Amelogenesis and MIH – the role of MMP20 and KLK4

Abstract

Enamel formation is a highly coordinated process. The enamel protein matrix, secreted by the ameloblasts, contains three main proteins: amelogenin, enamelin and ameloblastin. These proteins are specific to enamel and are degrade by specific proteases, MMP20 and KLK4. Human mutations in genes coding for the enamel proteinases cause variable degrees of hypomineralization. In MIH permanent molars the mineral content is very low and the protein content may reach 30-40% in volume. We should look at the function of MMP20 and KLK4 during the first two years of life in order to understand the findings in MIH.

Amelogenesis

Dental enamel is the hardest tissue in the human body, and although it starts as a tissue rich in proteins, by the time of eruption of the tooth in the oral cavity only less than 1% of its weight is proteins. It is composed of 96% carbonated apatite, 3% water and 1% proteins. Although the organic matter is a minor component of mature enamel it plays a very important role in the mechanical toughening of this tissue [1]. Enamel development (amelogenesis) can be described by four defined stages: presecretory, secretory, transition and maturation. The stages are defined by the morphology of the ameloblasts. The ameloblasts are a single cell layer that covers the developing enamel and is responsible for enamel composition. The ameloblasts are responsible for secreting enamel matrix proteins and proteinases, inducing mineral ribbons to form, and organizing them into rod and interred pattern. Presecretory stage: Prior to mineral formation, deposition of predentin by odontoblasts at the future DEJ occurs [2]. Predentin is composed primarily of collagen but also contain noncollagenous proteins. Predentin is the first to mineralize [3]. After initial dentin mineralization, differentiating preameloblasts extend cytoplasmatic projections through the basement membrane that remove it, and then the ameloblasts begin secreting enamel matrix proteins which rapidly initiate mineralization [4]. Secretory stage: The preameloblasts transform into secretory stage ameloblasts. Enamel matrix proteins are secreted, and the first enamel crystals formed grow between the dentin crystals. Secretory stage enamel is protein rich and has a soft cheese-like consistency. The ameloblasts secrete large amounts of enamel matrix proteins as they move away from the dentin surface. In association with newly secreted proteins, long thin mineral ribbons form rapidly. The parallel crystallite ribbons, approximately 10-40,000 [5] will eventually form into a rod (prism), and each ameloblasts is responsible for creating one enamel prism. Mineral crystallites developing between the rods (interrod) may have more limited lengths, but they are always positioned spatially to be at angles relative to rod crystallites [4]. During the secretory stage ameloblasts secrete three structural proteins (amelogenin, ameloblastin and enamelin) and one proteinase (matrix metalloproteinase-20, MMP20, Enamelysin). Amelogenin comprises approximately 80-90% of the organic matter, and ameloblastin and enamelin comprise 5% and 3-5% respectively [6]. MMP20 is present in trace amounts. Enamel crystals will form in the absence of amelogenin but will not form if either ameloblastin or enamelin is absent. By the end of the secretory stage the enamel layer has achieved its full thickness and 30% minerals. Transition stage:  The ameloblasts smooth off the enamel surface with a final coating of aprismatic enamel. Maturation stage: The ameloblasts actively secrete kallikrein- related peptidase-4 (KLK-4) to help remove the mass of previously secreted and partially hydrolyzed matrix proteins, so that the rod and interrod crystallites can expand in volume. The initial enamel ribbons are only a few apatitic [Ca₁₀ (PO₄)₆(OH)₂] unit cells in thickness and a length that nay extend through the entire thickness of the enamel layer [7]. After the enamel rods have formed, an area exists between the rod and interod enamel that contains a thin organic matrix with no crystals, the rod sheath [8] (Figs 1-2).

MMP20 and KLK4

MMP20 (Enamelysin) has been shown to be expressed by secretory stage ameloblasts. It is the only proteinase present in the enamel matrix during the secretory stage [9,10].

The main function of Mmp20 is to cleave the most abundant enamel matrix protein, amelogenin [10]. It is also likely responsible for generating an enamelin cleavage product. MMP20 will also cleave the KLK4 propeptide to produce catalytically active KLK4 [11]. Human MMP20 is expressed from a gene on chromosome 11q22-q23. Seven different human MMP20 mutations are known to cause autosomal recessive hypomaturation or hypoplastic-hypomaturation Amelogenesis Imperfecta (AI). The teeth are normal in size but the enamel layer does not contrast well with dentin on radiographs and the enamel tends to chip away [12,13]. The lack of functional MMP20 in mammalian species without enamel (baleen whales), demonstrate that MMP20 is essential for enamel formation but is not essential for any other biological function. KLK4 is the fourth member of a cluster of 15 serine protease genes that comprise the human kallikrein locus near the telomere on the long arm of chromosome 19. KLK4 is expressed during the early maturation stage when the enamel proteins are reabsorbed from the hardening enamel [14]. The only essential function of KLK4 is in enamel development. KLK4 is activated by MMP20 and by a cysteine aminopeptidase [15]. KLK4 is necessary to break up aggregates of accumulated enamel protein cleavage products in the deeper regions of the enamel layer. Two different KLK4 mutations in human are known to cause autosomal recessive hypomaturation AI. The enamel is normal in size but chipped from the dentine. KLK4 is essential for enamel to achieve its final hardening form its only function, like MMP20, is in the dental enamel development.

MIH

Molar Incisor Hypomineralization (MIH), a defect of hypomineralized enamel, affects one or more permanent first molars with or without permanent incisors involvement [16]. It occurs when the ameloblasts are affected in the maturation stage and the result is enamel with less than 96% mineralization. Clinically, it present asymmetric severity with demarcated opacities that vary in color shade from white to yellow/brownish, with sharp demarcation between the affected and sound enamel [17]. The main characteristic of teeth with MIH is porous enamel that can be easily damaged due to masticatory forces (Fig. 3). Children with MIH could present more intense dental sensitivity due to temperature variations, due to combination of the chronic pulpitis and innervation of the region right under the hypomineralized area [18]. There is no conclusive data in relation to the etiology of MIH. A genetic research of MIH affected populations in Turkey and Brazil showed that the ENAMrs 3796704 marker was associated with MIH manifestation [19].

The mineral contain of hypomineralized enamel in molar was highly reduced (calcium 46.3% and phosphate 36.5% in normal enamel in comparison to calcium 30.99% and phosphate 14.53% in affected molar) [20] (Fig 4 and Table 1). The protein content in MIH affected molars: compared to sound enamel, brown enamel showed a 15–21-fold higher protein content, and yellow and chalky enamel showed about 8-fold higher protein content [21]. Initial morphological studies with polarized light microscopy demonstrated areas of porosity of varying degrees.

Table 1: Mineral content (%) of MIH molar

Recent microstructural analysis by means of scanning electron microscopy (SEM) identified a less well organized prism structure with voids present, compared to sound enamel whose prism structure is well arranged [22]. Transmission electron microscope analysis revealed that the hypomineralized areas in enamel were associated with marked changes in microstructure: loosely packed apatite crystals within prisms and wider sheath regions. These microstructural changes appear to occur during enamel maturation [23].

Based on the published data on MIH teeth, like reduced mineral content, higher protein content (8-20 folds more) and wider sheath regions in the enamel, we can postulate that the main problem in MIH teeth is reduced protein degradation. After secretory stage of amelogenesis the mineral content is 30%, while after the maturation stage the mineral content reaches 85%. So, it looks that MIH occurs during the maturation stage, when KLK4 is the main proteinase that degrade the protein content of the enamel and allow apatite crystals to grow in volume.

The global prevalence of MIH was reported between 2.9-44% [24]. In Israel, the prevalence of MIH, in an Arab cohort, was significantly higher in boys age 6-10 years old than in girls of the same age, 19.8% Vs 16.6%, (P<0.05) [25]. Analysis of MMP20 and KLK4 was performed in a cohort of 500 children aged 0-5 years old in an Arab population in Israel, the same population that was analyzed for MIH prevalence [26]. The concentration of MMP20 in boys was significantly reduced (P<0.05) in comparison with girls and the results for KLK4 were similar. When we compared age groups, less than 2 years to 2-5 years, the concentration of KLK4 in the younger age group was significantly higher. The age of 2 years is significant since the MIH affects only two thirds of the crown of first permanent molars, part of the crown that develops and mineralizes during the first two years after birth. The significant differences in the concentration of MMP20 between boys and girls may be related to the differences in MIH prevalence between boys and girls. Reduced concentration of MMP20 in boys may explain the higher prevalence of MIH in boys in the Arab population in Israel.

The next step is to clinically examine the children that were analyzed for MMP20 and KLK4 concentration, 2-3 years after, in order to allow eruption of the molar teeth, and to try to correlate between the clinical findings of MIH and proteinases concentrations.

Conclusions:

• Amelogenesis begins by creating a protein matrix by the ameloblasts that mineralized as the ameloblasts move upward.
• The proteins in the enamel are specialized and can be detected only in the enamel during amelogenesis.
• The proteases that degrade the enamel proteins are MMP20 and KLK4.
• In MIH the mineral content of the enamel is reduced and the protein content is very high.
• It may be postulated that MIH is caused by malfunction of the enamel proteases.
• The research should focus on the genes that regulate the proteases.

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