Ameloblast-lineage Cells Research Articles

Loss of Autophagy Disrupts Stemness of Ameloblast-Lineage Cells in Aging

Autophagy is one of the intracellular degradation pathways and maintains cellular homeostasis, regulating the stress response, cell proliferation, and signal transduction. To elucidate the role of autophagy in the maintenance of dental epithelial stem cells and the subsequent enamel formation, we analyzed autophagy-deficient mice in epithelial cells (Atg7f/f;KRT14-Cre mice), focusing on the influence of aging and stress environments. We also performed in vitro cell and organ culture experiments with an autophagy inhibitor. In young Atg7f/f;KRT14-Cre mice, morphological change was not obvious in maxillary incisors, except for the remarkable cell death in the stratum intermedium of the transitional stage. However, under stress conditions of hyperglycemia, the incisor color changed to white in diabetes Atg7f/f;KRT14-Cre mice. Regarding dental epithelial stem cells, the shape of the apical bud region of the incisor became irregular with age, and odontoma was formed in aged Atg7f/f;KRT14-Cre mice. In addition, the shape of apical bud culture cells of Atg7f/f;KRT14-Cre mice became irregular and enlarged atypically, with epigenetic changes during culture, suggesting that autophagy deficiency may induce tumorigenesis in dental epithelial cells. The epigenetic change and upregulation of p21 expression were induced by autophagy inhibition in vivo and in vitro. These findings suggest that autophagy is important for the regulation of stem cell maintenance, proliferation, and differentiation of ameloblast-lineage cells, and an autophagy disorder may induce tumorigenesis in odontogenic epithelial cells.

Perfluorooctanoic acid-induced cell death via the dual roles of ROS-MAPK/ERK signaling in ameloblast-lineage cells

Perfluorooctanoic acid (PFOA) is an artificial fluorinated organic compound that has generated increased public attention due to its potential health hazards. Unsafe levels of PFOA exposure can affect reproduction, growth and development. During tooth enamel development (amelogenesis), environmental factors including fluoride can cause enamel hypoplasia. However, the effects of PFOA on ameloblasts and tooth enamel formation remain largely unknown. In the present study we demonstrate several PFOA-mediated cell death pathways (necrosis/necroptosis, and apoptosis) and assess the roles of ROS-MAPK/ERK signaling in PFOA-mediated cell death in mouse ameloblast-lineage cells (ALC).ALC cells were treated with PFOA. Cell proliferation and viability were analyzed by MTT assays and colony formation assays, respectively. PFOA suppressed cell proliferation and viability in a dose dependent manner. PFOA induced both necrosis (PI-positive cells) and apoptosis (cleaved-caspase-3, γH2AX and TUNEL-positive cells). PFOA significantly increased ROS production and up-regulated phosphor-(p)-ERK. Addition of ROS inhibitor N-acetyl cysteine (NAC) suppressed p-ERK and decreased necrosis, and increased cell viability compared to PFOA alone, whereas NAC did not change apoptosis. This suggests that PFOA-mediated necrosis was induced by ROS-MAPK/ERK signaling, but apoptosis was not associated with ROS. Addition of MAPK/ERK inhibitor PD98059 suppressed necrosis and increased cell viability compared to PFOA alone. Intriguingly, PD98059 augmented PFOA-mediated apoptosis. This suggests that p-ERK promoted necrosis but suppressed apoptosis. Addition of the necroptosis inhibitor Necrostatin-1 restored cell viability compared to PFOA alone, while pan-caspase inhibitor Z-VAD did not mitigate PFOA-mediated cell death. These results suggest that 1) PFOA-mediated cell death was mainly caused by necrosis/necroptosis by ROS-MAPK/ERK signaling rather than apoptosis, 2) MAPK/ERK signaling plays the dual roles (promoting necrosis and suppressing apoptosis) under PFOA treatment. This is the initial report to indicate that PFOA could be considered as a possible causative factor for cryptogenic enamel malformation. Further studies are required to elucidate the mechanisms of PFOA-mediated adverse effects on amelogenesis.

New Ameloblastoma Cell Lines Enable Preclinical Study of Targeted Therapies

Ameloblastoma (AB) is an odontogenic tumor that arises from ameloblast-lineage cells. Although relatively uncommon and rarely metastatic, AB tumors are locally invasive and destructive to the jawbone and surrounding structures. Standard-of-care surgical resection often leads to disfigurement, and many tumors will locally recur, necessitating increasingly challenging surgeries. Recent genomic studies of AB have uncovered oncogenic driver mutations, including in the mitogen-activated protein kinase (MAPK) and Hedgehog signaling pathways. Medical therapies targeting those drivers would be a highly desirable alternative or addition to surgery; however, a paucity of existing AB cell lines has stymied clinical translation. To bridge this gap, here we report the establishment of 6 new AB cell lines—generated by “conditional reprogramming”—and their genomic characterization that reveals driver mutations in FGFR2, KRAS, NRAS, BRAF, PIK3CA, and SMO. Furthermore, in proof-of-principle studies, we use the new cell lines to investigate AB oncogene dependency and drug sensitivity. Among our findings, AB cells with KRAS or NRAS mutation (MAPK pathway) are exquisitely sensitive to MEK inhibition, which propels ameloblast differentiation. AB cells with activating SMO-L412F mutation (Hedgehog pathway) are insensitive to vismodegib; however, a distinct small-molecule SMO inhibitor, BMS-833923, significantly reduces both downstream Hedgehog signaling and tumor cell viability. The novel cell line resource enables preclinical studies and promises to speed the translation of new molecularly targeted therapies for the management of ameloblastoma and related odontogenic neoplasms.

Melatonin-Medicated Neural JNK3 Up-Regulation Promotes Ameloblastic Mineralization.

Introduction: Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates. It functions have been reported in previous stomatological studies as anti-inflammation, antioxidant, osseointegration of dental implants and stimulation to dental pulp stem cells differentiation, but its role in ameloblastic differentiation and mineralization has been rarely studied. Objective: To reveal the effects of melatonin on the mineralization of ameloblast lineage cells (ALCs), and to identify the change in gene expression and the potential mechanism based on ribonucleic acid sequencing (RNA-seq) analysis. Method: ALCs were induced in melatonin-conditioned medium. After 7-days culture, Western blot, real-time PCR, alkaline phosphatase (ALP) activity test, RNA-seq were accordingly used to detect the change in molecular level. After 1-month odontogenic induction in melatonin medium, Alizarin Red-S (ARS) staining showed the changes of mineral nodules. Differentially expressed genes (DEGs), enrichment of functions and signaling pathways analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database were performed. The JNK3 antagonist (JNK3 inhibitor IX, SR3576) and β-arrestin1 (Arrb1) overexpression were applied to confirm the fluctuation of melatonin-medicated JNK3 and Arrb1 expression. Results: In this study, we found out melatonin contributed to the ameloblastic mineralization, from which we can observed the elevated expression of enamel matrix protein, and increased ALP activity and mineralized nodules formation. RNA-seq analysis showed the up-regulation of neural JNK3 and down-regulation of Arrb1 in ALCs. Meanwhile, phosphorylated JNK3 deficiency (phosphorylated JNK3 inhibitor---SR3576 added to culture medium) led to mineralization delay, and Arrb1 overexpression proved Arrb1 takes bridge between melatonin receptors (MTNR) and JNK3 in MAPK signaling pathway.

Calcium mitigates fluoride-induced kallikrein 4 inhibition via PERK/eIF2α/ATF4/CHOP endoplasmic reticulum stress pathway in ameloblast-lineage cells

ObjectivesThe present study aimed to investigated the effect and mechanism of Ca2+ treatment on fluoride in ameloblast-lineage cells (ALCs). Materials and methodsThe effects of fluoride and different Ca2+ levels treatment on the proliferative activity, cell apoptosis, cell cycle, intracellular free Ca2+, were firstly determined. Kallikrein 4 (KLK4), glucose-responsive protein 78 (GRP78), Protein kinase R -like endoplasmic reticulum kinase (PERK), the α subunit of eukaryotic initiation factor 2 (eIF2α), activating transcription factor 4 (ATF4), CCAAT enhancer-binding protein homologous protein (CHOP), were investigated in ALCs. ResultsThe proliferative activity was obviously inhibited under concentrations of single fluoride high than 1 mM, and indicated highest proliferation at single 2.5 mM Ca2+ concentration in ALC cells. In addition, we found that single fluoride markedly induced intracellular free Ca2+ increasing, G2/M phase arrest, apoptosis. GRP78 and endoplasmic reticulum stress pathway of PERK/eIF2α/ATF4/CHOP were significantly increased, while the proliferation and KLK4 were markedly reduced in ALCs. Ca2+ additional treatment can obviously reverse the effect of fluoride-induced apoptosis and inhibition of KLK4. The effect of GRP78 and endoplasmic reticulum stress pathway of PERK/eIF2α/ATF4/CHOP were also alleviated under Ca2+ additional treatment in ALCs. More important, the results of 2.5 mmol/L Ca2+ treatment on the proliferation, cell cycle and apoptosis suggest this concentration is relatively better to mediate the intracellular Ca2+ homeostasis in ALCs. ConclusionsIn sum, Ca2+-supplementation exerts antagonistic the toxic effects on fluoride and this inhibitory effect suggests the potential implications for Ca2+-supplementation on fluorosis.

Roles of heterogeneous nuclear ribonucleoprotein L in enamel organ development and the differentiation of ameloblasts

ObjectiveWe aimed to explore the role of Heterogeneous Nuclear Ribonucleoprotein L(hnRNP L) in enamel organ development through hnRNP L conditional knockout mice and knockdown of hnRNP L expression in mouse ameloblast-lineage cells (mALCs) MethodsWe created K14cre-mediated hnRNP L conditional knockout mice (hnRNP LK14/fl) and silenced the expression of hnRNP L in mALCs to investigate the role of hnRNP L in enamel organ development. ResultsWe found that hnRNP LK14/fl mice presented enamel organ development defects with reduced number of inner enamel epithelium (IEE) cells. The proliferation and differentiation of the IEE cells/ameloblasts were suppressed. The cell proliferation and mineralization ability were also decreased after hnRNP L knockdown. Further studies showed that Bone Morphogenetic Protein (BMP) signaling pathway was attenuated after the knockdown of hnRNP L expression both in vivo and in vitro. ConclusionsThese findings suggest that hnRNP L plays a critical role in enamel organ development by promoting the IEE cell/ameloblast proliferation and differentiation. BMP signaling pathway may be involved in the process.

Functional Expression of Sodium-Dependent Glucose Transporter in Amelogenesis

Glucose is an essential source of energy for mammalian cells and is transported into the cells by glucose transporters. There are 2 types of glucose transporters: one is a passive glucose transporter, GLUT (SLC2A), and the other is a sodium-dependent active glucose transporter, SGLT (SLC5A). We previously reported that the expression of GLUTs during tooth development is precisely and spatiotemporally controlled and that the glucose uptake mediated by GLUT1 plays a crucial role in early tooth morphogenesis and tooth size determination. This study aimed to clarify the localization and roles of SGLT1 and SGLT2 in murine ameloblast differentiation by using immunohistochemistry, immunoelectron microscopy, an in vitro tooth organ culture experiment, and in vivo administration of an inhibitor of SGLT1/2, phloridzin. SGLT1, which has high affinity with glucose, was immunolocalized in the early secretory ameloblasts and the ruffle-ended ameloblasts in the maturation stage. However, SGLT2, which has high glucose transport capacity, was observed in the stratum intermedium, papillary layer, and ameloblasts at the maturation stage and colocalized with Na+-K+-ATPase. The inhibition of SGLT1/2 by phloridzin in the tooth germs induced the disturbance of ameloblast differentiation and enamel matrix formation both in vitro (organ culture) and in vivo (mouse model). The expression of SGLT1 and SGLT2 was significantly upregulated in hypoxic conditions in the ameloblast-lineage cells. These findings suggest that the active glucose uptake mediated by SGLT1 and SGLT2 is strictly regulated and dependent on the intra- and extracellular microenvironments during tooth morphogenesis and that the appropriate passive and active glucose transport is an essential event in amelogenesis.

Combination of Runx2 and Cbfβ upregulates Amelotin gene expression in ameloblasts by directly interacting with cis‑enhancers during amelogenesis.

Amelotin (Amtn) is a recently identified enamel protein secreted by ameloblasts at late stage of enamel development. Runt‑related transcription factor 2 (Runx2) in combination with the coactivator core‑binding factor β (Cbfβ) regulates the early stages of tooth development. The aim of the present study was to investigate the role of Runx2 in the regulation of Amtn gene expression in ameloblasts. Immunohistochemistry was performed and the results revealed that Runx2 protein was predominantly expressed in the nuclei of ameloblasts during the transition stage and the maturation stage of enamel development, whereas Cbfβ was expressed in ameloblasts from the secretory stage to the maturation stage. Reverse transcription‑quantitative polymerase chain reaction results demonstrated that Runx2 knockdown decreased Amtn expression in ameloblast‑lineage cells and co‑expression of Runx2 and Cbfβ in ameloblast lineage cells induced an upregulation in Amtn gene expression. Two putative Runx2‑binding sites within the Amtn promoter were identified using bioinformatics analysis. Results of an electrophoretic mobility shift assay and chromatin immunoprecipitation indicated that Runx2/Cbfβ bound to specific DNA sequences. Site‑directed mutagenesis of the Runx2 binding sites within the Amtn promoter resulted in decreased basal promoter activity and did not affect the overexpressed Runx2/Cbfβ. The results of the present study suggest that Runx2 upregulates Amtn gene expression via binding directly to Runx2 sites within the Amtn promoter during amelogenesis.

Role of protein delta homolog 1 in the proliferation and differentiation of ameloblasts.

Protein delta homolog 1 (DLK1) regulates the odontoblastic differentiation of human dental pulp stem cells. It was hypothesized that DLK1 may exert regulatory effects on epithelial-mesenchymal interactions in tooth development. The present study investigated the expression of DLK1 during the development of mouse enamel and its role in the proliferation and differentiation of ameloblast-lineage cells (ALCs). DLK1 expression was upregulated in ameloblasts in the first mandibular molar during the entire process of enamel development. The mRNA and protein levels of DLK1 were significantly upregulated following ameloblastic induction in ALCs. In addition, overexpression of DLK1 promoted the proliferation of ALCs, inhibited ameloblastic differentiation, upregulated the expression of amelogenin and enamelin, and downregulated the expression of odontogenic ameloblast-associated protein and kallikrein 4. The results of the present study suggested that DLK1 may be a potent regulator of ameloblast proliferation and differentiation, and may regulate enamel formation during tooth development.

The crucial role of the TRPM7 kinase domain in the early stage of amelogenesis

Transient receptor potential melastatin-7 (TRPM7) is a bi-functional protein containing a kinase domain fused to an ion channel. TRPM7 is highly expressed in ameloblasts during tooth development. Here we show that TRPM7 kinase-inactive knock-in mutant mice (TRPM7 KR mice) exhibited small enamel volume with opaque white-colored incisors. The TRPM7 channel function of ameloblast-lineage cells from TRPM7 KR mice was normal. Interestingly, phosphorylation of intracellular molecules including Smad1/5/9, p38 and cAMP response element binding protein (CREB) was inhibited in ameloblasts from TRPM7 KR mice at the pre-secretory stage. An immunoprecipitation assay showed that CREB was bound to TRPM7, suggesting that direct phosphorylation of CREB by TRPM7 was inhibited in ameloblast-lineage cells from TRPM7 KR mice. These results indicate that the function of the TRPM7 kinase domain plays an important role in ameloblast differentiation, independent of TRPM7 channel activity, via phosphorylation of CREB.

Fluoride Alters Klk4 Expression in Maturation Ameloblasts through Androgen and Progesterone Receptor Signaling.

Fluorosed maturation stage enamel is hypomineralized in part due to a delay in the removal of matrix proteins to inhibit final crystal growth. The delay in protein removal is likely related to reduced expression of kallikrein-related peptidase 4 (KLK4), resulting in a reduced matrix proteinase activity that found in fluorosed enamel. Klk4 transcription is known to be regulated in other cell types by androgen receptor (AR) and progesterone receptors (PR). In this study, we determined the possible role of fluoride in down-regulation of KLK4 expression through changes in AR and PR. Immunohistochemical localization showed that both AR and PR nuclear translocation was suppressed in fluoride exposed mice. However, when AR signaling was silenced in mouse ameloblast-lineage cells (ALCs), expression of both Pgr and Klk4 were increased. Similar to the effect from AR silencing, fluoride also upregulated Pgr in ALCs, but downregulated Klk4. This finding suggests that though suppression of AR transactivation by fluoride increases Prg expression, inhibition of PR transactivation by fluoride has a much greater effect, ultimately resulting in downregulation of Klk4 expression. These findings indicate that in ameloblasts, PR has a dominant role in regulating Klk4 expression. We found that when AR was retained in the cytoplasm in the presence of fluoride, that co-localized with heat shock protein 90 (HSP90), a well-known chaperone for steroid hormone receptors. HSP90 also known to regulate TGF-β signaling. Consistent with the effect of fluoride on AR and HSP90, we found evidence of reduced TGF-β signaling activity in fluorosed ameloblasts as reduced immunolocalization of TGFB1 and TGFBR-2 and a significant increase in Cyclin D1 mRNA expression, which also possibly contributes to the reduced AR signaling activity. In vitro, when serum was removed from the media, aluminum was required for fluoride to inhibit the dissociation of HSP90 from AR. In conclusion, fluoride related downregulation of Klk4 is associated with reduced nuclear translocation of AR and PR, and also reduced TGF-β signaling activity, all of which are regulated by HSP90. We suggest that a common mechanism by which fluoride affects AR, PR, and TGF-β signaling is through inhibiting ATP-dependent conformational cycling of HSP90.

4-phenylbutyrate Mitigates Fluoride-Induced Cytotoxicity in ALC Cells.

Chronic fluoride over-exposure during pre-eruptive enamel development can cause dental fluorosis. Severe dental fluorosis is characterized by porous, soft enamel that is vulnerable to erosion and decay. The prevalence of dental fluorosis among the population in the USA, India and China is increasing. Other than avoiding excessive intake, treatments to prevent dental fluorosis remain unknown. We previously reported that high-dose fluoride induces endoplasmic reticulum (ER) stress and oxidative stress in ameloblasts. Cell stress induces gene repression, mitochondrial damage and apoptosis. An aromatic fatty acid, 4-phenylbutyrate (4PBA) is a chemical chaperone that interacts with misfolded proteins to prevent ER stress. We hypothesized that 4PBA ameliorates fluoride-induced ER stress in ameloblasts. To determine whether 4PBA protects ameloblasts from fluoride toxicity, we analyzed gene expression of Tgf-β1, Bcl2/Bax ratio and cytochrome-c release in vitro. In vivo, we measured fluorosis levels, enamel hardness and fluoride concentration. Fluoride treated Ameloblast-lineage cells (ALC) had decreased Tgf-β1 expression and this was reversed by 4PBA treatment. The anti-apoptotic Blc2/Bax ratio was significantly increased in ALC cells treated with fluoride/4PBA compared to fluoride treatment alone. Fluoride treatment induced cytochrome-c release from mitochondria into the cytosol and this was inhibited by 4PBA treatment. These results suggest that 4PBA mitigates fluoride-induced gene suppression, apoptosis and mitochondrial damage in vitro. In vivo, C57BL/6J mice were provided fluoridated water for six weeks with either fluoride free control-chow or 4PBA-containing chow (7 g/kg 4PBA). With few exceptions, enamel microhardness, fluorosis levels, and fluoride concentrations of bone and urine did not differ significantly between fluoride treated animals fed with control-chow or 4PBA-chow. Although 4PBA mitigated high-dose fluoride toxicity in vitro, a diet rich in 4PBA did not attenuate dental fluorosis in rodents. Perhaps, not enough intact 4PBA reaches the rodent ameloblasts necessary to reverse the effects of fluoride toxicity. Further studies will be required to optimize protocols for 4PBA administration in vivo in order to evaluate the effect of 4PBA on dental fluorosis.

Beta-catenin is essential for ameloblast movement during enamel development.

Beta-catenin is a multifunctional protein that plays key roles in cadherin-based cell adherens junctions and in the Wnt signaling pathway. The canonical Wnt/β-catenin pathway can regulate transcription factors that control cell movement/invasion. We investigated whether β-catenin regulates ameloblast movement through canonical Wnt signaling. The morphological and physical properties of enamel were assessed in enamel from control and β-catenin conditional knockout (cKO) mice. Ameloblast-lineage cells (ALC) were used to investigate the potential roles of β-catenin in cell migration and in E-cadherin expression. Compared with controls, incisors from β-catenin cKO mice were short, blunt, and where enamel was present, it was soft and malformed. Scanning electron microscopy revealed a dysplastic rod pattern within the enamel of incisors from β-catenin cKO mice, and Vickers microhardness measurements confirmed that mice with β-catenin ablated from their enamel organ had enamel that was significantly softer than normal. Amelogenesis was disrupted in the absence of β-catenin and the ameloblasts did not differentiate properly. We further demonstrated that migration of ALCs was inhibited in vitro and that E-cadherin expression was significantly up-regulated when ALCs were treated with the β-catenin inhibitor, ICG-001. Beta-catenin ablation causes enamel malformation in mice and this phenotype may occur, in part, by a lack of ameloblast differentiation and/or movement necessary to form the decussating enamel rod structure.

P38/JNK signaling pathway mediates the fluoride-induced down-regulation of Fam83h

Background/AimThe similar clinical and pathological feature in fluorosis and amelogenesis imperfect with FAM83H mutations imply that excess fluoride could have effects on the expression of FAM83H and could elaborate this process by some signal pathways regulation. The present study aims to investigate the effects of fluoride on Fam83h expression and try to explore the molecular signaling regulation between them as well as the association of high concentration fluoride with mineralization in ameloblast lineage cells. MethodsProtein expression and signaling pathways of mouse ameloblast-like LS8 cells, exposed to fluoride or MAPK inhibitors, were compared to control cells without exposure. Fam83h, proteins of MAPK signal pathways (ERK, P38 and JNK) were examined by Quantitative real-time PCR and/or Western-blot. ALP activity and ALP staining were used to detect the mineralization in the cells with exposure during 7-day mineralization inducing differentiation. ResultsThe results showed that Fam83h protein level in LS8 cells decreased in the presence of fluoride and MAPK inhibitors. Down-regulation of Fam83h by fluoride was related to suppression of JNK and P38 phosphorylation, and the descending degree of P38 was more obvious. Fluoride and MAPK inhibitors treatment significantly decreased the mineralization level in LS8 cells. ConclusionThe findings suggest that JNK and P38 could be key regulatory element for Fam83h expression, and that LS8 cells can respond to fluoride by down-regulating Fam83h expression through the regulation of JNK and p38 signaling pathways.

Isolation and characterization of embryonic ameloblast lineage cells derived from tooth buds of fetal miniature swine.

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

Wnt-RhoA Signaling Pathways in Fluoride-Treated Ameloblast-Lineage Cells

This study examined the effect of sodium fluoride (NaF) on the Wnt and RhoA signaling pathways in murine ameloblast-lineage cells (ALCs) to better understand the developmental mechanisms of dental fluorosis. Wnt and Rho pathway activities were investigated when ALCs were treated with 1.5 m<smlcap>M</smlcap> NaF, dickkopf-related protein-1 (Dkk-1), secreted frizzled related-protein-2 (sFRP-2), β-catenin siRNA dominant negative RhoA (RhoA^DN) plasmid and Y-27632. Wnt pathway activity was investigated via RT-PCR, Western blot and Topflash luciferase assay. The activity of the RhoA pathway was analyzed via Rho pull-down assay and immunoprecipitation. The differentiation of ALCs was analyzed by alkaline phosphatase assay. Western blot and Topflash luciferase assay results verified that both the Wnt and Rho pathways were upregulated by 1.5 m<smlcap>M</smlcap> NaF. Wnt was discovered to be located upstream from the Rho pathway, as confirmed by treatment with Wnt pathway cell receptor inhibitors Dkk-1 and sFRP-2, leading to a decrease in RhoA and ROCK activity. Inhibition of the Rho pathway with RhoA^DN plasmid and Y-27632 caused upregulation of Wnt pathway activity which could be further increased by 1.5 m<smlcap>M</smlcap> NaF. The increased Wnt pathway activity was found to negatively regulate ALC differentiation. These data suggest that fluoride could induce the cross-talk between Wnt and RhoA signaling pathways, and these responses are predicted to contribute to the development of enamel fluorosis.

Fluorosed mouse ameloblasts have increased SATB1 retention and Gαq activity.

Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF3 led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.

Transforming growth factor-β1 regulates expression of the matrix metalloproteinase 20 (Mmp20) gene through a mechanism involving the transcription factor, myocyte enhancer factor-2C, in ameloblast lineage cells

Matrix metalloproteinase-20 (Mmp20) plays an essential role in amelogenesis during tooth development and is regulated by transforming growth factor-β1 (TGF-β1) in mouse ameloblast lineage cells (ALCs). The objective of this study was to explore the role of myocyte enhancer factor-2C (MEF2C), a key transcription factor in craniofacial development, in TGF-β1-induced Mmp20 gene expression. We investigated Mmp20 expression in ALCs over-expressing MEF2C and in ALCs with MEF2C knocked down. We also analyzed activity of the Mmp20 promoter using a transient reporter gene-expression assay in cultured ALCs. Putative transcription factor-binding sites for MEF2C and TGF-β1 on the Mmp20 promoter were analyzed with bioinformatics tools and examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The expression of Mmp20 was induced, in a dose-dependent manner, by MEF2C over-expression, and TGF-β1-induced Mmp20 expression was blocked by MEF2C knockdown in ALCs. There was a TGF-β1/MEF2C-responsive region, including a putative MEF2-binding site, between base pairs -356 and -73 of the Mmp20 promoter. Mutation of the putative MEF2-binding site significantly reduced Mmp20 promoter activity upon activation with MEF2C or TGF-β1. In conclusion, TGF-β1-induced Mmp20 expression in ALCs was regulated through the MEF2-binding site on the Mmp20 promoter and thus mediated by the MEF2C signaling pathway.

Inductive Ability of Human Developing and Differentiated Dental Mesenchyme

The development of cell-based therapeutic strategies to bioengineer tooth tissue is a promising approach for the treatment of lost or damaged tooth tissue. The lack of a readily available cell source for human dental epithelial cells (ECs) severely constrains the progress of tooth bioengineering. Previous studies in model organisms have demonstrated that developing dental mesenchyme can instruct nondental epithelium to differentiate into enamel-forming epithelium. In this study, we characterized the ability of fetal and adult human dental mesenchyme to promote differentiation of human embryonic stem cell (hESC)-derived ECs (ES-ECs) into ameloblast-lineage cells. ES-ECs were co-cultured either with human fetal dental mesenchymal cells (FDMCs) or with adult dental mesenchymal cells (ADMCs) in either a three-dimensional culture system, or in the renal capsules of SCID mice. When co-cultured with FDMCs in vitro, ES-ECs polarized and expressed amelogenin. Tooth organ-like structures assembled with epithelium and encased mesenchyme and developing enamel-like structures could be detected in the complexes resulting from in vitro and ex vivo co-culture of ES-ECs and FDMCs. In contrast, co-cultured ES-ECs and ADMCs formed amorphous spherical structures and occasionally formed hair. Transcription factors were significantly upregulated in FDMCs compared to ADMCs including MSX1, GLI1, LHX6, LHX8,LEF1 and TBX1. In summary, FDMCs but not ADMCs had the capacity to induce differentiation of ES-ECs into ameloblast lineage cells. Further characterization of the functional differences between these two types of dental mesenchyme could enable reprogramming of ADMCs to enhance their odontogenic inductive competence.

Bcl11b transcription factor plays a role in the maintenance of the ameloblast-progenitors in mouse adult maxillary incisors

Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11bKO/KO mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11bKO/KO mice. Here, we examined compound heterozygous Bcl11bS826G/KO mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11bS826G/KO incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11bS826G/KO incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial–mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells.