which rapidly initiate mineralization[4]

23 Secretory Stage Thepreameloblasts transform into secre-tory stage ameloblasts by elongating into tall columnar cellsand by forming Tomesrsquo processes at their apical ends nearestthe forming enamelThe Tomesrsquo process is a conical structurethat points toward the forming enamelmatrix Enamelmatrixproteins are primarily secreted from one side of the Tomesrsquoprocess (secretory face) and all ameloblasts within a rowsecrete protein from the same side of their Tomesrsquo processesThe first enamel crystals (ribbons) formed grow betweenthe dentin crystals perhaps by mineralizing around dentinproteins such as collagen These crystal ribbons elongate atthe mineralization front where enamel proteins are secreted[22] Secretory stage enamel is protein rich and has a softcheese-like consistency

The ameloblasts start secreting large amounts of enamelmatrix proteins as they move away from the dentin surfaceso that the nascent enamel layer can thicken In associationwith newly secreted proteins long thin mineral ribbons formrapidly normal to the secretory surface of the ameloblastsThe parallel crystallite ribbons approximately 10000 to40000 [23] will eventually form into a rod (prism) and eachameloblast is responsible for creating one enamel prism allof which collectively form a highly ordered 3D structureSoon after the initial formation of crystallite ribbons theameloblasts develop their apical Tomesrsquo processesThis estab-lishes a two-compartmental system where proteins destinedto form interrod enamel tend to exit near the ldquobaserdquo of theprocess whereas those involved in rod formation tend toexit from the ldquotiprdquo (secretory face) of the process Duringthis time protein cleavage products are either reabsorbedby the ameloblasts or may accumulate between the rod andinterrod enamel Mineral crystallites forming within the rodwill growprogressively in 119888-axis length parallel to one anotheras ameloblasts move away from the dentin surface Mineralcrystallites developing between the rods (interrod) may havemore limited lengths but they are always positioned spatiallyto be at angles relative to rod crystallites [4]

During the secretory stage ameloblasts not only moveaway from the dentin as the enamel thickens but they alsomove in groups that slide by one another and this movementculminates in the characteristic decussating enamel prismpattern observed in rodent incisors [24] or the entwinedgnarled prism pattern seen in human molars [25] As thisoccurs ameloblasts secrete four different proteins into the

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Documents

Review Article Dental Enamel Development: Proteinases and ...downloads.hindawi.com/archive/2013/684607.pdf · Review Article Dental Enamel Development: Proteinases and Their Enamel