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1.
J Biol Chem ; 299(5): 104638, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36963497

RESUMO

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.


Assuntos
Diferenciação Celular , Proteínas Ligadas por GPI , Odontoblastos , Odontogênese , Animais , Camundongos , Proteínas Morfogenéticas Ósseas/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicosilfosfatidilinositóis/metabolismo , Proteínas Ligadas por GPI/metabolismo , Microdomínios da Membrana/metabolismo , Odontoblastos/citologia , Odontoblastos/metabolismo , Domínios Proteicos
2.
Pflugers Arch ; 476(9): 1411-1421, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39101996

RESUMO

Hypoxia is relevant to several physiological and pathological processes and this also applies for the tooth. The adaptive response to lowering oxygen concentration is mediated by hypoxia-inducible factors (HIFs). Since HIFs were shown to participate in the promotion of angiogenesis, stem cell survival, odontoblast differentiation and dentin formation, they may play a beneficial role in the tooth reparative processes. Although some data were generated in vitro, little is known about the in vivo context of HIFs in tooth development. In order to contribute to this field, the mouse mandibular first molar was used as a model.The expression and in situ localisation of HIFs were examined at postnatal (P) days P0, P7, P14, using RT-PCR and immunostaining. The expression pattern of a broad spectrum of hypoxia-related genes was monitored by customised PCR Arrays. Metabolic aspects were evaluated by determination of the lactate level and mRNA expression of the mitochondrial marker Nd1.The results show constant high mRNA expression of Hif1a, increasing expression of Hif2a, and very low expression of Hif3a during early postnatal molar development. In the examined period the localisation of HIFs in the nuclei of odontoblasts and the subodontoblastic layer identified their presence during odontoblastic differentiation. Additionally, the lower lactate level and higher expression of mitochondrial Nd1 in advanced development points to decreasing glycolysis during differentiation. Postnatal nuclear localisation of HIFs indicates a hypoxic state in specific areas of dental pulp as oxygen demands depend on physiological events such as crown and root dentin mineralization.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Polpa Dentária , Subunidade alfa do Fator 1 Induzível por Hipóxia , Dente Molar , Animais , Polpa Dentária/metabolismo , Camundongos , Dente Molar/metabolismo , Dente Molar/crescimento & desenvolvimento , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Odontoblastos/metabolismo , Redes e Vias Metabólicas , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Repressoras , Proteínas Reguladoras de Apoptose
3.
J Cell Biochem ; 125(7): e30577, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38720665

RESUMO

Odontoblast differentiation is a key process in dentin formation. Mouse dental papilla cells (mDPCs) are pivotal in dentinogenesis through their differentiation into odontoblasts. Odontoblast differentiation is intricately controlled by transcription factors (TFs) in a spatiotemporal manner. Previous research explored the role of RUNX2 and KLF4 in odontoblast lineage commitment, respectively. Building on bioinformatics analysis of our previous ATAC-seq profiling, we hypothesized that KLF4 potentially collaborates with RUNX2 to exert its biological role. To investigate the synergistic effect of multiple TFs in odontoblastic differentiation, we first examined the spatiotemporal expression patterns of RUNX2 and KLF4 in dental papilla at the bell stage using immunostaining techniques. Notably, RUNX2 and KLF4 demonstrated colocalization in preodontoblast. Further, immunoprecipitation and proximity ligation assays verified the interaction between RUNX2 and KLF4 in vitro. Specifically, the C-terminus of RUNX2 was identified as the interacting domain with KLF4. Functional implications of this interaction were investigated using small hairpin RNA-mediated knockdown of Runx2, Klf4, or both. Western blot analysis revealed a marked decrease in DSPP expression, an odontoblast differentiation marker, particularly in the double knockdown condition. Additionally, alizarin red S staining indicated significantly reduced mineralized nodule formation in this group. Collectively, our findings highlight the synergistic interaction between RUNX2 and KLF4 in promoting odontoblast differentiation from mDPCs. This study contributes to a more comprehensive understanding of the regulatory network of TFs governing odontoblast differentiation.


Assuntos
Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Papila Dentária , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like , Odontoblastos , Fator 4 Semelhante a Kruppel/metabolismo , Odontoblastos/metabolismo , Odontoblastos/citologia , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Papila Dentária/citologia , Papila Dentária/metabolismo
4.
Development ; 148(2)2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33323370

RESUMO

The control of size and shape is an important part of regulatory process during organogenesis. Tooth formation is a highly complex process that fine-tunes the size and shape of the tooth, which are crucial for its physiological functions. Each tooth consists of a crown and one or more roots. Despite comprehensive knowledge of the mechanism that regulates early tooth crown development, we have limited understanding of the mechanism regulating root patterning and size during development. Here, we show that Ror2-mediated non-canonical Wnt signaling in the dental mesenchyme plays a crucial role in cell proliferation, and thereby regulates root development size in mouse molars. Furthermore, Cdc42 acts as a potential downstream mediator of Ror2 signaling in root formation. Importantly, activation of Cdc42 can restore cell proliferation and partially rescue the root development size defects in Ror2 mutant mice. Collectively, our findings provide novel insights into the function of Ror2-mediated non-canonical Wnt signaling in regulating tooth morphogenesis, and suggest potential avenues for dental tissue engineering.


Assuntos
Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Raiz Dentária/embriologia , Raiz Dentária/metabolismo , Via de Sinalização Wnt , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Masculino , Mesoderma/embriologia , Camundongos , Camundongos Mutantes , Morfogênese , Odontoblastos/citologia , Odontoblastos/metabolismo , Raiz Dentária/citologia
5.
Cell Mol Biol (Noisy-le-grand) ; 70(3): 22-28, 2024 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-38650160

RESUMO

This study aimed to evaluate the physiological role of NAMPT associated with MDPC-23 odontoblast cell proliferation. Cell viability was measured using the (DAPI) staining, caspase activation analysis and immunoblotting were performed. Visfatin promoted MDPC-23 odontoblast cell growth in a dose-dependent manner. Furthermore, the up-regulation of Visfatin promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers in MDPC-23 cells. However, FK-866 cell growth in a dose-dependent manner induced nuclear condensation and fragmentation. FK-866-treated cells showed H&E staining and increased apoptosis compared to control cells. The expression of anti-apoptotic factors components of the mitochondria-dependent intrinsic apoptotic pathway significantly decreased following FK-866 treatment. The expression of pro-apoptotic increased upon FK-866 treatment. In addition, FK-866 activated caspase-3 and PARP to induce cell death. In addition, after treating FK-866 for 72 h, the 3/7 activity of MDPC-23 cells increased in a concentration-dependent manner, and the IHC results also confirmed that Caspase-3 increased in a concentration-dependent. Therefore, the presence or absence of NAMPT expression in dentin cells was closely related to cell proliferation and formation of extracellular substrates.


Assuntos
Apoptose , Proliferação de Células , Nicotinamida Fosforribosiltransferase , Odontoblastos , Nicotinamida Fosforribosiltransferase/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Odontoblastos/efeitos dos fármacos , Odontoblastos/citologia , Odontoblastos/metabolismo , Animais , Camundongos , Linhagem Celular , Citocinas/metabolismo , Caspase 3/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Acrilamidas/farmacologia , Odontogênese/efeitos dos fármacos
6.
Cell Mol Biol (Noisy-le-grand) ; 70(6): 135-141, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38836669

RESUMO

Epigenetic change has been found to play an important role in cell differentiation and regulation and the dental pulp stem cell in tissue engineering is gaining attention due to the ability of cells to differentiate into odontoblast and other cells. This study evaluated the influence of poly L- lactic acid with hydroxyapatite-coated with polyaniline scaffold (PLLA/HA/PANI) on dental pulp stem cell (DPSC) proliferation and differentiation. After scaffold preparation and DPSCs seeding, the cells proliferation and differentiation were evaluated by immunocytochemistry assay and cell viability was measured by cytotoxicity / MTT assay. The results showed (PLLA/HA/PANI) scaffold facilitates DPSC proliferation and differentiation with gene expression. This finding underscores the promise of this biomaterial combination as a scaffold for dental tissue regeneration and application.


Assuntos
Materiais Biocompatíveis , Diferenciação Celular , Proliferação de Células , Polpa Dentária , Durapatita , Odontoblastos , Osteoblastos , Células-Tronco , Alicerces Teciduais , Polpa Dentária/citologia , Humanos , Diferenciação Celular/efeitos dos fármacos , Odontoblastos/citologia , Odontoblastos/efeitos dos fármacos , Odontoblastos/metabolismo , Alicerces Teciduais/química , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Proliferação de Células/efeitos dos fármacos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Durapatita/química , Durapatita/farmacologia , Compostos de Anilina/farmacologia , Compostos de Anilina/química , Poliésteres/química , Poliésteres/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Engenharia Tecidual/métodos
7.
Int J Hyperthermia ; 41(1): 2369749, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38925872

RESUMO

PURPOSE: Dentin hypersensitivity (DH) is a prevalent condition, but long-term effective treatments are scarce. Differentiation of odontoblast-like cells is promising for inducing tertiary dentinogenesis and ensuring sustained therapeutic efficacy against DH. This study examined the effects and mechanism of action of mild heat stress (MHS) on the differentiation of odontoblast-like MDPC-23 cells. METHODS: We used a heating device to accurately control the temperature and duration, mimicking the thermal microenvironment of odontoblast-like cells. Using this device, the effects of MHS on cell viability and differentiation were examined. Cell viability was assessed using the MTT assay. The expression and nucleoplasmic ratio of the yes-associated protein (YAP) were examined by western blotting and immunofluorescence. The gene expression levels of heat shock proteins (HSPs) and dentin matrix protein-1 (DMP1) were measured using qPCR. Dentin sialophosphoprotein (DSPP) expression was evaluated using immunofluorescence and immunoblotting. Verteporfin was used to inhibit YAP activity. RESULTS: Mild heat stress (MHS) enhanced the odontoblast differentiation of MDPC-23 cells while maintaining cell viability. MHS also increased YAP activity, as well as the levels of HSP25 mRNA, HSP70 mRNA, HSP90α mRNA, DMP1 mRNA, and DSPP protein. However, after YAP inhibition, both cell viability and the levels of HSP90α mRNA, DMP1 mRNA, and DSPP protein were reduced. CONCLUSION: YAP plays a crucial role in maintaining cell viability and promoting odontoblast differentiation of MDPC-23 cells under MHS. Consequently, MHS is a potential therapeutic strategy for DH, and boosting YAP activity could be beneficial for maintaining cell viability and promoting odontoblast differentiation.


Assuntos
Diferenciação Celular , Resposta ao Choque Térmico , Odontoblastos , Proteínas de Sinalização YAP , Odontoblastos/metabolismo , Animais , Proteínas de Sinalização YAP/metabolismo , Camundongos , Linhagem Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sobrevivência Celular
8.
Oral Dis ; 30(6): 3745-3760, 2024 09.
Artigo em Inglês | MEDLINE | ID: mdl-38409677

RESUMO

OBJECTIVES: A zinc-finger transcription factor family comprising specificity proteins (SPs) and Krüppel-like factor proteins (KLFs) plays an important role in dentin development and regeneration. However, a systematic regulatory network involving SPs/KLFs in odontoblast differentiation has not yet been described. This review examined the expression patterns of SP/KLF gene family members and their current known functions and mechanisms in odontoblast differentiation, and discussed prospective research directions for further exploration of mechanisms involving the SP/KLF gene family in dentin development. MATERIALS AND METHODS: Relevant literature on SP/KLF gene family members and dentin development was acquired from PubMed and Web of Science. RESULTS: We discuss the expression patterns, functions, and related mechanisms of eight members of the SP/KLF gene family in dentin development and genetic disorders with dental problems. We also summarize current knowledge about their complementary or synergistic actions. Finally, we propose future research directions for investigating the mechanisms of dentin development. CONCLUSIONS: The SP/KLF gene family plays a vital role in tooth development. Studying the complex complementary or synergistic interactions between SPs/KLFs is helpful for understanding the process of odontoblast differentiation. Applications of single-cell and spatial multi-omics may provide a more complete investigation of the mechanism involved in dentin development.


Assuntos
Diferenciação Celular , Fatores de Transcrição Kruppel-Like , Odontoblastos , Odontoblastos/metabolismo , Humanos , Diferenciação Celular/genética , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Sp/genética , Fatores de Transcrição Sp/metabolismo , Dentina/metabolismo , Odontogênese/genética , Odontogênese/fisiologia
9.
Differentiation ; 133: 88-97, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37579565

RESUMO

This study investigated the expression of sortilin 1 (SORT1) in cultured human dental pulp-derived stem cells (hDPSCs) and its role in their odontoblastic differentiation. Permanent teeth were extracted from five patients, and the dental pulp was harvested for explant culture. Fluorescence-activated cell sorting was used to analyze the outgrowth of adherent cells and cells that had migrated from the tissue margin. SORT1 expression was detected in hDPSCs simultaneously expressing the mesenchymal stem cell markers CD44 and CD90. The odontoblastic differentiation potential of SORT1-positive hDPSCs was examined via staining for alkaline phosphatase (ALP), an early odontoblastic differentiation marker. ALP staining was more intense in SORT1-positive than in SORT1-negative hDPSCs. Consistently, the expression of mRNA encoding SORT1 and p75NTR, a binding partner of SORT1, increased in SORT1-positive hDPSCs during odontoblastic differentiation. In addition, pro-nerve growth factor (NGF), a ligand for SORT1-p75NTR co-receptor, promoted ALP expression in SORT1-positive hDPSCs, and the interaction between SORT1 and p75NTR was detected using a coimmunoprecipitation assay. The function of SORT1 in odontoblastic differentiation was examined via RNA interference using shRNA targeting SORT1. ALP staining intensity in SORT1/shRNA-transfected cells was markedly lower than in control/shRNA-transfected cells. SORT1 knockdown decreased JUN phosphorylation and recruitment of phosphorylated JUN to the ALP promoter. Collectively, these results indicate that SORT1 is involved in the odontoblastic differentiation of hDPSCs through the JUN N-terminal kinases (JNK)/JUN signaling pathway and that the binding of SORT1 and p75NTR plays an important role in this process.


Assuntos
Polpa Dentária , Odontoblastos , Humanos , Odontoblastos/metabolismo , Células-Tronco , RNA Interferente Pequeno/farmacologia , Diferenciação Celular/genética , Células Cultivadas
10.
Differentiation ; 134: 52-60, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37898102

RESUMO

Epithelial-mesenchymal interactions occur during tooth development. The dental epithelium (DE) is regarded as the signal center that regulates tooth morphology. However, the mechanism by which DE regulates the differentiation of mesenchyme-derived dental papilla (DP) into odontoblasts remains unclear. Using miniature pigs as a model, we analyzed the expression profiles of the DE and DP during odontoblast differentiation using high-throughput RNA sequencing. The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is one of the most enriched pathways in both DE and DP. The PI3K/AKT pathway was first activated in the inner enamel epithelium but not in the DP on embryonic day 50. This pathway was then activated in the odontoblast layer on embryonic day 60. We showed that AKT activation promoted odontoblast differentiation of DP cells. We further demonstrated that activation of PI3K/AKT signaling in the DE effectively increased the expression levels of AKT and dentin sialophosphoprotein in DP cells. Additionally, we found that DE cells secreted collagen type IV alpha 6 chain (COL4A6) downstream of epithelial AKT signaling to positively regulate mesenchymal AKT levels. Therefore, our data suggest that PI3K/AKT signaling from the DE to the DP promotes odontoblast differentiation via COL4A6 secretion.


Assuntos
Odontoblastos , Proteínas Proto-Oncogênicas c-akt , Animais , Suínos , Odontoblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Diferenciação Celular/genética , Epitélio
11.
Odontology ; 112(1): 125-137, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37493885

RESUMO

Perfect intercellular junctions are key for odontoblast barrier function. However, whether Partitioning defective-3 (Par3) is expressed in odontoblasts and its potential effects on odontoblast junctions are unknown. Herein, we investigated the effect of Par3 on cellular junctions and the biological behavior of odontoblast-lineage cells (OLCs). Whole-transcriptome sequencing was used to analyze the effects of Par3 on OLCs and the underlying molecular mechanism. Par3 was detected under physiological and inflammatory conditions in OLCs. To investigate the regulatory effect of Par3 on junctions between mouse OLCs, the effects of Par3 downregulation on the proliferation, migration, cycle and apoptosis of OLCs were detected by 5-ethyl-2'-deoxyuridine (EdU) and Transwell assays and flow cytometry. Western blotting and alizarin red S and alkaline phosphatase (ALP) staining were used to observe the effect of Par3 downregulation on OLC mineralization. Whole-transcriptome sequencing was used to investigate the biological role of Par3 in OLCs and potential molecular mechanisms. Par3 was located along the odontoblast layer in the rat pulp tissue and in the cytoplasm of OLCs. Par3 expression was downregulated under inflammatory conditions. The OLC junctions were discontinuous, and total Zona occluden-1 (ZO-1) expression and expression of ZO-1 at the membrane in OLCs were reduced after Par3 silencing (P < 0.05). Expression of a junction-related protein (ZO-1) was downregulated after the downregulation of Par3 (P < 0.05), and ZO-1 moved from the cell membrane to the cytoplasm. OLC proliferation and migration were enhanced, but apoptosis and mineralization were inhibited in shPar3-transfected cells (P < 0.05). Sequencing identified 2996 differentially expressed genes (DEGs), which were mainly enriched in the response to stimuli and binding. Downregulation of Par3 could overactivate the PI3k-AKT pathway by promoting AKT phosphorylation (P < 0.05). Downregulation of Par3 may disrupt junctions between OLCs by affecting ZO-1 expression and distribution and promote OLC proliferation and migration but inhibit OLC mineralization. Par3 may interact with 14-3-3 proteins for PI3K-AKT pathway activation to affect OLC junctions and function.


Assuntos
Odontoblastos , Fosfatidilinositol 3-Quinases , Camundongos , Ratos , Animais , Odontoblastos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/farmacologia , Linhagem Celular , Junções Intercelulares , Diferenciação Celular
12.
Int J Mol Sci ; 25(11)2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38891829

RESUMO

It is remarkable how teeth maintain their healthy condition under exceptionally high levels of mechanical loading. This suggests the presence of inherent mechanical adaptation mechanisms within their structure to counter constant stress. Dentin, situated between enamel and pulp, plays a crucial role in mechanically supporting tooth function. Its intermediate stiffness and viscoelastic properties, attributed to its mineralized, nanofibrous extracellular matrix, provide flexibility, strength, and rigidity, enabling it to withstand mechanical loading without fracturing. Moreover, dentin's unique architectural features, such as odontoblast processes within dentinal tubules and spatial compartmentalization between odontoblasts in dentin and sensory neurons in pulp, contribute to a distinctive sensory perception of external stimuli while acting as a defensive barrier for the dentin-pulp complex. Since dentin's architecture governs its functions in nociception and repair in response to mechanical stimuli, understanding dentin mechanobiology is crucial for developing treatments for pain management in dentin-associated diseases and dentin-pulp regeneration. This review discusses how dentin's physical features regulate mechano-sensing, focusing on mechano-sensitive ion channels. Additionally, we explore advanced in vitro platforms that mimic dentin's physical features, providing deeper insights into fundamental mechanobiological phenomena and laying the groundwork for effective mechano-therapeutic strategies for dentinal diseases.


Assuntos
Dentina , Dentina/fisiologia , Dentina/metabolismo , Humanos , Animais , Odontoblastos/fisiologia , Odontoblastos/metabolismo , Odontoblastos/citologia , Mecanotransdução Celular/fisiologia , Fenômenos Biomecânicos , Polpa Dentária/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia
13.
Int J Mol Sci ; 25(13)2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38999956

RESUMO

The transforming growth factor ß (TGFß) superfamily is a master regulator of development, adult homeostasis, and wound repair. Dysregulated TGFß signaling can lead to cancer, fibrosis, and musculoskeletal malformations. We previously demonstrated that TGFß receptor 2 (Tgfbr2) signaling regulates odontoblast differentiation, dentin mineralization, root elongation, and sensory innervation during tooth development. Sensory innervation also modulates the homeostasis and repair response in adult teeth. We hypothesized that Tgfbr2 regulates the neuro-pulpal responses to dentin injury. To test this, we performed a shallow dentin injury with a timed deletion of Tgfbr2 in the dental pulp mesenchyme of mice and analyzed the levels of tertiary dentin and calcitonin gene-related peptide (CGRP) axon sprouting. Microcomputed tomography imaging and histology indicated lower dentin volume in Tgfbr2cko M1s compared to WT M1s 21 days post-injury, but the volume was comparable by day 56. Immunofluorescent imaging of peptidergic afferents demonstrated that the duration of axon sprouting was longer in injured Tgfbr2cko compared to WT M1s. Thus, CGRP+ sensory afferents may provide Tgfbr2-deficient odontoblasts with compensatory signals for healing. Harnessing these neuro-pulpal signals has the potential to guide the development of treatments for enhanced dental healing and to help patients with TGFß-related diseases.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Polpa Dentária , Dentina , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais , Animais , Polpa Dentária/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Camundongos , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/genética , Dentina/metabolismo , Camundongos Knockout , Odontoblastos/metabolismo
14.
Int J Mol Sci ; 25(12)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38928274

RESUMO

Epigenetic modulation, including histone modification, alters gene expression and controls cell fate. Histone deacetylases (HDACs) are identified as important regulators of dental pulp cell (DPC) mineralisation processes. Currently, there is a paucity of information regarding the nature of histone modification and HDAC expression in the dentine-pulp complex during dentinogenesis. The aim of this study was to investigate post-translational histone modulation and HDAC expression during DPC mineralisation and the expression of Class I/II HDACs during tooth development and in adult teeth. HDAC expression (isoforms -1 to -6) was analysed in mineralising primary rat DPCs using qRT-PCR and Western blot with mass spectrometry being used to analyse post-translational histone modifications. Maxillary molar teeth from postnatal and adult rats were analysed using immunohistochemical (IHC) staining for HDACs (1-6). HDAC-1, -2, and -4 protein expression increased until days 7 and 11, but decreased at days 14 and 21, while other HDAC expression increased continuously for 21 days. The Class II mineralisation-associated HDAC-4 was strongly expressed in postnatal sample odontoblasts and DPCs, but weakly in adult teeth, while other Class II HDACs (-5, -6) were relatively strongly expressed in postnatal DPCs and adult odontoblasts. Among Class I HDACs, HDAC-1 showed high expression in postnatal teeth, notably in ameloblasts and odontoblasts. HDAC-2 and -3 had extremely low expression in the rat dentine-pulp complex. Significant increases in acetylation were noted during DPC mineralisation processes, while trimethylation H3K9 and H3K27 marks decreased, and the HDAC-inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced H3K27me3. These results highlight a dynamic alteration in histone acetylation during mineralisation and indicate the relevance of Class II HDAC expression in tooth development and regenerative processes.


Assuntos
Polpa Dentária , Dentina , Dentinogênese , Histona Desacetilases , Animais , Acetilação , Ratos , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Dentina/metabolismo , Polpa Dentária/metabolismo , Polpa Dentária/citologia , Polpa Dentária/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , Histonas/metabolismo , Dente Molar/metabolismo , Dente Molar/crescimento & desenvolvimento , Odontoblastos/metabolismo , Masculino
15.
J Biol Chem ; 298(8): 102220, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35780838

RESUMO

WW domain-containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2-PTEN-KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo.


Assuntos
Dentinogênese , Fatores de Transcrição Kruppel-Like , Odontoblastos , PTEN Fosfo-Hidrolase , Ubiquitina-Proteína Ligases , Animais , Diferenciação Celular , Dentina/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Knockout , Odontoblastos/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
16.
Biochem Biophys Res Commun ; 650: 47-54, 2023 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-36773339

RESUMO

Iroquois homeobox (Irx) genes are TALE-class homeobox genes that are evolutionarily conserved across species and have multiple critical cellular functions in fundamental tissue development processes. Previous studies have shown that Irxs genes are expressed during tooth development. However, the precise roles of genes in teeth remain unclear. Here, we demonstrated for the first time that Irx3 is an essential molecule for the proliferation and differentiation of odontoblasts. Using cDNA synthesized from postnatal day 1 (P1) tooth germs, we examined the expression of all Irx genes (Irx1-Irx6) by RT-PCR and found that all genes except Irx4 were expressed in the tooth tissue. Irx1-Irx3 a were expressed in the dental epithelial cell line M3H1 cells, while Irx3 and Irx5 were expressed in the dental mesenchymal cell line mDP cells. Only Irx3 was expressed in both undifferentiated cell lines. Immunostaining also revealed the presence of IRX3 in the dental epithelial cells and mesenchymal condensation. Inhibition of endogenous Irx3 by siRNA blocks the proliferation and differentiation of mDP cells. Wnt3a, Wnt5a, and Bmp4 are factors involved in odontoblast differentiation and were highly expressed in mDP cells by quantitative PCR analysis. Interestingly, the expression of Wnt5a (but not Wnt3a or Bmp4) was suppressed by Irx3 siRNA. These results suggest that Irx3 plays an essential role in part through the regulation of Wnt5a expression during odontoblast proliferation and differentiation.


Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Odontoblastos/metabolismo , Genes Homeobox , Diferenciação Celular , Proliferação de Células
17.
J Pineal Res ; 74(4): e12865, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36864655

RESUMO

Tooth development is a complex process that is tightly controlled by circadian rhythm. Melatonin (MT) is a major hormonal regulator of the circadian rhythm, and influences dentin formation and odontoblastic differentiation during tooth development; however, the underlying mechanism remains elusive. This study investigated how MT regulates odontoblastic differentiation, with a special focus on its regulation of mitochondrial dynamics. In rat dental papilla cells (DPCs), we found that MT promotes odontoblastic differentiation concurrently with enhanced mitochondrial fusion, while disruption of mitochondrial fusion by depleting optic atrophy 1 (OPA1) impairs MT-mediated differentiation and mitochondrial respiratory functions. Through RNA sequencing, we discovered that MT significantly upregulated malic enzyme 2 (ME2), a mitochondrial NAD(P)+ -dependent enzyme, and identified ME2 as a critical MT downstream effector that orchestrates odontoblastic differentiation, mitochondrial fusion, and respiration functions. By detecting the spatiotemporal expression of ME2 in developing tooth germs, and using tooth germ reconstituted organoids, we also provided in vivo and ex vivo evidence that ME2 promotes dentin formation, indicating a possible involvement of ME2 in MT-modulated tooth development. Collectively, our findings offer novel understandings regarding the molecular mechanism by which MT affects cell differentiation and organogenesis, meanwhile, the critical role of ME2 in MT-regulated mitochondrial functions is also highlighted.


Assuntos
Melatonina , Animais , Ratos , Diferenciação Celular , Polpa Dentária , Melatonina/metabolismo , Dinâmica Mitocondrial , Odontoblastos/metabolismo , Respiração , Malato Desidrogenase/metabolismo
18.
Int Endod J ; 56(4): 432-446, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36462163

RESUMO

AIM: The physiological effects and cellular mechanism of 25-hydroxycholesterol (25-HC), which is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase (CH25H) expressed under inflammatory conditions, are still largely unknown during odontoclastogenesis. This study aimed to evaluate 25-HC-induced odontoclastogenesis and its cellular mechanisms in odontoblast-like MDPC-23 cells. METHODOLOGY: To investigate 25-HC-induced odontoclastogenesis of MDPC-23 cells and its cellular mechanism, haemotoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, dentine resorption assay, zymography, reactive oxygen species (ROS) detection, immunocytochemistry, and nuclear translocation were performed. The experimental values are presented as mean ± standard deviation and were compared using analysis of variance, followed by post hoc multiple comparisons (Tukey's test) using SPSS software version 22 (IBM Corp.). A p-value <.05 was considered statistically significant. RESULTS: Lipopolysaccharide or receptor activator of nuclear factor-κB ligand (RANKL) induced the synthesis of 25-HC via the expression of CH25H in MDPC-23 cells (p < .01). Multinucleated giant cells with morphological characteristics and TRAP activity of the odontoclast were increased by 25-HC in MDPC-23 cells (p < .01). Moreover, 25-HC increased dentine resorption through the expression and activity of matrix metalloproteinases in MDPC-23 cells. It not only increased the expression of odontoclastogenic biomarkers but also translocated cytosolic nuclear factor-κB (NF-κB) to the nucleus in MDPC-23 cells. Additionally, 25-HC not only increased the production of ROS (p < .01), expression of inflammatory mediators (p < .01), pro-inflammatory cytokines, receptor activator of NF-κB (RANK), and RANKL but also suppressed the expression of osteoprotegerin (OPG) in MDPC-23 cells. In contrast, CDDO-Me, a chemical NF-κB inhibitor, decreased TRAP activity (p < .01) and downregulated the expression of the odontoclastogenic biomarkers, including RANK and RANKL, in MDPC-23 cells. CONCLUSION: 25-HC induced odontoclastogenesis by modulating the RANK-RANKL-OPG axis via NF-κB activation in MDPC-23 cells. Therefore, these findings provide that 25-HC derived from cholesterol metabolism may be involved in the pathophysiological etiological factors of internal tooth resorption.


Assuntos
NF-kappa B , Odontoblastos , Diferenciação Celular , NF-kappa B/metabolismo , Odontoblastos/metabolismo , Osteoclastos , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima , Animais , Camundongos
19.
Int J Mol Sci ; 24(13)2023 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-37445839

RESUMO

Human dental pulp stem cells (hDPSCs) possess remarkable self-renewal and multilineage differentiation ability. PER2, an essential circadian molecule, regulates various physiological processes. Evidence suggests that circadian rhythm and PER2 participate in physiological functions of DPSCs. However, the influence of PER2 on DPSCs' differentiation remains largely unknown. This study aimed to explore the effect and potential mechanism of PER2 on hDPSCs' differentiation. Dental pulp tissues were extracted, and hDPSCs were cultured for in vitro and in vivo experiments. Dorsal subcutaneous transplantation was performed in 6-week-old male BALB/c mice. The hDPSCs' odontoblastic/osteogenic differentiation was assessed, and mitochondrial metabolism was evaluated. The results indicated PER2 expression increasing during hDPSCs' odontoblastic/osteogenic differentiation. Gain- and loss-of function studies confirmed that PER2 promoted alkaline phosphatase (ALP) activity, mineralized nodules deposition, mRNA expression of DSPP, DMP1, COL1A1 and protein expression of DSPP and DMP1 in hDPSCs. Furthermore, PER2 enhanced collagen deposition, osteodentine-like tissue formation and DSPP expression in vivo. Mitochondrial metabolic evaluation aimed to investigate the mechanism of PER2-mediated hDPSC odontoblastic/osteogenic differentiation, which showed that PER2 increased ATP synthesis, elevated mitochondrial membrane potential and changed expression of proteins regulating mitochondrial dynamics. This study demonstrated that PER2 promoted hDPSCs' odontoblastic/osteogenic differentiation, which involved mitochondrial metabolic change.


Assuntos
Polpa Dentária , Osteogênese , Animais , Camundongos , Humanos , Masculino , Osteogênese/genética , Polpa Dentária/metabolismo , Odontoblastos/metabolismo , Diferenciação Celular/genética , Células-Tronco/metabolismo , Células Cultivadas , Proliferação de Células , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo
20.
Int J Mol Sci ; 24(11)2023 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-37298716

RESUMO

Dentin regeneration is the preferred method used to preserve dental pulp vitality after pulp exposure due to caries. Red light-emitting diode irradiation (LEDI), which is based on photobiomodulation (PBM), has been used to promote hard-tissue regeneration. However, the underlying mechanism still needs elucidation. This study aimed to explore the mechanism involved in red LEDI affecting dentin regeneration. Alizarin red S (ARS) staining revealed that red LEDI induced mineralization of human dental pulp cells (HDPCs) in vitro. We further distinguished the cell proliferation (0-6 d), differentiation (6-12 d), and mineralization (12-18 d) of HDPCs in vitro and treated cells either with or without red LEDI in each stage. The results showed that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, increased mineralized nodule formation around HDPCs. Western blot also indicated that red LEDI treatment in the mineralization stage, but not the proliferation or differentiation stages, upregulated the expression of dentin matrix marker proteins (dentin sialophosphoprotein, DSPP; dentin matrix protein 1, DMP1; osteopontin, OPN) and an intracellular secretory vesicle marker protein (lysosomal-associated membrane protein 1, LAMP1). Therefore, the red LEDI might enhance the matrix vesicle secretion of HDPCs. On the molecular level, red LEDI enhanced mineralization by activating the mitogen-activated protein kinase (MAPK) signaling pathways (ERK and P38). ERK and P38 inhibition reduced mineralized nodule formation and the expression of relevant marker proteins. In summary, red LEDI enhanced the mineralization of HDPCs by functioning to produce a positive effect in the mineralization stage in vitro.


Assuntos
Polpa Dentária , Odontoblastos , Humanos , Polpa Dentária/metabolismo , Odontoblastos/metabolismo , Diferenciação Celular , Proliferação de Células , Sistema de Sinalização das MAP Quinases , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Fosfatase Alcalina/metabolismo , Fosfoproteínas/metabolismo
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