RESUMEN
Fibroblast growth factor 2 (FGF2) is a crucial factor in odontoblast differentiation and dentin matrix deposition, which facilitates pulpodentin repair and regeneration. Nevertheless, the specific biological function of FGF2 in odontoblastic differentiation remains unclear because it is controlled by complex signalling pathways. This study aimed to investigate the mechanism underlying the effect of FGF2 on osteo/odontogenic differentiation of stem cells from the apical papilla (SCAP). SCAP were pretreated with conditioned media containing FGF2 for 1 week, followed by culturing in induced differentiation medium for another week. RNA sequencing (RNA-seq) combined with quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the pathways affected by FGF2 in SCAP. Osteo/odontogenic differentiation of SCAP was determined using Alizarin red S staining, alkaline phosphatase staining, RT-qPCR, and western blotting. Pretreatment with FGF2 for 1 week increased the osteo/odontogenic differentiation ability of SCAP. RNA-seq and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that phosphatidylinositol 3-kinase (PI3K)/AKT signalling is involved in the osteogenic function of FGF2. RT-qPCR results indicated that SCAP expressed FGF receptors, and western blotting showed that p-AKT was reduced in FGF2-pretreated SCAP. The activation of the PI3K/AKT pathway partially reversed the stimulatory effect of FGF2 on osteo/odontogenic differentiation of SCAP. Our findings suggest that pretreatment with FGF2 enhances the osteo/odontogenic differentiation ability of SCAP by inhibiting the PI3K/AKT pathway.
Asunto(s)
Diferenciación Celular , Papila Dental , Factor 2 de Crecimiento de Fibroblastos , Odontogénesis , Osteogénesis , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Células Madre , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Diferenciación Celular/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Papila Dental/citología , Papila Dental/metabolismo , Humanos , Odontogénesis/efectos de los fármacos , Células Madre/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Células CultivadasRESUMEN
BACKGROUND: Circular RNA (circRNA) is a key player in regulating the multidirectional differentiation of stem cells. Previous research by our group found that the blue light-emitting diode (LED) had a promoting effect on the osteogenic/odontogenic differentiation of human stem cells from apical papilla (SCAPs). This research aimed to investigate the differential expression of circRNAs during the osteogenic/odontogenic differentiation of SCAPs regulated by blue LED. MATERIALS AND METHODS: SCAPs were divided into the irradiation group (4 J/cm2) and the control group (0 J/cm2), and cultivated in an osteogenic/odontogenic environment. The differentially expressed circRNAs during osteogenic/odontogenic differentiation of SCAPs promoted by blue LED were detected by high-throughput sequencing, and preliminarily verified by qRT-PCR. Functional prediction of these circRNAs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the circRNA-miRNA-mRNA networks were also constructed. RESULTS: It showed 301 circRNAs were differentially expressed. GO and KEGG analyses suggested that these circRNAs were associated with some signaling pathways related to osteogenic/odontogenic differentiation. And the circRNA-miRNA-mRNA networks were also successfully constructed. CONCLUSION: CircRNAs were involved in the osteogenic/odontogenic differentiation of SCAPs promoted by blue LED. In this biological process, circRNA-miRNA-mRNA networks served an important purpose, and circRNAs regulated this process through certain signaling pathways.
Asunto(s)
Diferenciación Celular , Papila Dental , Luz , Odontogénesis , Osteogénesis , ARN Circular , Células Madre , ARN Circular/genética , ARN Circular/metabolismo , Humanos , Osteogénesis/genética , Diferenciación Celular/genética , Células Madre/metabolismo , Células Madre/citología , Odontogénesis/genética , Papila Dental/citología , Papila Dental/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Ontología de Genes , Células Cultivadas , Perfilación de la Expresión Génica/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Regulación de la Expresión Génica/efectos de la radiación , Luz AzulRESUMEN
Methyltransferase-like 3 (METTL3) is a crucial element of N6-methyladenosine (m6A) modifications and has been extensively studied for its involvement in diverse biological and pathological processes. In this study, we explored how METTL3 affects the differentiation of stem cells from the apical papilla (SCAPs) into odonto/osteoblastic lineages through gain- and loss-of-function experiments. The m6A modification levels were assessed using m6A dot blot and activity quantification experiments. In addition, we employed Me-RIP microarray experiments to identify specific targets modified by METTL3. Furthermore, we elucidated the molecular mechanism underlying METTL3 function through dual-luciferase reporter gene experiments and rescue experiments. Our findings indicated that METTL3+/- mice exhibited significant root dysplasia and increased bone loss. The m6A level and odonto/osteoblastic differentiation capacity were affected by the overexpression or inhibition of METTL3. This effect was attributed to the acceleration of pre-miR-665 degradation by METTL3-mediated m6A methylation in cooperation with the "reader" protein YTHDF2. Additionally, the targeting of distal-less homeobox 3 (DLX3) by miR-665 and the potential direct regulation of DLX3 expression by METTL3, mediated by the "reader" protein YTHDF1, were demonstrated. Overall, the METTL3/pre-miR-665/DLX3 pathway might provide a new target for SCAP-based tooth root/maxillofacial bone tissue regeneration.
Asunto(s)
Diferenciación Celular , Proteínas de Homeodominio , Metiltransferasas , MicroARNs , Células Madre , Factores de Transcripción , Animales , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Diferenciación Celular/genética , Papila Dental/citología , Papila Dental/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Metilación , Metiltransferasas/metabolismo , Metiltransferasas/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células Madre/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Papila Dental , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel , Odontoblastos , Factor 4 Similar a Kruppel/metabolismo , Odontoblastos/metabolismo , Odontoblastos/citología , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Papila Dental/citología , Papila Dental/metabolismoRESUMEN
The coordination between odontoblastic differentiation and directed cell migration of mesenchymal progenitors is necessary for regular dentin formation. The synthesis and degradation of hyaluronan (HA) in the extracellular matrix create a permissive niche that directly regulates cell behaviors. However, the role and mechanisms of HA degradation in dentin formation remain unknown. In this work, we present that HA digestion promotes odontoblastic differentiation and cell migration of mouse dental papilla cells (mDPCs). Hyaluronidase 2 (HYAL2) is responsible for promoting odontoblastic differentiation through degrading HA, while hyaluronidase 1 (HYAL1) exhibits negligible effect. Silencing Hyal2 generates an extracellular environment rich in HA, which attenuates F-actin and filopodium formation and in turn inhibits cell migration of mDPCs. In addition, activating PI3K/Akt signaling significantly rescues the effects of HA accumulation on cytodifferentiation. Taken together, the results confirm the contribution of HYAL2 to HA degradation in dentinogenesis and uncover the mechanism of the HYAL2-mediated HA degradation in regulating the odontoblastic differentiation and migration of mDPCs.
Asunto(s)
Diferenciación Celular , Movimiento Celular , Papila Dental , Ácido Hialurónico , Hialuronoglucosaminidasa , Odontoblastos , Animales , Hialuronoglucosaminidasa/metabolismo , Hialuronoglucosaminidasa/genética , Ratones , Ácido Hialurónico/metabolismo , Odontoblastos/metabolismo , Odontoblastos/citología , Papila Dental/citología , Papila Dental/metabolismo , Transducción de Señal , Proteínas Ligadas a GPI/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Células Cultivadas , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.
Asunto(s)
Proteínas de la Matriz Extracelular , ARN de Interacción con Piwi , Animales , Ratones , Diferenciación Celular/genética , Células Cultivadas , Papila Dental/química , Papila Dental/metabolismo , Pulpa Dental/química , Proteínas de la Matriz Extracelular/metabolismo , Odontoblastos , Transducción de Señal , Proteína Smad1/metabolismoRESUMEN
Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, ß-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, ß-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury.
Asunto(s)
Histonas , Tubulina (Proteína) , Ratas , Animales , Histonas/metabolismo , Nestina/genética , Nestina/metabolismo , Diferenciación Celular , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Células Madre/metabolismo , Neurogénesis , Papila Dental/metabolismo , Células Cultivadas , OsteogénesisRESUMEN
OBJECTIVE: To analyze the effect of P11-4 self-assembly peptide on cell viability and osteogenic capacity of SCAPs through mineral deposition and gene expression of osteogenic markers. METHODS: SCAPs were seeded in contact with P11-4 (10 µg/ml, 100 µg/ml and 1 mg/ml) solution. Cell viability was evaluated using a colorimetric assay MTT: 3-(4,5-dimethyl-thiazolyl-2)-2,5- diphenyltetrazolium bromide) in an experimental time of 24, 48 and 72 h (n = 7). Mineral deposition and quantification provided by the cells was tested using the Alizarin Red staining and Cetylpyridinium Chloride (CPC), respectively, after 30 days (n = 4). Gene expression of Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP) and Osteocalcin (OCN) was quantified using quantitative polymerase chain reaction (RT-qPCR), at 3 and 7 days with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, and relative gene expression was measured using the ΔΔCq method. Data were analyzed using Kruskall-Wallis followed by multiple comparisons, and T-test for gene expression with α=0.05. RESULTS: All tested concentrations (10 µg/ml, 100 µg/ml and 1 mg/ml) were not cytotoxic at time 24 and 48 h. After 72 h, a slight decrease in cell viability was observed for the lowest concentration (10 µg/ml). The concentration of 100 µg/ml P11-4 showed the highest mineral deposition. However, qPCR analysis of P11-4 (10 µg/ml) showed upregulation of RUNX2 and OCN at 3 days, with downregulation of ALP at 3 and 7d CONCLUSION: P11-4 did not affect cell viability, induced mineral deposition in SCAPs, and upregulated the expression of RUNX2 and OCN genes at 3 days, while downregulating ALP expression at 3 and 7 days. CLINICAL SIGNIFICANCE: Based on the results obtained in this study it can be stated that self-assembling peptide P11-4 is a potential candidate to induce mineralization on dental stem cells for regenerative purposes and also for a clinical use as a capping agent without compromising the cells health.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal , Osteogénesis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Osteogénesis/genética , Papila Dental/metabolismo , Diferenciación Celular/genética , Células Madre/metabolismo , Proliferación Celular , Células CultivadasRESUMEN
OBJECTIVES: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation. MATERIALS AND METHODS: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms. RESULTS: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. CONCLUSIONS: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration.
Asunto(s)
Pulpa Dental , Histona Demetilasas con Dominio de Jumonji , Regeneración , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Papila Dental/metabolismo , Pulpa Dental/metabolismo , Osteogénesis/genética , ARN Interferente Pequeño , Células Madre , Humanos , Histona Demetilasas con Dominio de Jumonji/genéticaRESUMEN
Stem cells from the apical papilla (SCAP) are a promising resource for use in regenerative endodontic treatment (RET) that may be adversely affected by oral bacteria, which in turn can exert an effect on the success of RET. Our work aims to study the cytokine profile of SCAP upon exposure to oral bacteria and their supernatants-Fusobacterium nucleatum and Enterococcus faecalis-as well as to establish their effect on the osteogenic and immunogenic potentials of SCAP. Further, we target the presence of key proteins of the Wnt/ß-Catenin, TGF-ß, and NF-κB signaling pathways, which play a crucial role in adult osteogenic differentiation of mesenchymal stem cells, using the Western blot (WB) technique. The membrane-based sandwich immunoassay and transcriptomic analysis showed that, under the influence of F. nucleatum (both bacteria and supernatant), the production of pro-inflammatory cytokines IL-6, IL-8, and MCP-1 occurred, which was also confirmed at the mRNA level. Conversely, E. faecalis reduced the secretion of the aforementioned cytokines at both mRNA and protein levels. WB analysis showed that SCAP co-cultivation with E. faecalis led to a decrease in the level of the key proteins of the Wnt/ß-Catenin and NF-κB signaling pathways: ß-Catenin (p = 0.0068 *), LRP-5 (p = 0.0059 **), and LRP-6 (p = 0.0329 *), as well as NF-kB (p = 0.0034 **) and TRAF6 (p = 0.0285 *). These results suggest that oral bacteria can up- and downregulate the immune and inflammatory responses of SCAP, as well as influence the osteogenic potential of SCAP, which may negatively regulate the success of RET.
Asunto(s)
Papila Dental , Boca , Osteogénesis , Células Madre , Adulto , Bacterias , Diferenciación Celular/fisiología , Citocinas , Papila Dental/metabolismo , Humanos , Boca/microbiología , FN-kappa B/metabolismo , Osteogénesis/genética , Análisis por Matrices de Proteínas/métodos , ARN Mensajero , Células Madre/metabolismo , Transcriptoma , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16-20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 µM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 µM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP-Smad signaling pathway.
Asunto(s)
Catequina/análogos & derivados , Papila Dental/citología , Papila Dental/efectos de los fármacos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Odontogénesis/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Adolescente , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Catequina/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Papila Dental/metabolismo , Humanos , Células Madre Multipotentes/metabolismo , Regeneración/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Adulto JovenRESUMEN
During tooth development, dental papilla cells differentiate into odontoblasts with polarized morphology and cell function. Our previous study indicated that the C-Jun N-terminal kinase (JNK) pathway regulates human dental papilla cell adhesion, migration, and formation of focal adhesion complexes. The aim of this study was to further examine the role of the JNK pathway in dental papilla cell polarity formation. Histological staining, qPCR, and Western Blot suggested the activation of JNK signaling in polarized mouse dental papilla tissue. After performing an in vitro tooth germ organ culture and cell culture, we found that JNK inhibitor SP600125 postponed tooth germ development and reduced the polarization, migration and differentiation of mouse dental papilla cells (mDPCs). Next, we screened up-regulated polarity-related genes during dental papilla development and mDPCs or A11 differentiation. We found that Prickle3, Golga2, Golga5, and RhoA were all up-regulated, which is consistent with JNK signaling activation. Further, constitutively active RhoA mutant (RhoA Q63L) partly rescued the inhibition of SP600125 on cell differentiation and polarity formation of mDPCs. To sum up, this study suggests that JNK signaling has a positive role in the formation of dental papilla cell polarization.
Asunto(s)
Papila Dental/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Animales , Antracenos/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Papila Dental/citología , Papila Dental/patología , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Mutagénesis , Germen Dentario/crecimiento & desarrollo , Germen Dentario/metabolismo , Germen Dentario/patología , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
INTRODUCTION: SIRT4 is a mitochondrial sirtuin. Owing to its dependance on the cofactor nicotinamide adenine dinucleotide (NAD+), SIRT4 can act as a mitochondrial metabolic sensor of cellular energy status. We have previously shown that enhancement of mitochondrial functions is vital for the odontogenic diï¬ ;erentiation of dental papilla cells (DPCs) during dentinogenesis. However, whether SIRT4 serves as an effective regulator of DPC diï¬ ;erentiation by affecting mitochondrial functions remains unexplored. METHODS: Primary DPCs obtained from the first molar dental papilla of neonatal Sprague-Dawley rats were used in this study. The expression pattern of SIRT4 was observed by immunohistochemistry in the first molar of postnatal day 1 (P1) rats. The changes in SIRT4 expression during odontogenic DPC differentiation were evaluated using real-time quantitative polymerase chain reaction (PCR), western blotting, and immunofluorescence. DPCs with loss (small interfering RNA-mediated knockdown) and gain (plasmid transfection-induced overexpression) of SIRT4 function were used to explore the role of SIRT4 in odontogenic differentiation. Mitochondrial function assays were performed using ATP, reactive oxygen species (ROS), and NAD+/NADH kits to investigate the potential mechanisms involved in SIRT4-mediated dentinogenesis. RESULTS: In the present study, we found that SIRT4 expression increased in a time-dependent manner during odontogenic differentiation bothin vivo and in vitro. Sirt4 knockdown resulted in reduced odontogenic differentiation and mineralization, whereas an opposite effect was observed with SIRT4 overexpression. Furthermore, our results verified that in addition to reducing DPC differentiation, Sirt4 knockdown could also significantly reduce ATP levels, elevate the NAD+/NADH ratio, and increase ROS levels. CONCLUSION: SIRT4 regulates mitochondrial functions and the antioxidant capacity of DPCs, thereby influencing dentin formation and tooth development, a phenomenon that may provide a foundation for better understanding the specific molecular mechanisms underlying dentin regeneration.
Asunto(s)
Papila Dental/citología , Mitocondrias/metabolismo , Odontogénesis , Especies Reactivas de Oxígeno/metabolismo , Sirtuinas/metabolismo , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Papila Dental/metabolismo , Modelos Animales , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Sirtuinas/genéticaRESUMEN
The effects of the renin-angiotensin system (RAS) on stem cells isolated from human dental apical papilla (SCAPs) are completely unknown. Therefore, the aim of this study was to identify RAS components expressed in SCAPs and the effects of angiotensin (Ang) II and Ang-(1-7) on cell proliferation. SCAPs were collected from third molar teeth of adolescents and maintained in cell culture. Messenger RNA expression and protein levels of angiotensin-converting enzyme (ACE), ACE2, and Mas, Ang II type I (AT1) and type II (AT2) receptors were detected in SCAPs. Treatment with either Ang II or Ang-(1-7) increased the proliferation of SCAPs. These effects were inhibited by PD123319, an AT2 antagonist. While Ang II augmented mTOR phosphorylation, Ang-(1-7) induced ERK1/2 phosphorylation. In conclusion, SCAPs produce the main RAS components and both Ang II and Ang-(1-7) treatments induced cell proliferation mediated by AT2 activation through different intracellular mechanisms.
Asunto(s)
Angiotensina II/farmacología , Angiotensina I/farmacología , Proliferación Celular/efectos de los fármacos , Papila Dental/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Células Madre/efectos de los fármacos , Adolescente , Células Cultivadas , Papila Dental/metabolismo , Femenino , Humanos , Imidazoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Peptidil-Dipeptidasa A/metabolismo , Fosforilación/efectos de los fármacos , Piridinas/farmacología , ARN Mensajero/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Células Madre/metabolismoRESUMEN
Background: The formation of dentin-pulp involves complex epithelial-mesenchymal interactions between Hertwig's epithelial root sheath cells (HERS) and dental papilla cells (DPCs). Earlier studies have identified some of the regulatory molecules participating in the crosstalk between HERS and DPCs and the formation of dentin-pulp. In the present study we focused on the role of HERS-secreted exosomes in DPCs and the formation of dentin-pulp. Specifically, we hypothesized that exosome-like vesicles (ELVs) might mediate the function of HERS and trigger lineage-specific differentiation of dental mesenchymal cells. To test our hypothesis, we evaluated the potential of ELVs derived from a HERS cell line (ELVs-H1) in inducing in vitro and in vivo differentiation of DPCs. Methods: ELVs-H1 were characterized using transmission electron microscopy and dynamic light scattering. The proliferation, migration, and odontoblast differentiation of DPCs after treatment with ELVs-H1, was detected by CCK8, transwell, ALP, and mineralization assays, respectively. Real time PCR and western blotting were used to detect gene and protein expression. For in vivo studies, DPC cells were mixed with collagen gel combined with or without ELVs and transplanted into the renal capsule of rats or subcutaneously into nude mice. HE staining and immunostaining were used to verify the regeneration of dentin-pulp and expression of odontoblast differentiation markers. Results: ELVs-H1 promoted the migration and proliferation of DPCs and also induced odontogenic differentiation and activation of Wnt/ß-catenin signaling. ELVs-H1 also contributed to tube formation and neural differentiation in vitro. In addition, ELVs-H1 attached to the collagen gel, and were slowly released and endocytosed by DPCs, enhancing cell survival. ELVs-H1 together with DPCs triggered regeneration of dental pulp-dentin like tissue comprised of hard (reparative dentin-like tissue) and soft (blood vessels and neurons) tissue, in an in vivo tooth root slice model. Conclusion: Our data highlighted the potential of ELVs-H1 as biomimetic tools in providing a microenvironment for specific differentiation of dental mesenchymal stem cells. From a developmental perspective, these vesicles might be considered as novel mediators facilitating the epithelial-mesenchymal crosstalk. Their instructive potency might be exploited for the regeneration of dental pulp-dentin tissues.
Asunto(s)
Pulpa Dental/metabolismo , Dentina/metabolismo , Animales , Diferenciación Celular/fisiología , China , Papila Dental/metabolismo , Pulpa Dental/fisiología , Dentina/fisiología , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Exosomas/fisiología , Regeneración Tisular Guiada Periodontal/métodos , Humanos , Células Madre Mesenquimatosas , Ratones , Ratones Desnudos , Ratas , Regeneración/fisiología , Raíz del Diente/metabolismo , Vía de Señalización WntRESUMEN
Enhancing the functions of mesenchymal stem cells (MSCs) is considered a potential approach for promoting tissue regeneration. In the present study, we investigate the role of HOXC8 in regulating differentiation and migration by using stem cells of the apical papilla (SCAPs). Our results showed that overexpression of HOXC8 suppressed the osteo-/dentinogenic differentiation, as detected by measuring alkaline phosphatase activity, in vitro mineralization, and the expressions of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, bone sialoprotein, runt-related transcription factor 2, and osterix in SCAPs, and inhibited in vivo osteo-/dentinogenesis of SCAPs. In addition, knockdown of HOXC8 promoted the osteo-/dentinogenic differentiation potentials of SCAPs. Mechanically, HOXC8 enhanced KDM1A transcription by directly binding to its promoter. HOXC8 and KDM1A also inhibited the migration and chemotaxis abilities of SCAPs. To sum up, HOXC8 negatively regulated the osteo-/dentinogenic differentiation and migration abilities of SCAPs by directly enhancing KDM1A transcription and indicated that HOXC8 and KDM1A could serve as potential targets for enhancing dental MSC mediated tissue regeneration.
Asunto(s)
Diferenciación Celular/fisiología , Histona Demetilasas/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre/metabolismo , Diferenciación Celular/genética , Proliferación Celular/fisiología , Células Cultivadas , Papila Dental/metabolismo , Genes Homeobox/fisiología , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/fisiologíaRESUMEN
Gestational diabetes mellitus (GDM) is an important factor involved in the pathogenesis of organ development in the offspring. Here, we analyzed the effects of GDM on odontoblastic differentiation of dental papilla cells (DPCs) and dentin formation in offspring and investigated their underlying mechanisms. A GDM rat model was induced by intraperitoneal injection of streptozotocin and offspring were collected. The results showed that GDM significantly affected odontoblast differentiation and dentin formation in offspring tooth. GDM activated the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-ĸB) signaling pathway and inhibited SMAD1/5/9 signaling to modulate the odontoblastic differentiation of DPCs in offspring. Inhibition of TLR4 signaling by treated with TAK-242 significantly reverses the suppression of odonto-differentiation of DPCs in diabetic offspring. Taken together, these data indicate GDM activated the offspring DPCs TLR4/NF-ĸB signaling, which suppressed the SMAD1/5/9 phosphorylation and then inhibited odontoblasts differentiation and dentin formation.
Asunto(s)
Diferenciación Celular/genética , Papila Dental/crecimiento & desarrollo , Diabetes Gestacional/genética , Receptor Toll-Like 4/genética , Animales , Calcificación Fisiológica/genética , Proliferación Celular/efectos de los fármacos , Papila Dental/metabolismo , Pulpa Dental/crecimiento & desarrollo , Pulpa Dental/patología , Diabetes Gestacional/patología , Femenino , Humanos , FN-kappa B/genética , Odontoblastos/metabolismo , Fosforilación/genética , Embarazo , Ratas , Transducción de Señal/genética , Proteína Smad1 , Sulfonamidas/farmacologíaRESUMEN
Cellular differentiation is caused by highly controlled modifications in the gene expression but rarely involves a change in the DNA sequence itself. Histone acetylation is a major epigenetic factor that adds an acetyl group to histone proteins, thus altering their interaction with DNA and nuclear proteins. Illumination of the histone acetylation during dentinogenesis is important for odontoblast differentiation and dentinogenesis. In the current study, we aimed to discover the roles and regulation of acetylation at histone 3 lysine 9 (H3K9ac) and H3K27ac during dentinogenesis. We first found that both of these modifications were enhanced during odontoblast differentiation and dentinogenesis. These modifications are dynamically catalyzed by histone acetyltransferases (HATs) and deacetylases (HDACs), among which HDAC3 was decreased while p300 increased during odontoblast differentiation. Moreover, overexpression of HDAC3 or knockdown p300 inhibited odontoblast differentiation in vitro, and inhibition of HDAC3 and p300 with trichostatin A or C646 regulated odontoblast differentiation. Taken together, the results of our present study suggest that histone acetylation is involved in dentinogenesis and coordinated expression of p300- and HDAC3-regulated odontoblast differentiation through upregulating histone acetylation.
Asunto(s)
Papila Dental/citología , Dentinogénesis , Proteína p300 Asociada a E1A/metabolismo , Histona Desacetilasas/metabolismo , Histonas/química , Células Madre Mesenquimatosas/citología , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Papila Dental/metabolismo , Proteína p300 Asociada a E1A/genética , Histona Desacetilasas/genética , Células Madre Mesenquimatosas/metabolismo , RatonesRESUMEN
: Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O2] is low (3-8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Papila Dental/metabolismo , Células Madre/citología , Autofagia/fisiología , Diferenciación Celular/fisiología , Hipoxia de la Célula/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Integrina alfa6/metabolismo , Proteínas de la Membrana/metabolismo , Tercer Molar/citología , Osteogénesis/fisiología , Oxígeno/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Antígenos Embrionarios Específico de Estadio/metabolismo , Nicho de Células Madre/fisiologíaRESUMEN
Biological phenotypes of mesenchymal stem cells (MSCs) are regulated by a series of biochemical elements, including microRNAs, hormones and growth factors. Our previous study illustrated a significant role of miR-141-3p during the osteogenic differentiation of stem cells from apical papilla (SCAPs). Nevertheless, the functions of miR-141-3p in regulating the proliferative ability and senescence of SCAPs have not been determined. This study identified that overexpression of miR-141-3p inhibited the proliferative ability of SCAPs. Meanwhile, the senescence of SCAPs was ahead of time. Conversely, transfection of miR-141-3p inhibitor promoted the proliferative ability of SCAPs and delayed their senescence. Yes-associated protein (YAP) was predicted as the downstream target gene of miR-141-3p by online softwares (miRDB, miRTarBase, miRWalk, and TargetScan), and was further verified by dual-luciferase reporter gene assay. Additionally, knockdown of YAP inhibited the proliferation and accelerated the senescence of SCAPs. Collectively, these findings proposed a novel direction that miR-141-3p impeded proliferative ability and promoted senescence of SCAPs through post-transcriptionally downregulating YAP.