Cynthia Suggs

Human and Mouse Enamel Phenotypes Resulting from Mutation or Altered Expression of AMEL, ENAM, MMP20 and KLK4

Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions.

The Amelogenin C-Terminus Is Required for Enamel Development

The abundant amelogenin proteins are responsible for generating proper enamel thickness and structure, and most amelogenins include a conserved hydrophilic C-terminus. To evaluate the importance of the C-terminus, we generated transgenic mice that express an amelogenin lacking the C-terminal 13 amino acids (CTRNC). MicroCT analysis of TgCTRNC29 teeth (low transgene number) indicated that molar enamel density was similar to that of wild-type mice, but TgCTRNC18 molar enamel (high transgene number) was deficient, indicating that extra transgene copies were associated with a more severe phenotype. When amelogenin-null (KO) and TgCTRNC transgenic mice were mated, density and volume of molar enamel from TgCTRNCKO offspring were not different from those of KO mice, indicating that neither TgCTRNC18 nor TgCTRNC29 rescued enamel’s physical characteristics. Because transgenic full-length amelogenin partially rescues both density and volume of KO molar enamel, it was concluded that the amelogenin C-terminus is essential for proper enamel density, volume, and organization.

Transgenic Mice that Express Normal and Mutated Amelogenins

Amelogenin proteins are secreted by ameloblasts within the enamel organ during tooth development. To better understand the function of the 180-amino-acid amelogenin (M180), and to test the hypothesis that a single proline-to-threonine (P70T) change would lead to an enamel defect similar to amelogenesis imperfecta (AI) in humans, we generated transgenic mice with expression of M180, or M180 with the proline-to-threonine (P70T) mutation, under control of the Amelx gene regulatory regions. M180 teeth had a relatively normal phenotype; however, P70T mineral was abnormally porous, with aprismatic regions similar to those in enamel of male amelogenesis imperfecta patients with an identical mutation. When Amelx null females were mated with P70T transgenic males, offspring developed structures similar to calcifying epithelial odontogenic tumors in humans. The phenotype argues for dominant-negative activity for the P70T amelogenin, and for the robust nature of the process of amelogenesis.

Calretinin Expression in the Differential Diagnosis of Human Ameloblastoma and Keratocystic Odontogenic Tumor

Ameloblastoma is a benign, locally aggressive epithelial odontogenic tumor that has the potential to become malignant and produce metastasis to distant sites such as lungs and kidneys. The histologic presentation can be, in some instances, mistaken for keratocystic odontogenic tumor (KCOT) (formerly known as odontogenic keratocyst). The expression of calretinin [calbindin2 (CALB2)] was investigated on both ameloblastoma and KCOT. Nineteen cases of ameloblastoma and 17 cases of KCOT were stained with calretinin antiserum 18-0211 (Zymed, San Francisco, CA). All cases (100%) of ameloblastoma showed positive calretinin staining, restricted to the neoplastic epithelial component and none (0%) of the 17 KCOTs showed positive calretinin staining. Gene expression profiling of ameloblastomas showed CALB2 expressed in the basal cell layer of columnar cells resembling preameloblasts, in all 5 of the ameloblastomas evaluated. Taken together, the results of this study strongly support calretinin as a useful immunohistochemical marker for ameloblastoma and malignant ameloblastoma and it can also be used in the differential diagnosis of KCOT.

Enamelin Is Critical for Ameloblast Integrity and Enamel Ultrastructure Formation

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam−/− mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam−/− mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam−/− background did not fully recover enamel formation while a medium expresser in the Enam+/− background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation.

Partial Rescue of the Amelogenin Null Dental Enamel Phenotype

The amelogenins are the most abundant secreted proteins in developing dental enamel. Enamel from amelogenin (Amelx) null mice is hypoplastic and disorganized, similar to that observed in X-linked forms of the human enamel defect amelogenesis imperfecta resulting from amelogenin gene mutations. Both transgenic strains that express the most abundant amelogenin (TgM180) have relatively normal enamel, but strains of mice that express a mutated amelogenin (TgP70T), which leads to amelogenesis imperfecta in humans, have heterogeneous enamel structures. When Amelx null (KO) mice were mated with transgenic mice that produce M180 (TgM180), the resultant TgM180KO offspring showed evidence of rescue in enamel thickness, mineral density, and volume in molar teeth. Rescue was not observed in the molars from the TgP70TKO mice. It was concluded that a single amelogenin protein was able to significantly rescue the KO phenotype and that one amino acid change abrogated this function during development.

Rescue of the murine amelogenin null phenotype with two amelogenin transgenes

The amelogenin proteins are required for normal enamel development, and the most abundant amelogenins expressed from alternatively spliced mRNAs are M180 and leucine-rich amelogenin protein (LRAP). The X-Chromosomal Amelogenin (Amelx) null [knockout (KO)] mouse has an enamel defect similar to human X-linked amelogenesis imperfecta. The disorganized enamel layer in KO mice is 10-20% of the thickness of wild-type (WT) enamel and lacks prismatic structures. When the KO mice were mated with mice that express the transgene M180-87, (TgM180-87) partial rescue of the phenotype was observed such that enamel thickness, volume, and density increased. A second transgene was introduced by mating TgM180 KO mice with TgLRAP mice, and male offspring were characterized for genotype and tooth phenotype was evaluated by scanning electron microscopy. The molar enamel thickness of TgM180-LRAP KO mice was further increased, and the structure was improved, with a more defined decussation pattern compared with singly rescued mice. We conclude that TgM180 provides significant rescue of the KO phenotype. Although the effectiveness of the LRAP transgene, alone, to rescue is less obvious, the addition of the LRAP transgene to the M180 transgene in KO enamel leads to an added improvement in both amount and structure and thus these transgenes function in a complementary manner. Together, the two most abundant amelogenins lead to the formation of obvious enamel decussation patterns.

The leucine-rich amelogenin peptide alters the amelogenin null enamel phenotype

Introduction: The amelogenin proteins secreted by ameloblasts during dental enamel development are required for normal enamel structure. Amelx null (KO) mice have hypoplastic, disorganized enamel similar to that of human patients with mutations in the AMELX gene, and provide a model system for studies of the enamel defect amelogenesis imperfecta. Because many amelogenin proteins are present in developing enamel due to RNA alternative splicing and proteolytic processing, understanding the function of individual amelogenins has been challenging. Purpose: Our objective was to better understand the role of LRAP, a 59 amino acid leucine-rich amelogenin peptide, in the development of enamel. Approach: Teeth from transgenic mice that express LRAP under control of the Amelx regulatory regions were analyzed for mechanical properties, and transgenic males were mated with female KO mice. Male offspring with a null background that were transgene positive or transgene negative were compared to determine phenotypic differences using microcomputed tomography (microCT) and scanning electron microscopy (SEM). Results: Nanoindentation revealed no differences between LRAP transgenic and wild-type murine enamel. Using microCT, LRAPKO enamel volume and density measurements were similar to those from KO mice. However, in etched samples examined by SEM, the organization of the enamel rod pattern was altered by the presence of the LRAP transgene. Conclusions: The presence of LRAP leads to changes in enamel appearance compared to enamel from KO mice. Expression of a combination of amelogenin transgenes in KO mice may lead to rescue of the individual characteristics of normal enamel.

Microgenomics of Ameloblastoma

Gene expression profiles of human ameloblastoma microdissected cells were characterized with the purpose of identifying genes and their protein products that could be targeted as diagnostic and prognostic markers as well as for potential therapeutic interventions. Five formalin-fixed, decalcified, paraffin-embedded samples of ameloblastoma were subjected to laser capture microdissection, linear mRNA amplification, and hybridization to oligonucleotide human 41,000 RNA arrays and compared with universal human reference RNA, to determine the gene expression signature. Assessment of the data by Significance Analysis of Microarrays (SAM) and cluster analysis showed that 38 genes were highly expressed (two-fold increase) in all samples, while 41 genes were underexpressed (two-fold reduction). Elements of the sonic hedgehog pathway and Wingless type MMTV integration site family were validated by immunohistochemistry. We have identified the expression of multiple genes and protein products that could serve as potential diagnostic, prognostic, and therapeutic targets.

WNT5A Expression in Ameloblastoma and Its Roles in Regulating Enamel Epithelium Tumorigenic Behaviors

Odontogenic tumors originate from the remains of migrating enamel epithelium after the completion of normal tooth genesis. These enamel epithelium remnants exhibit the ability to recapitulate the events that occur during tooth formation. Several lines of evidence suggest that aberrance in the signaling pathways similar to the ones that are used during tooth development, including the WNT pathway, might be the cause of odontogenic tumorigenesis and maintenance. In this study we demonstrated that WNT5A expression was intense in both the epithelial component of ameloblastomas, the most common epithelial odontogenic tumor, and in this tumors likely precursor cell, the enamel epithelium located at the cervical loop of normal developing human tooth buds. Additionally, when WNT5A was overexpressed in enamel epithelium cells (LS-8), the clones expressing high levels of WNT5A (S) exhibited characteristics of tumorigenic cells, including growth factor independence, loss of anchorage dependence, loss of contact inhibition, and tumor formation in immunocompromised mice. Moreover, overexpression of WNT5A drastically increased LS-8 cell migration and actin reorganization when compared with controls. Suppression of endogenous WNT5A in LS-8 cells (AS) greatly impaired their migration and AS cells failed to form significant actin reorganization and membrane protrusion was rarely seen. Taken together, our data indicate that WNT5A signaling is important in modulating tumorigenic behaviors of enamel epithelium cells in ameloblastomas.