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 tumor's 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.

Primary failure of eruption and PTH1R: the importance of a genetic diagnosis for orthodontic treatment planning.

INTRODUCTION: Primary failure of eruption (PFE) is characterized by nonsyndromic eruption failure of permanent teeth in the absence of mechanical obstruction. Recent studies support that this dental phenotype is inherited and that mutations in PTH1R genes explain several familial cases of PFE. The objective of our study was to investigate how genetic analysis can be used with clinical diagnostic information for improved orthodontic management of PFE. METHODS: We evaluated a family (n = 12) that segregated an autosomal dominant form of PFE with 5 affected and 7 unaffected persons. Nine available family members (5 male, 4 female) were enrolled and subsequently characterized clinically and genetically. RESULTS: In this family, PFE segregated with a novel mutation in the PTH1R gene. A heterozygous c.1353-1 G>A sequence alteration caused a putative splice-site mutation and skipping of exon 15 that segregated with the PFE phenotype in all affected family members. CONCLUSIONS: A PTH1R mutation is strongly associated with failure of orthodontically assisted eruption or tooth movement and should therefore alert clinicians to treat PFE and ankylosed teeth with similar caution-ie, avoid orthodontic treatment with a continuous archwire.

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.

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.

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.

DLX3 c.561_562delCT mutation causes attenuated phenotype of tricho-dento-osseous syndrome.

The distal-less homeobox gene DLX3 is expressed in a variety of tissues including placenta, skin, hair, teeth, and bone. Mutation of DLX3 (c.571_574delGGGG) causes the tricho-dento-osseous syndrome (TDO), characterized by abnormal hair, teeth, and bone. Evaluation of a kindred segregating the DLX3 c.561_562delCT mutation revealed distinct changes in the hair, teeth, and bones as has been observed with the DLX3 c.571_574delGGGG mutation. Previously, the DLX3 c.561_562delCT mutation was associated with autosomal dominant amelogenesis imperfecta with taurodontism. The present study shows that the DLX3 c.560_561delCT mutation causes an attenuated TDO phenotype with less severe hair, tooth, and bone manifestations compared with individuals having the DLX3 c.571_574delGGGG mutation. Careful phenotyping of individuals with allelic DLX3 mutations reveals marked differences in phenotypic severity indicating that the carboxy-terminus of the DLX3 protein is critical in determining its function during development in these different tissues.

Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium.

Formation of tooth enamel is a very complex process in which a specific set of proteins secreted by ameloblasts play a primordial role. As part of a screening procedure to identify novel proteins secreted by EO (enamel organ) cells of rat incisors, we isolated a partial cDNA fragment (EO-017) that is the homologue of the recently described mouse Amtn (amelotin) gene [Iwasaki, Bajenova, Somogyi-Ganss, Miller, Nguyen, Nourkeyhani, Gao, Wendel and Ganss (2005) J. Dent. Res. 84, 1127-1132]. Presented herein is the cloning of rat and pig full-length cDNAs with their deduced protein sequences. Detailed expression profiling by Northern-blot analysis and RT (reverse transcriptase)-PCR on rat and mouse tissues revealed highest expression in the mandible, more specifically in the maturation stage of the EO. Among all tissues tested, low expression was detected only in periodontal ligament, lung, thymus and gingiva. In silico analyses revealed that the Amtn gene is highly conserved in seven other mammals, but is absent from fish, birds and amphibians. The Amtn protein is enriched in proline, leucine, glutamine and threonine (52% of total) and contains a perfectly conserved protein kinase CK2 phosphorylation site. Transient transfection experiments in HEK-293 cells (human embryonic kidney cells) showed that secreted Amtn is post-translationally modified possibly through O-linked oligosaccharides on threonine residues. In concordance with its predominant expression site, immunofluorescence localization within the rat and mouse mandibles revealed Amtn localized to the basal lamina of maturation stage ameloblasts of incisors and unerupted molars. Intense Amtn protein expression was also detected in the internal basal lamina of junctional epithelium in molars. The peculiar and unique cellular localization of Amtn suggests a role in cell adhesion.

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.

Remineralization potential of a fluoridated carbamide peroxide whitening gel.

PURPOSE: Little knowledge exists regarding the potential remineralizing benefits of adding fluoride to carbamide peroxide-based whitening gels. The aim of this project was to evaluate whether a whitening system with fluoride will remineralize previously demineralized enamel. MATERIALS AND METHODS: Twenty-four extracted teeth were sectioned into quadrants labeled A to D. Tooth quadrants in groups A, B, and D were demineralized with a lactic acid, methyl-cellulose gel system to mimic incipient carious lesions. Group C was neither demineralized nor treated. Group D was demineralized, but not treated with a whitening gel. Groups A and B were exposed to one of two commercially prepared 10% carbamide peroxide whitening gels: one that was fluoride free (group A) and one that contained fluoride (0.463%NaF) (group B). Remineralization was evaluated histologically and analyzed statistically using paired t tests accepting p < 0.05 as significant. RESULTS: Shade comparisons showed equal whitening efficacy of both gel A and B. Paired t tests show a significant reduction in lesion depth after treatment with the fluoride containing gel (B: mean lesion depth = 100 microm; p < 0.01), while there was no difference for the gel lacking fluoride (A: mean lesion depth = 110 microm). CONCLUSION: Under the conditions of this study, the addition of fluoride to a tooth whitening system does not affect the gel's whitening efficacy. The addition of fluoride could provide remineralization properties to the gel. As tooth whitening therapies continue to grow and evolve, a strong focus should rest on improving these materials in ways that will provide ever greater patient benefits. Coupling the esthetic benefits of whitening with the preventive benefits of fluoride is a natural step in this direction. CLINICAL SIGNIFICANCE: Therapuetic quantities of fluoride can impact remineralization properties of commercially available tooth whitening gels without altering whitening properties.

Human enamel phenotype associated with amelogenesis imperfecta and a kallikrein-4 (g.2142G>A) proteinase mutation.

Kallikrein-4 is known to be highly expressed during the maturation stage of enamel formation and is thought to be critical for the final phase of crystallite growth. The purpose of this study was to evaluate the enamel phenotype in humans with a known KLK-4 mutation (g.2142G>A). Primary teeth from two individuals with a known KLK-4 mutation were evaluated using amino acid analysis and light and electron microscopy. Light microscopy showed the enamel was of normal thickness but opaque throughout its width compared with normal enamel. Electron microscopy showed enamel affected by the KLK-4 mutation had a normal prismatic structure and generally had a well-organized and discernable crystallite composition. In some areas, globular structures were present where crystallites were not discernable or appeared to have an altered morphology. The KLK-4 mutant enamel had an increased protein content compared with normal enamel. Human enamel formed with a lack of functioning KLK-4 proteinase is altered primarily in the completeness of crystallite growth, while enamel thickness and prism structure remains essentially normal. Collectively, these studies suggest that the KLK-4 proteinase is essential for the final crystallite growth of enamel but is not critical for crystallite orientation, prism formation or enamel thickness.

MEPE/OF45, a new dentin/bone matrix protein and candidate gene for dentin diseases mapping to chromosome 4q21.

Substantial progress has been made regarding the molecular etiology of the dentin diseases dentinogenesis imperfecta types II and III, and dentin dysplasia type II. Genetic linkage studies have identified the critical loci for these diseases on human chromosome 4q21. Located within an overlapping segment of these disease loci is a dentin/bone gene cluster that includes osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein, and a new gene MEPE also known as OF45, renamed osteoregulin. In this paper, we report the location of MEPE/OF45 in relationship to the other members of the dentin/bone gene cluster as well as the genomic organization of the human gene. For the first time, MEPE/OF45 expression was shown in dental tissue, in particular odontoblasts, by reverse-transcription polymerase chain reaction (RT-PCR) amplification and characterization of a partial mouse cDNA. Our data provide the first evidence that MEPE/OF45 is expressed during odontogenesis and should be considered as a candidate gene for dentin structural diseases mapping to human chromosome 4q21.