Higher expression of WNT5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

WNT5A is a secreted signaling protein that promotes migration and invasion of oral squamous cell carcinoma (OSCC) cells through activation of non‐canonical WNT signaling. Here, we examined expression of WNT5A, β‐catenin, and E‐cadherin by immunohistochemistry in 21 human diagnostic incision biopsies that each had regions of oral mucosa with a normal appearance adjacent to the affected tissue, dysplasia, and OSCC. We also investigated the effect of recombinant WNT5A (rWNT5A) on expression of the cell‐adhesion proteins E‐cadherin and β‐catenin by western blot analysis. No expression of WNT5A protein was present in oral mucosa with a normal appearance or in mild grade dysplasia. However, expression of WNT5A increased along with increasing grade of dysplasia, and the highest expression was detected in OSCCs. Expression of membranous β‐catenin and of E‐cadherin was lower, whereas expression of cytoplasmic β‐catenin was higher, in OSCCs than in non‐cancerous regions. However, there was no correlation between expression of WNT5A and expression of either β‐catenin or E‐cadherin. Furthermore, treatment of OSCC cells with rWNT5A had no effect on the expression of β‐catenin or E‐cadherin. Taken together with previous results, we conclude that WNT5A influences the progression of OSCC without affecting the canonical WNT/β‐catenin pathway and without down‐regulating E‐cadherin. WNT5A may have potential as a biological marker for malignant transformation of dysplasia to OSCC.     

Ameloblasts require active RhoA to generate normal dental enamel

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up‐regulated. Transgenic mice were generated that express a dominant‐negative RhoA transgene in ameloblasts using amelogenin gene‐regulatory sequences. Transgenic and wild‐type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F‐actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho‐associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E‐cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E‐cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.     

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7^fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.     

Enamel and dental anomalies in latent‐transforming growth factor beta‐binding protein 3 mutant mice

Latent‐transforming growth factor beta‐binding protein 3 (LTBP‐3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor‐β (TGF‐β). To investigate the role of LTBP‐3 in tooth formation we performed micro‐computed tomography (micro‐CT), histology, and scanning electron microscopy analyses of adult Ltbp3‐/‐ mice. The Ltbp3‐/‐ mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation‐stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous‐like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP‐3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF‐β signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3‐/‐ mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.     

Lentivirus‐mediated gene silencing of beta‐catenin inhibits growth of human tongue cancer cells

J Oral Pathol Med (2011) 40 : 643–650 Background: Beta‐catenin is one of the key components of Wnt signaling pathway. Increased level of this protein has been proved to be associated with enhanced cellular proliferation and the development of many kinds of cancers. But its role in the carcinogenesis in human tongue squamous cell carcinoma, one of the most common carcinomas of the human oral cavity, remains poorly characterized. Methods: In this study, we used lentivirus‐mediated RNA interference (RNAi) targeted against beta‐catenin to determine the effects of decreasing the high constitutive level of this protein in human tongue carcinoma cell line Tca8113. Results: Our studies demonstrated that RNAi directly against beta‐catenin markedly decreased beta‐catenin gene expression and inhibited cellular proliferation as reflected in the reduced growth of tongue cancer cells both in vitro and in nude mice. Conclusions: RNA interference (RNAi) targeting against beta‐catenin can induce cell growth suppression of tongue cancer and may have the potential as a therapeutic modality to treat human tongue cancer.     

Distribution and structure of the initial dental enamel formed in incisors of young wild‐type and Tabby mice

Mouse incisor enamel can be divided into four layers: an inner prism‐free layer; an inner enamel with prism decussation; outer enamel with parallel prisms; and a superficial prism‐free layer. We wanted to study how this complex structural organization is established in the very first enamel formed in wild‐type mice and also in Tabby mice where enamel coverage varies considerably. Unworn incisors from young female wild‐type and Tabby mice were ground, etched, and analyzed using scanning electron microscopy. In both wild‐type and Tabby mice, establishment of the enamel structural characteristics in the initially formed enamel proceeded as follows, going from the incisal tip in an apical direction: (i) a zone with prism‐free enamel, (ii) a zone with occasional prisms most often inclined incisally, and (iii) a zone where prism decussation was gradually established in the inner enamel. The distribution of enamel in Tabby mice exhibited considerable variability. The sequence of initial enamel formation in mouse incisors mimics development from a primitive (prism‐free) structure to an evolved structure. It is suggested that genes controlling enamel distribution are not associated with genes controlling enamel structure. The control of ameloblast configuration, life span, organization in transverse rows, and movement is important for establishing the characteristic mature pattern of mouse incisor enamel.     

Cyclosporine A Enhances Gingival β‐Catenin Stability via Wnt Signaling

Background : Cyclosporine A (CsA) increases β‐catenin messenger RNA (mRNA) and protein expression. The present study demonstrates that Wnt/β‐catenin signaling inhibits β‐catenin degradation in the gingiva. Methods: Forty 5‐week‐old male Sprague‐Dawley rats were assigned to two study groups after healing from right maxillary molar extractions. The rats in the experimental group were fed 30 mg/kg CsA daily for 4 weeks, whereas the control rats were fed mineral oil. At the end of the study, all rats were sacrificed, and the gingivae were obtained. The gingival morphology after CsA treatment was evaluated by histology, and the genes related to Wnt/β‐catenin signaling were initially screened by microarray. Polymerase chain reaction, Western blotting, and immunohistochemistry were used to examine the mRNA and protein expression of proliferating cell nuclear antigen, cyclin D1, E‐cadherin, β‐catenin, Dvl‐1, glycogen synthase kinase‐3β, axin‐1, and adenomatous polyposis coli (APC). Phosphoserine and ubiquitinylated β‐catenin were detected after immunoprecipitation. Results: In rats treated with CsA, overgrowth of gingivae was observed, and altered expression of genes related to Wnt/β‐catenin signaling was detected by the microarray. The gingival mRNA and protein expression profiles for genes associated with Wnt/β‐catenin signaling further confirmed the effect of CsA: β‐catenin and Dvl‐1 expression increased, but APC and axin‐1 expression decreased. Western blotting and immunohistochemistry showed decreases in β‐catenin serine phosphorylation (33/37) and ubiquitinylation in the gingivae of CsA‐treated rats. Conclusion: CsA‐enhanced gingival β‐catenin stability may be involved in gene upregulation or β‐catenin degradation via the Wnt/β‐catenin pathway.     

Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino-enamel junction, and a decussating enamel rod pattern

Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.     

Differential expression of canonical and non-canonical Wnt ligands in ameloblastoma

J Oral Pathol Med (2012) 41 : 332–339 Background: Canonical and non‐canonical Wnt signaling pathways modulate diverse cellular processes during embryogenesis and post‐natally. Their deregulations have been implicated in cancer development and progression. Wnt signaling is essential for odontogenesis. The ameloblastoma is an odontogenic epithelial neoplasm of enamel organ origin. Altered expressions of Wnts‐1, ‐2, ‐5a, and ‐10a are detected in this tumor. The activity of other Wnt members remains unclarified. Materials and Methods: Canonical (Wnts‐1, ‐2, ‐3, ‐8a, ‐8b, ‐10a, and ‐10b), non‐canonical (Wnts‐4, ‐5a, ‐5b, ‐6, 7a, ‐7b, and ‐11), and indeterminate groups (Wnts‐2b and ‐9b) were examined immunohistochemically in 72 cases of ameloblastoma (19 unicystic [UA], 35 solid/multicystic [SMA], eight desmoplastic [DA], and 10 recurrent [RA]). Results: Canonical Wnt proteins (except Wnt‐10b) were heterogeneously expressed in ameloblastoma. Their distribution patterns were distinctive with some overlap. Protein localization was mainly membranous and/or cytoplasmic. Overexpression of Wnt‐1 in most subsets (UA = 19/19; SMA = 35/35; DA = 5/8; RA = 7/10) (P < 0.05), Wnt‐3 in granular cell variant (n = 3/3), and Wnt‐8b in DA (n = 8/8) was key observations. Wnts‐8a and ‐10a demonstrated enhanced expression in tumoral buddings and acanthomatous areas. Non‐canonical and indeterminate Wnts were absent except for limited Wnt‐7b immunoreactivity in UA (n = 1/19) and SMA (n = 1/35). Stromal components expressed variable Wnt positivity. Conclusion: Differential expression of Wnt ligands in different ameloblastoma subtypes suggests that the canonical and non‐canonical Wnt pathways are selectively activated or repressed depending on the tumor cell differentiation status. Canonical Wnt pathway is most likely the main transduction pathway while Wnt‐1 might be the key signaling molecule involved in ameloblastoma tumorigenesis.     

Functional analysis of a novel missense mutation in AXIN2 associated with non‐syndromic tooth agenesis

Tooth agenesis is a congenital anomaly frequently seen in humans. Several genes have been associated with non‐syndromic tooth agenesis, including msh homeobox 1 (MSX1), paired box 9 (PAX9), axis inhibition protein 2 (AXIN2), ectodysplasin A (EDA), and wingless‐type MMTV integration site family member 10A (WNT10A). In this study, we investigated a Chinese family with non‐syndromic tooth agenesis. A novel missense mutation (c.C1978T) in AXIN2 was identified in affected members. The mutation results in a His660Tyr substitution located between the Axin beta‐catenin binding domain and the DIX domain of the axis inhibition protein 2 (AXIN2). We analysed this novel AXIN2 mutant, together with two reported AXIN2 mutants [c.1966C>T (p.Arg656Stop) and c.1994delG (p.Leu688Stop)] that cause colorectal cancer with and without oligodontia, to study the effect of the mutant p.His660Tyr on the Wnt/β‐catenin signaling pathway and to compare the molecular pathogenesis of different AXIN2 mutants in tooth agenesis and carcinogenesis. Further in vitro experiments indicated that the mutant p.His660Tyr caused inhibition of the Wnt/β‐catenin pathway, and the mutants p.Arg656Stop and p.Leu688Stop resulted in over‐activation of the Wnt/β‐catenin pathway. In line with previous AXIN2 mutation studies, we suggest that AXIN2 mutations with different levels of severity may have distinct effects on the Wnt pathway and the phenotype of disease. Our study provides functional evidence supporting the notion that both inhibition and over‐activation of the Wnt pathway may lead to tooth agenesis.     

Mutational analysis of Wnt signaling molecules in ameloblastoma with aberrant nuclear expression of β‐catenin

Background: To clarify the genetic background of ameloblastoma, expression of β‐catenin, and mutational status of genes involved in Wnt signaling pathway were investigated. Methods: We analyzed β‐catenin and cyclin D1 in 18 cases of ameloblastoma by immunohistochemical staining, and searched for mutations in CTNNB1 (gene for β‐catenin), APC, AXIN1, and AXIN2 by polymerase chain reaction (PCR) and direct sequencing method. Result: We detected membranous and occasionally cytoplasmic expression of β‐catenin in 16 of 18 cases (89%), and nuclear expression of β‐catenin principally in the peripheral columnar cells in 11 of 18 cases (61%). In nine of the 18 cases (50%), we detected the expression of cyclin D1 principally in the peripheral columnar cells. However, there was no correlation between nuclear expressions of β‐catenin and cyclin D1. No missense mutations were found in CTNNB1, APC, AXIN1, and AXIN2 in all cases except for silent mutation and already‐known single nucleotide polymorphism. Conclusion: Mutations in CTNNB1, APC, AXIN1, and AXIN2 are not implicated in nuclear accumulation of β‐catenin, and that the expression of cyclin D1 is accelerated independently of β‐catenin in ameloblastomas. Other Wnt signaling members or alternative pathways involved in the degradation of β‐catenin should be subject of further investigation.     

Homeobox protein MSX‐1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer‐binding factor 1 via Wnt/β‐catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage

Homeobox protein MSX‐1 (hereafter referred to as MSX‐1) is essential for early tooth‐germ development. Tooth‐germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX‐1 beyond this stage. Here, we investigated the roles of MSX‐1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β‐catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer‐binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β‐catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S‐phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1‐phase progression. We therefore conclude that MSX‐1 maintains cell proliferation by regulating transition of cells from G1‐phase to S‐phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β‐catenin signaling pathway.     

Buffering of protons released by mineral formation during amelogenesis in mice

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non‐fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator‐deficient (Cftr null), anion exchanger‐2‐deficient (Ae2a,b null), and amelogenin‐deficient (Amelx null) mice, was determined by quantitative X‐ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated‐matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl^?) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl^? content, the stronger the negative effects. In Amelx‐null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein‐4‐deficient (Klk4‐null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.     

A method of calculating the speed of movement of ameloblasts during rat incisor amelogenesis.

Ameloblast migration rates during secretion of enamel in rat incisors were calculated from measurements of (1) certain angular and linear parameters pertaining to the prism pattern, (2) the length of the zones of enamel secretion, (3) the impeded eruption rate of rat incisors, and (4) the diameters of the circles of which the rat incisors are parts. It is assumed that the ameloblasts move along the path of the prisms, that the incisors of adult rats erupt along the are of a stationary circle, and that the length of the zones of enamel secretion does not change with age. From data in the literature, the transverse component of ameloblast migration was calculated to be 9.9 /gmm/day in the upper incisor and 13.4μm/day in the lower. If adjacent rows of ameloblasts move in opposite directions, each ameloblast will in 1 day slide past five ameloblasts of the adjacent row in the upper incisor and past seven ameloblasts in the lower incisor. The results obtained for the component of ameloblast migration from the dentine surface were 9.0μm/day in the upper incisor and 12.3 μm/day in the lower, which are below the 16 /gmm/day enamel apposition rate of other workers. Due to the gingival inclination of enamel prisms, the ameloblasts move gingivally faster than the tooth erupts. This movement is faster during secretion of outer enamel than that of inner enamel. The increased inclination of prisms in the outer enamel zone may be effected by an increase in the gingivo-basal diameter of ameloblasts as they enter the zone of outer enamel secretion.     

Wnt-RhoA signaling is involved in dental enamel development

Transgenic mice that express dominant-negative RhoA (RhoA(DN) ) in ameloblasts have hypoplastic enamel with defects in molar cusps. β-catenin and Wnt5a were up-regulated in enamel organs of RhoA(DN) transgenic mice, which indicated that both canonical and non-canonical Wnt pathways are implicated in the process of enamel defect formation. It was hypothesized that expression of RhoA(DN) in ameloblasts interfered with normal enamel development through the pathways that were induced by fluoride. The Wnt and RhoA pathways were further investigated in an ameloblast-lineage cell line (ALC) by treatment with sodium fluoride (NaF). The activities of RhoA and Rho-associated protein kinase (ROCK) II decreased significantly by 8-12 hours, similar to decreased activity in RhoA(DN) transgenic mice. Both canonical and non-canonical Wnt pathways were activated by treatment with NaF, which was verified by western blotting and the β-catenin-TCF/LEF (T cell factor lymphanoid/enhancer factor) reporter gene (TOPflash) assay. β-catenin localization to both cytoplasm and nucleus was up-regulated in NaF-treated ALC, while Gsk-3β, the negative regulator of the Wnt pathway, showed a decreased pattern of expression. The current results indicate that both Wnt and RhoA pathways are implicated in fluoride-induced signaling transductions in the ALC as well as in the development of enamel defects in RhoA(DN) transgenic mice.     

Enamel alterations in serotonin 2B receptor knockout mice

The role of the serotonin 2B receptor (5-HT(2B) R) in enamel formation and mineralization was explored in adult 5HT(2B) R knockout (KO) mice compared with wild-type (WT) mice. In the molar, quantitative data obtained by micro-computed tomography imaging showed that the overall volume of the enamel layer was firmly reduced in KO mice. Defective mineralization was ascertained by energy-dispersive X-ray microanalysis. We also observed, using scanning electron microscopy, that parazones in the KO mice included two or three helicoidally twisted rods within Hunter-Schreger bands, instead of a single rod, as found in the WT mice. Minor disturbances were also detected in the incisors of KO mice. Structural modifications, thinner enamel crystallites, and porosities observed in KO mice indicate that the 5-HT(2B) R-mediated signaling pathways as part of the enamel formation process. These data provide a basis for evaluating the role of 5-HT(2B) R in ameloblast functions. Defects observed in the mineralization and structure of enamel in KO mice highlight that the 5-HT(2B) R interferes with the mechanisms directing amelogenesis.     

Immunoprofile of the adenomatoid odontogenic tumor

This study was focused on the immunohistochemical profile of the adenomatoid odontogenic tumor. A Pub/Medline search revealed a number of immunohistochemical studies including cytokeratin profiles, extracellular matrix proteins, Integrins, ameloblast‐associated proteins resorption regulators (RANK, RANKL), p53, PCNA, MDM2 protein, cyclin D1, Ki‐67, Bcl‐2 metallothionein, metalloproteinases, D56 hepatocyte growth factor, c‐met, DNA methyltransferase, podoplanin, TGF‐βI, Smad‐2/3, Smad‐I‐5/‐8, Smad 4, beta‐ catenin, calretinin, and clonality. Careful interpretation of the findings indicates that the adenomatoid odontogenic tumor may be more of a hamartomatous than neoplastic nature.