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Home , Ameloblast, Amelogenesis, Amelogenin

Dr. Gábor Varga

Department of Oral Biology February, 2016 Amelogenesis - introduction

• Amelogenesis as a part of formation

• Secretory phase of amelogenesis

• Maturation phase of amelogenesis

• Proteins involved in amelogenesis Molar Pulp Horn longitudinal section

the enamel covers the Tooth development

LAMINA BUD STAGE CAP STAGE BELL STAGE ERUPTION activation during tooth development

Epithelium

Mesenchyme Tooth development – details 1 Tooth development – details 2 Section of tooth – enamel and dentin formation Formal and structural changes of during enamel formation

1 2 3 4 5 6 7 1. morphogenetic, 2. inductive, 3. early secretory, 4 secretory, 5. maturation - ruffle-ended, 6. maturation – smooth-ended, 7. protective

Amelogenesis

 1st - Secretory phase

. Secretion of proteins . Foundation of the mineral structure

 2nd – Maturation phase

. Reabsorption of proteins and water removal . Secretion of mineral ions The initiation of enamel formation on the surface of the already formed, unstructured mantle-dentin zománc=enamel . The arrangement of ameloblasts during enamel formation

Outer enamel

Stellate reticulum

Stratum intermedium

Ameloblasts

Enamel matrix Fully differentiated secretory ameloblasts Secretory ameloblasts and surrounding cells Ultrastucture of secretory ameloblasts Tomes process sorrounded by freshly produced enamel

SG – secretory granule, PZ – prismatic (rod) enamel, IPZ – interprismatic (interrod) enamel Enamel structure Proximal Secretory ameloblasts – formation of prismatic enamel N (PE) and interprismatic enamel

GA (IPE) N: Nucleus SG GA: Golgo apparatus SG: Secretory granule TP: Tomes process Sh: Sheath region PE: Prismatic enamel IPE: Interprismatic enamel TP

TP

IPE

PE

Distal

PE PE Sh Sh IPE Three dimensional arrangement of crystal rods (prismatic enamel) in the vincinity of Tomes processes Parallel running crystallites (Kr) in the early phase of enamel development Amelogenesis

• 1st - Secretory phase

• 2nd – Maturation phase 2.a. reabsorption of proteins and water removal 2.b. secretion of mineral ions

Papillary layer (PL) cells between the capillaries and the maturation ameloblasts (MA). Multiple Transitions

Basement membrane

Enamel

Dentin Maturation phenotypes: ruffle-ended and smooth-ended maturation ameloblasts cycle back and forth during the maturation phase

G Za & Zo M

M E

Ruffle-ended and smooth-ended maturation ameloblasts cycle back and forth during the maturation phase. Cycling of the two phenotypes involves extensive remodeling of the distal cytoplasm and junctional complexes at both ends of the cells. The Golgi complexes (G) and the lysosomal (L) apparatus are well developed in both cell configurations. Zonula adherens (Za) and zonula occludens (Zo) shift from distal position in the ruffle-ended ameloblasts to a proximal position in the smooth-ended ameloblasts. Mitochondria (M) are located primarily in the distal cytoplasm. Endosomes (E) containing enamel matrix are present both in the ruffle-ended and smooth-ended ameloblasts. The ruffle ended surface primarily supports electrolyte exchange while the smooth ended form is for cell recovery and protein absorption. The two types of ameloblasts during the absorptive phase

Mineral secretion Protein and water absorption Ultrastructure of ruffle-ended maturation ameloblasts Ameloblast transporters

H+ ~ H+-ATPase Tight junction CO2 CO2 CO + H O (?)PMCA Ca2+ 2 2 + CA − H 2HCO3 PAT-1 NHE1 Na+ Cl− Na+ NBCe1 − CaCC 2HCO3 (?) − - - HCO3 Cl (HCO3 ) AE2 − CFTR Cl + + K TASK2 (?) Na + (?) NKCC1 K+ Na 2Cl− H+ NHE3 (?) + Na-K- 3Na + + ~ Na ATPase 2K 2+ + NCKX4 + Ca / K (?) Maxi-K K Na+ Ca2+ NCX

Na+ Basolateral Apical membrane membrane

2+ 2- + 10 Ca + 6 HPO4 + 2 H2O ↔ Ca10(PO4)6(OH)2+ 8 H Ameloblast calcium transport elements

H+ ~ Tight junction CO2 CO2

CO2 + H2O PMCA Ca2+ + CA − H 2HCO3 Na+ Cl− Na+ − 2HCO3 (?) − - - HCO3 Cl (HCO3 ) − Cl + + K Na + K+ Na 2Cl− H+ + 3Na + + ~ Na 2K 2+ + NCKX4 + Ca / K K Na+ Ca2+ NCX

Na+ Basolateral Apical membrane membrane

2+ 2- + 10 Ca + 6 HPO4 + 2 H2O ↔ Ca10(PO4)6(OH)2+ 8 H Ameloblast bicarbonate transport elements

H+ ~ H+-ATPase Tight junction CO2 CO2 CO + H O Ca2+ 2 2 + CA − H 2HCO3 PAT-1 NHE1 Na+ Cl− Na+ NBCe1 − CaCC 2HCO3 (?) − - - HCO3 Cl (HCO3 ) AE2 − CFTR Cl + + K TASK2 (?) Na + (?) NKCC1 K+ Na 2Cl− H+ NHE3 (?) + Na-K- 3Na + + ~ Na ATPase 2K 2+ + + Ca / K (?) Maxi-K K Na+ Ca2+

Na+ Basolateral Apical membrane membrane

2+ 2- + 10 Ca + 6 HPO4 + 2 H2O ↔ Ca10(PO4)6(OH)2+ 8 H Ameloblast bicarbonate transport elements Hypothetic model for pH regulation by ruffle ended ameloblasts to neutralize liberated H+

+ - + Na 2 HCO3 + 2 H 2 H2O + 2 CO2

Car 9 Nbce Bicarbonate is generated by carbonic anhydrase 2 Basolateral H2O + CO2 (Car2) and is exchanged by the pendrin (PD) anion Car 2 exchanger in the apical membrane for Cl-. Cl- is + - H HCO3 Nhe1 imported basolaterally by Ae2 and conducted into Na+ the enamel by CFTR. - HCO3 Ae2 Cl- Car 2 = carbonic anhydrase 2 (cytosolic), Car 9 = carbonic anhydrase 9 (membrane-bound), Apical Cftr = cystic fibrosis transmembrane conductance regulator - Cl Ae2 = anion exchanger 2, Cftr PD Nbce1 = sodium bicarbonate exchanger 1, Nhe1 = sodium hydrogen exchanger 1, - - HCO - P D= pendrin: HCO /Cl exchanger, Cl- 3 3 CO2 █ = tight junction H+ Apatite formation H2O Proposed pathway of enamel protein reabsorption and digestion by ruffle-ended ameloblasts A

1 2 3 4 5 6 7

B

G Tj M

M End End pH cycling in rodent incisior ameloblasts pH cycling in rodent incisior ameloblasts

Damkier at al. Bone, 60, 2014, 227 - 234 Hypothesis on the dynamics of phosphate equilibrium in solution and enamel crystal (A) and the effect of ameloblasts on phosphate dynamics in the RA phase (B) and SA phase (C).

Damkier at al. Bone, 60, 2014, 227 - 234 Rod enamel (prismatic enamel, PZ) in cross section electron microscopy picture Cross sectional scanning electron microscopy picture following acidic treatment Longitudinal sectional electron scanning microscopy of the enamel – rods are well visible Amelogenesis - enamel proteins

• • • • Amelotin (Ben Ganss, Toronto) • • Osterix (Ben Ganss, Toronto) • Proteinases (enamelysin - MMP-20 kallikrein 4 – KLK4) • Phosphatases

Proteins with know function are in bold

Amelogenin Concept of the role of in the mineralization of enamel The hydrophobic amelogenesis form globular aggregates (nanospheres) on secretion into the extracellular space. The nanospheres form lattices that regulate the spacing and the orientation of the C-axis of the newly forming enamel crystallites

Amelogenin secretion Crystals 1 grows in thickness

Assembly Hydrophylic 2 Platelike crystallites anionic terminals resorption 5 exposed of hydroxyapatite Proteinase-2 degrades the nanospheres

Nanospheres act 4 Nanospheres as spacers 3 Proteinase- 1 hydrophobic between (enamelysin) crystallites removes hydrophylic tails Disorder scores of amino acid sequences of proteins participating in Disorder frequency of amino acid chains of proteins participating in various biological functions Distribution of amelogenin and ameloblastin in enamel matrix Defect of amelogenesis in ameloblastin- null mice Role of ameloblastin in the regulation of ameloblast function

Ameloblast

Amelogenin Msx2 Amelogenin p75

Trks

? p21, p27 Enamel crystal Regulation of cell cycle

Receptor?

Ameloblastin in the enamel matrix Amelogenesis imperfecta The human amelogenin gene Amelogenin mutation leading to hypoplasia - loss of three amino acid and substitution of another one Structure of the X-chromosomal copy of the human amelogenin gene

Hypoplastic Hypomineralization Amelogenesis imperfecta Amelogenesis imperfecta (X-linked) (X-linked)

Genomic 1 2 3 4 5 6 7 1 2 3 4 5 6 7 sequence 56 66 48 42 45 435 160 56 66 48 42 45 435 160 9 bp deletion 5 kb deletion

1 2 3 4 5 6 7 1 2 3 4 5 6 7 mRNA

Predicted protein

Phenotype Thin enamel Poorly mineralized enamel The bar segments represent the introns and the boxes (1 through 7) correspond to the exons. The nucleotide numbers are indicated below the exons. (Adapted from Simmer et al.) Two mutations of the amelogenin gene that cause amelogenesis imperfecta Amelogenin (AMELX) mutations causing X-linked amelogenesis imperfecta How are changes in the AMELX gene related amelogenesis imperfecta? • One copy of the amelogenin gene is located on each of the sex chromosomes (the X and Y chromosomes). The AMELX gene, which is located on the , makes almost all of the body's amelogenin. The copy of the amelogenin gene on the , AMELY, makes very little amelogenin and is not needed for enamel formation. • At least 15 mutations in the AMELX gene have been identified in people with X- linked forms of amelogenesis imperfecta. (X-linked disorders are caused by mutations in on the X chromosome.) Some AMELX mutations lead to the production of an abnormal version of the amelogenin protein that can interfere with the formation and organization of enamel crystals. Other AMELX mutations prevent one copy of the gene from producing any amelogenin protein at all. Enamel cannot form properly without an adequate amount of amelogenin • Males have a single copy of the X chromosome in each cell. Males who inherit a defective copy of the AMELX gene have very little amelogenin and develop almost no enamel to cover and protect their teeth. Females have two copies of the X chromosome in each cell. Females who inherit one altered copy of the AMELX gene are less severely affected because they have a normal copy of the gene on the other X chromosome to produce amelogenin. Their may have structural defects such as a distinctive pattern of vertical grooves. No symptoms other than abnormal enamel development have been reported in people with AMELX mutations.

Enamelin mutations causing autosomal dominant amelogenesis imperfecta Enamelysin (MMP20) and kallikrein 4 (KLK4) mutations causing autosomal recessive amelogenesis imperfecta Proteins of enamel involved in Amelogenesis Imperfecta

Amelogenin: (product of AMELX and AMELY genes located on the X and Y chromosomes) is the most abundant protein in developing enamel [26, 27]. While its exact role in enamel formation is not fully understood, it is thought to be crucial for regulating the size and shape of the mineralizing enamel crystallites. Multiple human mutations in the AMELX gene are associated with different AI types. There are no known AMELY mutations. A transgenic mouse lacking expression of this gene has only a very thin covering of enamel that lacks a prismatic structure [28].

Ameloblastin: (product of AMBS gene located on chromosome 4) is another enamel associated protein that appears to be the second most abundant enamel matrix protein [29]. The function of this protein is mónot completely known but it may regulated ameloblast differentiation and formation. It is considered a likely candidate for being associated with some AI types.

Enamelin: (product of ENAM gene located on chromosome 4) is secreted by amelobasts in relatively low amounts. It is speculated that this protein could interact with amelogenin or other enamel matrix proteins and be important in determining growth of the length of enamel crystallites. Three different mutations ENAM gene mutations are associated with different AI types.

Enamelysin: (MMP20 gene located on chromosome 11) is a proteinase that cleaves amelogenin and is thought to be the major proteinase involved in processing the enamel matrix proteins [32, 33]. The enamelysin knockout mouse has a reduced enamel thickness and the enamel lacks a prismatic structure.

Kalikrein 4: (KLK4 gene located on chromosome 19) is a proteinase that is secreted predominantly during the maturation stage of enamel development [34]. This aggressive proteinase could be responsible for processing any proteins not cleaved by enamelysin. Period of amelogenesis in the permanent teeth of human .

Bars: from beginning to completion. Amelogenesis - summary

• Amelogenesis as a part of tooth formation

• Secretory phase of amelogenesis

• Maturation phase of amelogenesis

• Proteins involved in amelogenesis