ABSTRACT
Fluoride is a double-edged sword. It was widely used for early caries prevention while excessive intake caused a toxicology effect, affected enamel development, and resulted in dental fluorosis. The study aimed to evaluate the protective effect and mechanism of Epigallocatechin-3-gallate (EGCG) on the apoptosis induced by fluoride in ameloblast-like cells. We observed that NaF triggered apoptotic alterations in cell morphology, excessive NaF arrested cell cycle at the G1, and induced apoptosis by up-regulating Bax and down-regulating Bcl-2. NaF activated the insulin-like growth factor receptor (IGFR), and phosphatidylinositol-3-hydroxylase (p-PI3K), while dose-dependently down-regulating the expression of Forkhead box O1 (FoxO1). EGCG supplements reversed the changes in LS8 morphology, the cell cycle, and apoptosis induced by fluoride. These results indicated that EGCG possesses a protective effect against fluoride toxicity. Furthermore, EGCG suppressed the activation of p-PI3K and the down-regulation of FoxO1 caused by fluoride. Collectively, our findings suggested that EGCG attenuated fluoride-induced apoptosis by inhibiting the PI3K/FoxO1 signaling pathway. EGCG may serve as a new alternative method for dental fluorosis prevention, control, and treatment.
Subject(s)
Ameloblasts , Apoptosis , Catechin , Fluorides , Phosphatidylinositol 3-Kinases , Signal Transduction , Catechin/analogs & derivatives , Catechin/pharmacology , Apoptosis/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Animals , Fluorides/toxicity , Fluorides/pharmacology , Ameloblasts/drug effects , Ameloblasts/metabolism , Signal Transduction/drug effects , Forkhead Box Protein O1/metabolism , Cell Line , Mice , Sodium Fluoride/toxicity , Fluorosis, DentalABSTRACT
BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.
Subject(s)
Calcium , Dietary Supplements , Fluorosis, Dental , Animals , Male , Mice , Activating Transcription Factor 6/metabolism , Adenine/analogs & derivatives , Ameloblasts/metabolism , Ameloblasts/pathology , Ameloblasts/drug effects , Anoctamin-1/metabolism , Anoctamin-1/antagonists & inhibitors , Anoctamin-1/genetics , Calcium/metabolism , Disease Models, Animal , eIF-2 Kinase/metabolism , eIF-2 Kinase/genetics , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/metabolism , Fluorides/toxicity , Fluorides/adverse effects , Fluorosis, Dental/pathology , Fluorosis, Dental/metabolism , Fluorosis, Dental/etiology , Indoles , Mice, Inbred C57BL , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
Fluoride (F) and sulfur dioxide (SO2) contamination is recognized as a public health concern worldwide. Our previous research has shown that Co-exposure to F and SO2 can cause abnormal enamel mineralization. Ameloblastin (AMBN) plays a crucial role in the process of enamel mineralization. However, the process by which simultaneous exposure to F and SO2 influences enamel formation by regulating AMBN expression still needs to be understood. This study aimed to establish in vivo and in vitro models of F-SO2 Co-exposure and investigate the relationship between AMBN and abnormal enamel mineralization. By overexpressing/knocking out the Fibroblast Growth Factor 9 (FGF9) gene, we investigated the impact of FGF9-mediated Mitogen-Activated Protein Kinase (MAPK) signaling on AMBN synthesis to elucidate the mechanism underlying the induction of abnormal enamel mineralization by F-SO2 Co-exposure in rats. The results showed that F-SO2 exposure damaged the structure of rat enamel and ameloblasts. When exposed to F or SO2, gradual increases in the protein expression of FGF9 and phosphorylated p38 mitogen-activated protein kinase (p-P38) were observed. Conversely, the protein levels of AMBN, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) were decreased. AMBN expression was significantly correlated with FGF9, p-ERK, and p-JNK expression in ameloblasts. Interestingly, FGF9 overexpression reduced the levels of p-ERK and p-JNK, worsening the inhibitory effect of F-SO2 on AMBN. Conversely, FGF9 knockout increased the phosphorylation of ERK and JNK, partially reversing the F-SO2-induced downregulation of AMBN. Taken together, these findings strongly demonstrate that FGF9 plays a critical role in F-SO2-induced abnormal enamel mineralization by regulating AMBN synthesis through the JNK and ERK pathways.
Subject(s)
Dental Enamel , Fibroblast Growth Factor 9 , Fluorides , MAP Kinase Signaling System , Sulfur Dioxide , Animals , Fibroblast Growth Factor 9/genetics , Fibroblast Growth Factor 9/metabolism , Rats , Fluorides/toxicity , MAP Kinase Signaling System/drug effects , Dental Enamel/drug effects , Sulfur Dioxide/toxicity , Male , Rats, Sprague-Dawley , Dental Enamel Proteins/genetics , Dental Enamel Proteins/metabolism , Tooth Calcification/drug effects , Ameloblasts/drug effects , Ameloblasts/metabolismABSTRACT
Most cells use calcium (Ca2+) as a second messenger to convey signals that affect a multitude of biological processes. The ability of Ca2+ to bind to proteins to alter their charge and conformation is essential to achieve its signaling role. Cytosolic Ca2+ (cCa2+) concentration is maintained low at ~100 nM so that the impact of elevations in cCa2+ is readily sensed and transduced by cells. However, such elevations in cCa2+ must be transient to prevent detrimental effects. Cells have developed a variety of systems to rapidly clear the excess of cCa2+ including Ca2+ pumps, exchangers and sequestering Ca2+ within intracellular organelles. This Ca2+ signaling toolkit is evolutionarily adapted so that each cell, tissue, and organ can fulfill its biological function optimally. One of the most specialized cells in mammals are the enamel forming cells, the ameloblasts, which also handle large quantities of Ca2+. The end goal of ameloblasts is to synthesize, secrete and mineralize a unique proteinaceous matrix without the benefit of remodeling or repair mechanisms. Ca2+ uptake into ameloblasts is mainly regulated by the store operated Ca2+ entry (SOCE) before it is transported across the polarized ameloblasts to reach the insulated enamel space. Here we review the ameloblasts Ca2+ signaling toolkit and address how the common electronegative non-metal fluoride can alter its function, potentially addressing the biology of dental fluorosis.
Subject(s)
Ameloblasts/metabolism , Calcification, Physiologic/drug effects , Calcium/metabolism , Dental Sac/metabolism , Epithelial Cells/metabolism , Fluorides/pharmacology , Ameloblasts/drug effects , Animals , Dental Sac/drug effects , Epithelial Cells/drug effects , HumansABSTRACT
TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.
Subject(s)
Ameloblasts/metabolism , Calcium/metabolism , TRPM Cation Channels/metabolism , Ameloblasts/cytology , Ameloblasts/drug effects , Anilides/pharmacology , Animals , Cell Line , Humans , Hydrogen-Ion Concentration , Incisor/cytology , Ion Channel Gating/drug effects , Ion Transport/drug effects , Mibefradil/pharmacology , Mice , Models, Biological , Naltrexone/analogs & derivatives , Naltrexone/pharmacology , Rats , Thiadiazoles/pharmacologyABSTRACT
The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-ß1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-ß1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-ß1 stimulation. These results suggested that TGF-ß1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.
Subject(s)
Ameloblasts/metabolism , Tooth Germ/metabolism , Transcription Factors, General/metabolism , Transcription, Genetic , Ameloblasts/drug effects , Animals , Cell Differentiation , Cell Line , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Developmental , Mice, Knockout , Morphogenesis , Phosphorylation , Rats , Signal Transduction , Smad Proteins, Receptor-Regulated/metabolism , Tooth Germ/cytology , Tooth Germ/drug effects , Transcription Factors, General/genetics , Transcription, Genetic/drug effects , Transforming Growth Factor beta1/pharmacologyABSTRACT
Amoxicillin is a common pediatric drug. However, to the best of our knowledge, the role of amoxicillin in enamel hypomineralization has not yet been fully elucidated. The aim of the present study was to assess the effects of amoxicillin on enamel mineralization, the morphology of ameloblasts, as well as the expression of kallikreinrelated peptidase 4 (KLK4), and the tight junction proteins, claudin 1 (CLDN1), claudin 4 (CLDN4) and occludin (OCLN), in ameloblasts of juvenile mice. A total of 36 3dayold Kunming mice were randomly divided into three groups. The mice were administered 0, 50 or 100 mg/kg amoxicillin by intragastric administration for 19 days. The surface morphology and calcium (Ca), phosphorous (P) and carbon contents of mandibular incisors and first molars were examined by scanning electron microscopy and energy dispersive Xray spectroscopy. Histological changes in the ameloblasts of mandibular incisors were analyzed by hematoxylin and eosin staining. The KLK4, CLDN1, CLDN4 and OCLN expression levels of ameloblasts were observed by immunohistochemical staining. The incidence of white patches in the incisor was 100% in the 100 mg/kg amoxicillintreated groups. A greater number of enamel defects were observed in the incisal/occlusal half of mandibular incisors/molars compared with in the cervical half in the amoxicillintreated groups. Following phosphoricacid treatment, the enamel rod and interrod were aligned in a disorderly manner in the amoxicillintreated groups. Amoxicillin decreased the Ca/P ratio in the enamel of mandibular incisors and molars. More intercellular spaces among maturation ameloblasts were observed in the amoxicillintreated groups. Amoxicillin decreased KLK4 and CLDN1, CLDN4 and OCLN expression in mature ameloblasts. The administration of amoxicillin in juvenile mice induced enamel hypomineralization, and the effects of amoxicillin on enamel hypomineralization may be mediated via multiple pathways.
Subject(s)
Ameloblasts/drug effects , Ameloblasts/metabolism , Amoxicillin/pharmacology , Tight Junction Proteins/metabolism , Animals , Body Weight/drug effects , Claudin-1/metabolism , Claudin-4/metabolism , Dental Enamel/drug effects , Dental Enamel/metabolism , Female , Immunohistochemistry , Kallikreins/metabolism , Mice , Microscopy, Electron, Scanning , Occludin/metabolismABSTRACT
Calcium (Ca2+) release-activated Ca2+ (CRAC) channels mediated by STIM1/2 and ORAI (ORAI1-3) proteins form the dominant store-operated Ca2+ entry (SOCE) pathway in a wide variety of cells. Among these, the enamel-forming cells known as ameloblasts rely on CRAC channel function to enable Ca2+ influx, which is important for enamel mineralization. This key role of the CRAC channel is supported by human mutations and animal models lacking STIM1 and ORAI1, which results in enamel defects and hypomineralization. A number of recent reports have highlighted the role of the chanzyme TRPM7 (transient receptor potential melastanin 7), a transmembrane protein containing an ion channel permeable to divalent cations (Mg2+, Ca2+), as a modulator of SOCE. This raises the question as to whether TRPM7 should be considered an alternative route for Ca2+ influx, or if TRPM7 modifies CRAC channel activity in enamel cells. To address these questions, we monitored Ca2+ influx mediated by SOCE using the pharmacological TRPM7 activator naltriben and the inhibitor NS8593 in rat primary enamel cells and in the murine ameloblast cell line LS8 cells stimulated with thapsigargin. We also measured Ca2+ dynamics in ORAI1/2-deficient (shOrai1/2) LS8 cells and in cells with siRNA knock-down of Trpm7. We found that primary enamel cells stimulated with the TRPM7 activator potentiated Ca2+ influx via SOCE compared to control cells. However, blockade of TRPM7 with NS8593 did not decrease the SOCE peak. Furthermore, activation of TRPM7 in shOrai1/2 LS8 cells lacking SOCE failed to elicit Ca2+ influx, and Trpm7 knock-down had no effect on SOCE. Taken together, our data suggest that TRPM7 is a positive modulator of SOCE potentiating Ca2+ influx in enamel cells, but its function is fully dependent on the prior activation of the ORAI channels.
Subject(s)
Calcium/metabolism , Dental Enamel/cytology , ORAI1 Protein/metabolism , TRPM Cation Channels/metabolism , Ameloblasts/drug effects , Ameloblasts/metabolism , Animals , Cell Line , Female , Male , Mice , Naltrexone/analogs & derivatives , Naltrexone/pharmacology , RNA, Small Interfering/metabolism , Rats, Sprague-DawleyABSTRACT
OBJECTIVES: To investigate the effect and mechanism of calcium on LS8 cell differentiation, especially on phosphatidylinositol 3 kinase (PI3K) /protein kinase B(AKT) pathway. MATERIALS AND METHODS: Ameloblast-like LS8 cell line was used and additional 0-3.5 mmol/L calcium chloride was treated for 24 h, 48 h. Cell viability and morphological changes, cell cycle and associated regulatory proteins were analyzed. RESULTS: No significant effects on morphological changes were observed. Decreased cell viability and increased S phase cells were accompanied by the significant decrease of cyclin A and cyclin B proteins, and significant increase of cyclin D protein in LS8 cells. Additionally, kallikrein-4 and amelotin expressions were significantly increased. Finally, the levels of PI3K, AKT, p-AKT and forkhead box O3 (FOXO3) significantly downregulated after calcium treatment in LS8 cells. CONCLUSIONS: Calcium inhibit proliferation and promotes differentiation in LS8 cells, this is closely related to the downregulation of PI3K/AKT signal in LS8 cells.
Subject(s)
Ameloblasts/enzymology , Calcium/pharmacology , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Ameloblasts/drug effects , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Down-Regulation , MiceABSTRACT
Fluoride overexposure is an environmental health hazard and can cause enamel and skeletal fluorosis. Previously we demonstrated that fluoride increased acetylated-p53 and its downstream target p21 in ameloblast-derived LS8 cells. However, p21 function in fluoride toxicity is not well characterized. This study seeks to gain a better understanding of how p53 down-stream mediators, p21 and MDM2, respond to fluoride toxicity. LS8 cells were treated with NaF with/without MG-132 (proteasome inhibitor) or Nutlin-3a (MDM2 antagonist). NaF treatment for 2-6 h increased phospho-p21, which can inhibit apoptosis. However, phospho-p21 and p21 were decreased by NaF at 24 h, even though p21 mRNA was significantly increased at this time point. MG-132 reversed the fluoride-mediated p21 decrease, indicating that fluoride facilitates p21 proteasomal degradation. MG-132 suppressed fluoride-induced caspase-3 cleavage, suggesting that the proteasome plays a pro-apoptotic role in fluoride toxicity. NaF increased phospho-MDM2 in vitro and in mouse ameloblasts in vivo. Nutlin-3a suppressed NaF-mediated MDM2-p21 binding to reverse p21 degradation which increased phospho-p21. This suppressed apoptosis after 24 h NaF treatment. These results suggest that MDM2-mediated p21 proteasomal degradation with subsequent phospho-p21 attenuation contributes to fluoride-induced apoptosis. Inhibition of MDM2-mediated p21 degradation may be a potential therapeutic target to mitigate fluoride toxicity.
Subject(s)
Ameloblasts/drug effects , Ameloblasts/metabolism , Apoptosis/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Proto-Oncogene Proteins c-mdm2 , Sodium Fluoride/toxicity , Ameloblasts/cytology , Animals , Cell Line , Cyclin-Dependent Kinase Inhibitor p21/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p21/physiology , Dental Enamel/cytology , Dental Enamel/drug effects , Dental Enamel/metabolism , Imidazoles/pharmacology , Leupeptins/pharmacology , Mice , Mice, Inbred C57BL , Piperazines/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolismABSTRACT
In the development of dental fluorosis, oxidative stress is considered as the key mechanism. Endoplasmic reticulum (ER) stress can induce oxidative stress and activate the important antioxidative factor nuclear factor erythroid 2-related factor 2 (Nrf2) in a PKR-like ER kinase (PERK)-dependent manner, but combining ER stress and oxidative stress, the role of PERK-Nrf2 signaling pathway involved in fluoride-regulated ameloblasts is not fully defined. Here, we studied the effect of fluoride on PERK-Nrf2 signaling pathway in mouse ameloblasts. We found that low-dose and continuous fluoride exposure increased binding immunoglobulin protein expression and activated PERK-activating transcription factor 4 signaling pathway. Meanwhile, the expression of Nrf2 and its target genes (glutamylcysteine synthetase and glutathione S-transferase-P1) enhanced following ER stress. Tunicamycin increased the expression of PERK, leading to Nrf2 nuclear import, and tauroursodeoxycholate suppressed Nrf2 activation through PERK during ER stress, indicating that PERK activation is required for Nrf2 nuclear entry. Furthermore, tert-butylhydroquinone triggered the overexpression of Nrf2 to reduce ER stress, but luteolin inhibited Nrf2 nuclear localization to elevate ER stress. In summary, this study proved that fluoride under certain dose can induce ER stress and promote Nrf2 nuclear import via PERK activation and suggested that antioxidation mechanism mediated by PERK-Nrf2 can alleviate fluoride-induced ER stress effectively.
Subject(s)
Ameloblasts/drug effects , Fluorides/toxicity , NF-E2-Related Factor 2/metabolism , eIF-2 Kinase/metabolism , Ameloblasts/metabolism , Animals , Cells, Cultured , Endoplasmic Reticulum Stress/drug effects , Mice , NF-E2-Related Factor 2/genetics , Oxidative Stress/drug effects , Signal Transduction/drug effects , eIF-2 Kinase/geneticsABSTRACT
Fluorine is one of the trace elements necessary for health. It has many physiological functions, and participates in normal metabolism. However, fluorine has paradoxical effects on the body. Many studies have shown that tissues and organs of humans and animals appear to suffer different degrees of damage after long-term direct or indirect exposure to more fluoride than required to meet the physiological demand. Although the aetiology of endemic fluorosis is clear, its specific pathogenesis is inconclusive. In the past 5 years, many researchers have conducted in-depth studies into the pathogenesis of endemic fluorosis. Research in the areas of fluoride-induced stress pathways, signalling pathways and apoptosis has provided further extensive knowledge at the molecular and genetic level. In this article, we summarize the main results.
Subject(s)
Apoptosis/drug effects , Fluorides/adverse effects , Fluorosis, Dental/epidemiology , Ameloblasts/drug effects , Ameloblasts/pathology , Fluorosis, Dental/etiology , Fluorosis, Dental/genetics , Fluorosis, Dental/pathology , Humans , Signal Transduction/drug effectsABSTRACT
Introduction: Preameloblast-conditioned medium (PA-CM), as a mixture of dental epithelium-derived factors, has been reported to regenerate dentin and periodontal tissues in vitro and in vivo. The aim of this study was to investigate the biological effect of Cpne7 on the proliferation, migration, and cementoblast differentiation of periodontal cells in vitro, and on the regeneration of periodontal tissue using periodontal defect model with canine in vivo. Materials and methods: The effect of Cpne7 on cell proliferation, migration, and cementoblast differentiation of periodontal cells were evaluated in vitro. A periodontal defect canine model was designed and the defects were divided into five groups: Group 1: No treatment (negative control), Group 2: Collagen carrier only, Group 3: PA-CM with collagen carrier (positive control), Group 4: PA-CM + CPNE7 Antibody (Ab) with collagen carrier, and Group 5: recombinant CPNE7 (rCPNE7) protein with collagen carrier. Results: Cpne7 was expressed in HERS cells and periodontal ligament (PDL) fibers. By real-time PCR, Cpne7 increased expression of Cap compared to the control. In the periodontal defect canine model, rCPNE7 or PA-CM regenerated periodontal complex, and the arrangement of the newly formed PDL-like fibers were perpendicular to the newly formed cementum and alveolar bone like Sharpey's fibers in natural teeth, while PA-CM + CPNE7 Ab showed irregular arrangement of the newly formed PDL-like fibers compared to the rCPNE7 or PA-CM group. Conclusion: These findings suggest that Cpne7 may have a functional role in periodontal regeneration by supporting periodontal cell attachment to cementum and facilitating physiological arrangement of PDL fibers.
Subject(s)
Membrane Proteins/metabolism , Periodontium/physiology , Regeneration , Adolescent , Ameloblasts/cytology , Ameloblasts/drug effects , Animals , Cell Differentiation/drug effects , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Cementogenesis/drug effects , Culture Media, Conditioned/pharmacology , Dogs , Humans , Mice , Periodontium/cytology , Recombinant Proteins/pharmacology , Regeneration/drug effects , Tooth/growth & development , Tooth/metabolism , Young AdultABSTRACT
PURPOSE: To investigate the amelogenesis-inductive effects of surface structures at the nanoscale. For this purpose, variable nanostructured titanium dioxide (TiO2) surfaces were used as a framework to regulate the amelogenic behaviors of ameloblasts with the administration of retinoic acid (RA)/dexamethasone (DEX). MATERIALS AND METHODS: TiO2 nanotubular (NT) surfaces were fabricated via anodization. Mouse ameloblast-like LS-8 cells were seeded and cultured on NT surfaces in the presence or absence of RA/DEX for 48 h. The amelogenic behaviors and extracellular matrix (ECM) mineralization of LS-8 cells on nanostructured Ti surfaces were characterized using field emission scanning electron microscope, laser scanning confocal microscope, quantitative polymerase chain reaction, MTT assay, and flow cytometry. RESULTS: TiO2 NT surfaces (tube size ~30 and ~80 nm) were constructed via anodization at 5 or 20 V and denoted as NT5 and NT20, respectively. LS-8 cells exhibited significantly increased spread and proliferation, and lower rates of apoptosis and necrosis on NT surfaces. The amelogenic gene expression and ECM mineralization differed significantly on the NT20 and the NT5 and polished Ti sample surfaces in standard medium. The amelogenic behaviors of LS-8 cells were further changed by RA/DEX pretreatment, which directly drove maturation of LS-8 cells. CONCLUSION: Controlling the amelogenic behaviors of ameloblast-like LS-8 cells by manipulating the nanostructure of biomaterials surfaces represents an effective tool for the establishment of a systemic framework for supporting enamel regeneration. The administration of RA/DEX is an effective approach for driving the amelogenesis and maturation of ameloblasts.
Subject(s)
Amelogenesis/drug effects , Cell Differentiation/drug effects , Dexamethasone/pharmacology , Nanostructures/chemistry , Titanium/pharmacology , Tretinoin/pharmacology , Ameloblasts/cytology , Ameloblasts/drug effects , Ameloblasts/metabolism , Amelogenesis/genetics , Animals , Apoptosis/drug effects , Calcification, Physiologic/drug effects , Cell Adhesion/drug effects , Cell Line , Cell Proliferation/drug effects , Cell Shape/drug effects , Cells, Cultured , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Gene Expression Regulation/drug effects , Mice , Surface PropertiesABSTRACT
Decreased expression and increased phosphorylation of Forkhead box o1 (Foxo1) in ameloblasts were observed both in vivo and in vitro when treated by fluoride. The present study aims to investigate the possible relationship between Foxo1 and enamel matrix proteinases, matrix metalloproteinase 20 (MMP20), and kallikrein 4 (KLK4), in NaF-treated ameloblasts. Ameloblast-like cells (LS8 cells) were exposed to NaF at selected concentration (0/2 mM) for 24 h. Gene overexpression and silencing experiments were used to up- and down-regulate Foxo1 expression. The expression levels of Foxo1, MMP20, and KLK4 were detected by quantitative real-time PCR and western blot. Dual luciferase reporter assay was performed to evaluate the regulation of Foxo1 on the transcriptional activity of KLK4 promoter. The results showed that KLK4 expression was decreased in LS8 cells treated by NaF, while MMP20 expression was not changed. Foxo1 activation led to significantly up-regulation of KLK4 in LS8 cells under NaF condition. Knockout of Foxo1 markedly decreased klk4 expression in mRNA level, and intensified inhibition occurred in LS8 cells when combined with NaF treatment. However, the variation trend of MMP20 was not clear. Dual luciferase reporter assay showed that Foxo1 activation enhanced the transcriptional activity of KLK4 promoter. These findings suggest that the decrease of Foxo1 expression induced by high fluoride was a cause for low KLK4 expression.
Subject(s)
Ameloblasts/drug effects , Forkhead Box Protein O1/genetics , Gene Expression/drug effects , Kallikreins/genetics , Sodium Fluoride/pharmacology , Ameloblasts/cytology , Ameloblasts/metabolism , Animals , Cell Line , Forkhead Box Protein O1/metabolism , Kallikreins/metabolism , Matrix Metalloproteinase 20/genetics , Matrix Metalloproteinase 20/metabolism , MiceABSTRACT
Cancer is the second most frequent cause of death in children. Because the prognosis for childhood malignancies has improved, attention has now focused on long-term consequences of cancer treatment. The immediate effects of chemotherapy on soft tissues have been well described; however, there is less information about long-term effects of chemotherapy on the development of dental tissues. To test the association between the effect of chemotherapy on enamel development, we examined two groups of rats: one that had received an intraperitoneal dose of 200 mg/kg of irinotecan, whereas the other (control) group had received vehicle only. Rats were killed at 6, 48 and 96 hr post-injection; the mandibles dissected out, fixed for histological evaluation and scanned for mineralization defects by Micro-CT. Our results showed structural changes in the ameloblast layer along with a significant reduction in mineralization and thickness of enamel at 96 hr after chemotherapy. These data demonstrate that irinotecan induces structural changes in forming enamel that become apparent after anticancer chemotherapy treatment.
Subject(s)
Ameloblasts/drug effects , Amelogenesis/drug effects , Antineoplastic Agents/adverse effects , Dental Enamel/drug effects , Incisor/drug effects , Irinotecan/adverse effects , Ameloblasts/pathology , Animals , Calcification, Physiologic/drug effects , Dental Enamel/diagnostic imaging , Dental Enamel/growth & development , Dental Enamel/pathology , Female , Incisor/diagnostic imaging , Incisor/growth & development , Incisor/pathology , Injections, Intraperitoneal , Mandible/diagnostic imaging , Mandible/drug effects , Mandible/growth & development , Mandible/pathology , Rats , Rats, Wistar , X-Ray MicrotomographyABSTRACT
Fluoride has toxic potential particularly for teeth, bones, and kidney. This study was aimed to investigate the NaF exposure effects on the growth of ameloblasts and kidney proximal tubular cells. Adult male healthy rats were used as experiment models, divided into control and NaF-induced groups. The expression of amelogenin, Bcl-2, and caspase-3 were significantly different in the control and NaF-induced group (P < 0.05). There was no correlation among these proteins in the control group but significant correlation in the NaF-induced group (r = 0.694). There was a significant correlation in proximal tubular cells, as seen from the increase of caspase-3 in the NaF-induced group (r = 0.715).
Subject(s)
Ameloblasts/cytology , Kidney Tubules, Proximal/cytology , Sodium Fluoride/pharmacology , Ameloblasts/drug effects , Ameloblasts/metabolism , Animals , Caspase 3/metabolism , Cell Proliferation/drug effects , Epithelial Cells/metabolism , Immunohistochemistry , Male , Proto-Oncogene Proteins c-bcl-2/metabolism , RatsABSTRACT
Supraoptimal intake of fluoride (F) induces structural defects in forming enamel, dentin and bone and increases the risk of bone fractures. In comparison to bone and dentin is formation of enamel most sensitive to low levels of F and the degree of enamel fluorosis depends on the mouse strain. What molecular mechanism is responsible for these differences in sensitivity is unclear. Maturation ameloblasts transport bicarbonates into enamel in exchange for Cl- to buffer protons released by forming apatites. We proposed that F-enhanced mineral deposition releases excess of protons that will affect mineralization in forming enamel. In this study we tested the hypothesis that increased sensitivity to F is associated with a reduced capacity of ameloblasts to buffer acids. Quantified electron probe microanalysis showed that enamel of F-sensitive C57Bl mice contained the same levels of Cl- as enamel of F-resistant FVB mice. Enamel of C57Bl mice was less mineral dense, contained less Ca but more Mg and K. Ameloblast modulation was much more impaired than in FVB mice. In enamel of FVB mice the levels of Mg correlated negative with Ca (r=-0.57, p=0.01) and with the Ca/P molar ratio (r=-0.32, p=0.53). In moderate and high acidic enamel the correlations between Mg and Ca/P ratio were strong (r=-0.75, p=0.08) to very strong negative (r=-0.98, p=0.0020), respectively. Correlations in enamel between F and Ca were (weak) negative but between F and Ca/P very high positive (r=+0.95, p=0.003) in high acidic enamel and less positive (r=0.45, p=0.27) in moderate acidic fluorotic enamel (r=0.45, p=0.27). Similar correlations between Mg and Ca/P or F and Ca/P were found in dentin and bone of fluorotic and Cftr null mice. These data are consistent with the concept that Mg delays but F increases maturation of crystals particularly when enamel is acidic. The sensitivity of forming enamel to F likely is due to the sensitivity of pH cycling to acidification of enamel associated with F-induced release of protons.
Subject(s)
Ameloblasts/drug effects , Ameloblasts/metabolism , Fluorides/pharmacology , Magnesium/metabolism , Animals , Bone and Bones/metabolism , Calcium/metabolism , Chlorides/metabolism , Dental Enamel/drug effects , Dental Enamel/metabolism , Dentin/metabolism , Hydrogen-Ion Concentration , Mice, Inbred C57BL , Minerals/metabolism , Potassium/metabolismABSTRACT
Fluoride is an environmental toxicant and induces dental fluorosis and oxidative stress. Lycopene (LYC) is an effective antioxidant that is reported to attenuate fluoride toxicity. To determine the effects of LYC on sodium fluoride (NaF) -induced teeth and ameloblasts toxicity, rats were treated with NaF (10 mg/kg) and/or LYC (10 mg/kg) by orally administration for 5 weeks; ameloblasts were treated with NaF (5 mM) and/or LYC (2 µM) for 6 h. We found that the concentrations of fluoride, malondialdehyde (MDA) and reactive oxygen species (ROS), gene expressions and activities of Caspase-9 and Caspase-3, and the gene expressions of Bax were significantly decreased, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX), the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated rats group; concentrations of MDA and ROS, gene expressions and activities of Caspase-9 and Caspase-3, and the gene expression of Bax, and ameloblasts apoptosis rate were significantly decreased, while the activities of SOD and GPX, the gene expression of Bcl-2 were significantly increased in the LYC + NaF-treated ameloblasts group. These results suggest that LYC significantly combated NaF-induced ameloblasts apoptosis and dental fluorosis by attenuation oxidative stress and down-regulation Caspase pathway.
Subject(s)
Ameloblasts/pathology , Apoptosis/drug effects , Carotenoids/pharmacology , Caspases/metabolism , Fluorosis, Dental/pathology , Oxidative Stress/drug effects , Protective Agents/pharmacology , Sodium Fluoride/toxicity , Ameloblasts/drug effects , Ameloblasts/enzymology , Animals , Caspases/genetics , Down-Regulation/drug effects , Fluorosis, Dental/enzymology , Glutathione Peroxidase/metabolism , Incisor/drug effects , Lycopene , Male , Malondialdehyde/metabolism , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Superoxide Dismutase/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.