The Experts below are selected from a list of 3657 Experts worldwide ranked by ideXlab platform
Zhi Chen - One of the best experts on this subject based on the ideXlab platform.
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sall1 regulates commitment of odontoblast lineages by interacting with runx2 to remodel open chromatin regions
Stem Cells, 2020Co-Authors: Yuxiu Lin, Yao Xiao, Chujiao Lin, Qian Zhang, Shu Zhang, Fei Pei, Huan Liu, Zhi ChenAbstract:Mouse dental papilla cells (mDPCs) derive from cranial neural crest cells and maintain mesenchymal stem cell characteristics. The differentiation of neural crest cells into Odontoblasts is orchestrated by transcription factors regulating the expression of genes whose enhancers are initially inaccessible. However, the identity of the transcription factors driving the emergence of odontoblast lineages remains elusive. In this study, we identified SALL1, a transcription factor that was particularly expressed in preOdontoblasts, polarizing Odontoblasts, and secretory Odontoblasts in vivo. Knockdown of Sall1 in mDPCs inhibited their odontoblastic differentiation. In order to identify the regulatory network of Sall1, RNA sequencing and an assay for transposase-accessible chromatin with high-throughput sequencing were performed to analyze the genome-wide direct regulatory targets of SALL1. We found that inhibition of Sall1 expression could decrease the accessibility of some chromatin regions associated with odontoblast lineages at embryonic day 16.5, whereas these regions remained unaffected at postnatal day 0.5, suggesting that SALL1 regulates the fate of mDPCs by remodeling open chromatin regions at the early bell stage. Specifically, we found that SALL1 could directly increase the accessibility of cis-regulatory elements near Tgf-β2 and within the Runx2 locus. Moreover, coimmunoprecipitation and proximal ligation assays showed that SALL1 could establish functional interactions with RUNX2. Taken together, our results demonstrated that SALL1 positively regulates the commitment of odontoblast lineages by interacting with RUNX2 and directly activating Tgf-β2 at an early stage.
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mdm2 promotes odontoblast like differentiation by ubiquitinating dlx3 and p53
Journal of Dental Research, 2020Co-Authors: H Zheng, Zhi Chen, Guobin Yang, Guohua YuanAbstract:Dentin is an important structural component of the tooth. Odontoblast differentiation is an essential biological process that guarantees normal dentin formation, which is precisely regulated by various proteins. Murine double minute 2 (Mdm2) is an E3 ubiquitin ligase, and it plays a pivotal role in the differentiation of different cell types, such as osteoblasts and myoblasts. However, whether Mdm2 plays a role in odontoblast differentiation remains unknown. Here, we investigated the spatiotemporal expression of Mdm2 by immunostaining and found that Mdm2 was highly expressed in the Odontoblasts and slightly in the dental papilla cells of mouse incisors and molars. Gene knockdown and overexpression experiments verified that Mdm2 promoted the odontoblast-like differentiation of mouse dental papilla cells (mDPCs). Intranuclear colocalization and physical interaction between Mdm2 and distal-less 3 (Dlx3), a transcription factor important for odontoblast differentiation, was found during the odontoblast-like differentiation of mDPCs by double immunofluorescence and immunoprecipitation. Mdm2 was proved to monoubiquitinate Dlx3, which enhanced the expression of Dlx3 target gene Dspp. In addition, p53, the canonical substrate of Mdm2, was validated to be also ubiquitinated but degraded by Mdm2 during the odontoblast-like differentiation of mDPCs. Gene knockdown experiments confirmed that p53 inhibited the odontoblast-like differentiation of mDPCs. p53 and Mdm2 double knockdown partially rescued the reduced odontoblast-like differentiation by knockdown of Mdm2 alone. Taken together, our study revealed that Mdm2 promoted the odontoblast-like differentiation of mDPCs by ubiquitinating both Dlx3 and p53. On one hand, the monoubiquitination of Dlx3 by Mdm2 led to upregulation of Dspp, which is a marker of the odontoblast differentiation. On the other hand, ubiquitination of p53 by Mdm2 resulted in its degradation, which eliminated the inhibitory effect of p53 on the odontoblast-like differentiation of mDPCs.
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sp1 promotes the odontoblastic differentiation of dental papilla cells
Development Growth & Differentiation, 2015Co-Authors: Jie Zhang, Guohua Yuan, Lu Zhang, Huan Liu, Heng Lin, Zhi ChenAbstract:Odontoblasts are a type of terminally differentiated and matrix-secreting cells that are responsible for dentinogenesis. The process of odontoblast differentiation is regulated by a variety of transcription factors. The transcription factor SP1 is known to play an essential regulatory role in cell proliferation and differentiation. The purpose of this study was to investigate the role of SP1 in odontoblastic differentiation. Immunohistochemistry verified that SP1 was specifically expressed in polarizing and secretory Odontoblasts in vivo. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence revealed that the expression of SP1 was significantly upregulated during odontoblastic differentiation of mDPC6T cells, a dental papilla cell line. Overexpression of SP1 significantly increased the expression of odontoblast-related genes, including DSPP, DMP1 and ALP, and promoted the formation of mineralized nodules. Meanwhile, knockdown of SP1 decreased the expression of these odontoblast-related genes and suppressed the formation of mineralized nodules. Our results demonstrate that SP1 promotes the odontoblastic differentiation and mineralization of dental papilla cells.
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klf4 promoted odontoblastic differentiation of mouse dental papilla cells via regulation of dmp1
Journal of Cellular Physiology, 2013Co-Authors: Guohua Yuan, Lu Zhang, Zhi ChenAbstract:Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of Odontoblasts. We have recently found that Kruppel-like factor 4 (Klf4) was expressed in the polarizing and elongating Odontoblasts, but the function of Klf4 in the differentiation of Odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of Odontoblasts by up-regulating some odontoblast-related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4 significantly up-regulated odontoblast-related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4 down-regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4—Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of Odontoblasts via the up-regulation of Dmp1. J. Cell. Physiol. 228: 2076–2085, 2013. © 2013 Wiley Periodicals, Inc.
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klf4 promoted odontoblastic differentiation of mouse dental papilla cells via regulation of dmp1
Journal of Cellular Physiology, 2013Co-Authors: Heng Lin, Guohua Yuan, Lu Zhang, Huan Liu, Qin Sun, Zhi ChenAbstract:Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of Odontoblasts. We have recently found that Kruppel-like factor 4 (Klf4) was expressed in the polarizing and elongating Odontoblasts, but the function of Klf4 in the differentiation of Odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of Odontoblasts by up-regulating some odontoblast-related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4 significantly up-regulated odontoblast-related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4 down-regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4-Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of Odontoblasts via the up-regulation of Dmp1.
Anne George - One of the best experts on this subject based on the ideXlab platform.
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DPP promotes odontogenic differentiation of DPSCs through NF-κB signaling
'Springer Science and Business Media LLC', 2021Co-Authors: Yinghua Chen, Adrienn Pethö, Amudha Ganapathy, Anne GeorgeAbstract:Abstract Dentin phosphophoryn synthesized and processed predominantly by the Odontoblasts, functions as both structural and signaling protein. Mechanistic studies revealed that DPP stimulation of DPSCs positively impacted the differentiation of DPSCs into functional Odontoblasts. Results show that NF-κB signaling and transcriptional activation of genes involved in odontoblast differentiation were influenced by DPP signaling. Specifically, RelA/p65 subunit of NF-κB was identified as being responsible for the initiation of the differentiation cascade. Confocal imaging demonstrated the nuclear translocation of p65 with DPP stimulation. Moreover, direct binding of nuclear NF-κB p65 subunit to the promoter elements of Runx2, Osx, OCN, MMP1, MMP3, BMP4 and PTX3 were identified by ChIP analysis. Pharmacological inhibition of the NF-κB pathway using TPCA-1, a selective inhibitor of IKK-2 and JSH-23, an inhibitor that prevents nuclear translocation and DNA binding of p65 showed impairment in the differentiation process. Functional studies using Alizarin-Red staining showed robust mineral deposits with DPP stimulation and sparse deposition with defective odontoblast differentiation in the presence of inhibitors. In vivo expression of NF-κB targets such as OSX, OCN, PTX3 and p65 in Odontoblasts and dental pulp cells from DSPP null mouse was lower when compared with the wild-type. Overall, the results suggest an important role for DPP-mediated NF-κB activation in the transcriptional regulation of early odontogenic markers that promote differentiation of DPSCs
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dentin matrix protein 4 a novel secretory calcium binding protein that modulates odontoblast differentiation
Journal of Biological Chemistry, 2007Co-Authors: Karthikeyan Narayanan, Tanvi Muni, Amsaveni Ramachandran, Anne GeorgeAbstract:Abstract Formation of calcified tissues is a well regulated process. In dentin, the Odontoblasts synthesize several biomolecules that function as nucleators or inhibitors of mineralization. To identify genes that are odontoblast-specific, a subtractive hybridization technique was employed that resulted in the identification of a previously undescribed novel gene synthesized by the Odontoblasts. Based on the nomenclature in our laboratory, this gene has been named dentin matrix protein 4 (DMP4). The protein encoded by mouse DMP4 cDNA contained 579 amino acids, including a 26-amino acid signal peptide. Analysis of the protein sequence demonstrated the presence of a Greek key calcium-binding domain and one conserved domain of unknown function in all the species examined thus far. Calcium binding property was confirmed by 45Ca binding assays and the corresponding change in conformation by far-ultraviolet circular dichroism. Northern analysis demonstrated high expression levels of a single 3-kb mRNA transcript in tooth, whereas low expression levels were detected in other tissues. In situ hybridization analysis showed high expression levels of DMP4 in Odontoblasts and low levels in osteoblasts and ameloblasts during tooth development. Gain and loss of function experiments demonstrated that DMP4 had the potential to differentiate mesenchymal precursor cells into functional odontoblast-like cells.
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two related low molecular mass polypeptide isoforms of amelogenin have distinct activities in mouse tooth germ differentiation in vitro
Journal of Bone and Mineral Research, 2004Co-Authors: Kevin Tompkins, Anne George, Keith Alvares, Arthur VeisAbstract:Embryonic mouse tooth germs were cultured in vitro in the presence of two related amelogenin isoforms to determine their effects on tooth development. Our results show that these individual proteins have specific but quite different effects on epithelial-derived ameloblasts versus mesenchymal-derived Odontoblasts. Introduction: Amelogenins, the main protein components of enamel matrix, have been shown to have signaling activity. Amelogenin isoforms differing only by the presence or exclusion of exon 4, designated ‘A+4’ (composed of exons 2, 3, 4, 5, 6d, and 7) and ‘A-4’ (composed of exons 2, 3, 4, 5, 6d, and 7) and ‘A-4’ (composed of exons 2, 3, 5, 6d, and 7), showed similar, but different, effects both in vitro and in vivo on postnatal teeth. Materials and Methods: Lower first molar tooth germs of E15/16 CD1 mice were microdissected and cultured in vitro in a semisolid media containing either 20% FBS, 2% FBS, or 2% FBS with either 1.5 nM ‘A+4’, ‘A-4’, or both for 6 days. Tooth germs were analyzed by H&E staining and immunohistochemistry for collagen I, dentin matrix protein 2, and DAPI nuclear staining. Results: Teeth cultured in media containing 20% FBS showed normal development with polarized ameloblasts, and Odontoblasts producing dentin matrix, and DMP2 expression in Odontoblasts and pre-ameloblasts. Culture in 2% FBS media resulted in no ameloblast polarization and modest odontoblast differentiation with scant dentin matrix. Tooth germs cultured with ‘A+4’ in 2% FBS media had well-polarized Odontoblasts with robust dentin production and concomitant ameloblast polarization. DMP2 expression was equal to or greater than seen in the 20% FBS culture condition. In cultures with ‘A-4’ in 2% FBS media, odontoblast polarization and dentin production was reduced compared with ‘A+4’. However, the pre-ameloblast layer was disorganized, with no ameloblast polarization occurring along the dentin surface. DMP2 expression was reduced in the Odontoblasts compared with the 20% FBS and ‘A+4’ conditions and was almost completely abrogated in the pre-ameloblasts. Conclusion: These data show different signaling activities of these closely related amelogenin isoforms on tooth development. Here we make the novel observation that ‘A−4’ has an inhibitory effect on ameloblast development, whereas ‘A+4’ strongly stimulates odontoblast development. We show for the first time that specific amelogenin isoforms have effects on embryonic tooth development in vitro and also hypothesize that DMP2 may play a role in the terminal differentiation of both ameloblasts and Odontoblasts.
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odontoblast cells immortalized by telomerase produce mineralized dentin like tissue both in vitro and in vivo
Journal of Biological Chemistry, 2002Co-Authors: Karthikeyan Narayanan, Gen He, Abdullah Almushayt, Amsaveni Ramachandran, Carla A. Evans, Anne GeorgeAbstract:Abstract The formation of dentin provides one well accepted paradigm for studying mineralized tissue formation. For the assembly of dentin, several cellular signaling pathways cooperate to provide neural crest-derived mesenchymal cells with positional information. Further, “cross-talk” between signaling pathways from the mesenchymal derived odontoblast cells and the epithelially derived ameloblasts during development is responsible for the formation of functional Odontoblasts. These intercellular signals are tightly regulated, both temporally and spatially. When isolated from the developing tooth germ, Odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast cell line would be a valuable reproducible tool for studying the modulatory effects involved in odontoblast differentiation as well as the molecular events involved in mineralized dentin formation. In this study an immortalized odontoblast cell line, which has the required biochemical machinery to produce mineralized tissuein vitro, has been generated. These cells were implanted into animal models to determine their in vivo effects on dentin formation. After implantation, we observed a multistep, programmed cascade of gene expression in the exogenous Odontoblasts as the dentin formed de novo. Some of the genes expressed include the dentin matrix proteins 1, 2, and 3, which are extracellular matrix molecules responsible for the ultimate formation of mineralized dentin. The biological response was also examined by histology and radiography and confirmed for mineral deposition by von Kossa staining. Thus, a transformed odontoblast cell line was created with high proliferative capacity that might ultimately be used for the regeneration and repair of dentin in vivo.
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differentiation of embryonic mesenchymal cells to odontoblast like cells by overexpression of dentin matrix protein 1
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Karthikeyan Narayanan, Rampalli Srinivas, Bruce Quinn, Amsaveni Ramachandran, Anne GeorgeAbstract:Abstract Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of Odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated Odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.
Yuko Miyagoesuzuki - One of the best experts on this subject based on the ideXlab platform.
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laminin α2 is essential for odontoblast differentiation regulating dentin sialoprotein expression
Journal of Biological Chemistry, 2004Co-Authors: Kenji Yuasa, Yoko Kamasaki, Eri Arikawahirasawa, Emiko Fukumoto, Aya Yamada, Hidemitsu Harada, Kan Saito, Kazuhiro Kanaoka, Yuko MiyagoesuzukiAbstract:Abstract Laminin α2 is subunit of laminin-2 (α2β1γ1), which is a major component of the muscle basement membrane. Although the laminin α2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin α2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin α2 is expressed in dental mesenchymal cells, especially in Odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in Odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin α2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.
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laminin α2 is essential for odontoblast differentiation regulating dentin sialoprotein expression
Journal of Biological Chemistry, 2004Co-Authors: Kenji Yuasa, Yoko Kamasaki, Eri Arikawahirasawa, Emiko Fukumoto, Aya Yamada, Hidemitsu Harada, Kan Saito, Kazuhiro Kanaoka, Yuko MiyagoesuzukiAbstract:Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in Odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in Odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.
Hayato Ohshima - One of the best experts on this subject based on the ideXlab platform.
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lymphoid enhancer binding factor 1 expression precedes dentin sialophosphoprotein expression during rat odontoblast differentiation and regeneration
Journal of Endodontics, 2013Co-Authors: Mitsushiro Nakatomi, Hiroko Idayonemochi, Hayato OhshimaAbstract:Abstract Introduction The molecular mechanisms behind odontoblast differentiation remain obscure. Lymphoid enhancer-binding factor 1 (Lef1) is a transcription factor that mediates Wnt signaling and has been suggested to regulate dentin sialophosphoprotein ( Dspp ) expression in vitro . This study aimed to clarify their precise relationship in the process of odontoblast differentiation in vivo . Methods The detailed spatiotemporal expression patterns of Lef1 and Dspp together with other known and putative odontoblast differentiation markers such as P21 and heat-shock protein 25 ( Hsp25 ) were examined by in situ hybridization and immunohistochemistry on paraffin sections of rat incisors and developing molars at postnatal days 1–100. To observe odontoblast regeneration following tooth injury, a cavity was prepared on the upper first molar of 10-week-old rats and the expressions of Lef1 and Dspp were investigated. Results Following undifferentiated state expressing none of these examined markers, preOdontoblasts begun to express P21 , Lef1 and Hsp25 according to their progress of differentiation, although Dspp was undetectable. Immature Odontoblasts commenced transcribing Dspp simultaneously with dentin calcification. Lef1, Dspp and Hsp25 were co-expressed in mature Odontoblasts. In contrast to continuously growing incisors, Lef1, Dspp and P21 were down-regulated in the resting Odontoblasts in molars when primary dentin formation was completed. Remarkably, Lef1 expression also preceded Dspp expression in newly differentiated odontoblast-like cells during the pulpal healing process after tooth injury. Conclusions Lef1 expression precedes Dspp expression without exception in both primary and reparative dentinogeneses. Our results suggest that Lef1 might play a key role in odontoblast differentiation through regulating Dspp expression.
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expression patterns of nestin and dentin sialoprotein during dentinogenesis in mice
Biomedical Research-tokyo, 2012Co-Authors: Angela Quispesalcedo, Hiroko Idayonemochi, Mitsushiro Nakatomi, Hayato OhshimaAbstract:Differentiated Odontoblasts could not be identified by one unique phenotypic marker, but the combination of expression of dentin phosphoprotein (Dpp), dentin sialoprotein (Dsp), dentin matrix protein 1 (Dmp1), and nestin may be valuable for the assessment of these cells. However, the findings using these proteins remain controversial. This study aimed to compare two odontoblast differentiation markers: nestin and Dsp in the process of dentinogenesis in mice. We performed immunohistochemistry and/or in situ hybridization technique for nestin and Dsp using 3-week-old incisors as well as postnatal 1-day- to 8-week-old molars. PreOdontoblasts began to express nestin and Dsp proteins and Dsp mRNA, which increased in their intensity according to the progress of odontoblast differentiation in both incisors and developing molars. Nestin was consistently expressed in the differentiated Odontoblasts even after the completion of dentin matrix deposition. The expression of Dsp mRNA coincided with the odontoblast secretory activity for dentin matrix deposition. In contrast, other pulpal cells, predentin matrix and dentinal tubules also showed a positive reaction for Dsp protein in addition to differentiated Odontoblasts. In conclusion, nestin is valuable as a differentiation marker for Odontoblasts, whereas Dsp mRNA is a functional marker for their secretory activity.
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an immunohistochemical study of the expression of heat shock protein 25 and cell proliferation in the dental pulp and enamel organ during odontogenesis in rat molars
Archives of Oral Biology, 2006Co-Authors: Naohiro Nakasone, Hiromasa Yoshie, Hayato OhshimaAbstract:Summary Objectives The aim of this study is to clarify the functional significance of heat-shock protein (HSP)-25 during tooth development. Design We compared the expression of HSP-25 in the dental epithelial and mesenchymal cells with their proliferative activity during odontogenesis in rat molars on postnatal days 1–100 by immunohistochemistry using anti-HSP-25 and anti-5-bromo-2′-deoxyuridine (BrdU) for cell proliferation assay. Results On day 1, BrdU-immunoreactive cells were densely located in the inner enamel epithelium in the cervical loop and inter-cusped areas and the dental pulp adjacent to them, whereas HSP-25-immunoractivity (IR) was restricted to the cusped area where Odontoblasts and ameloblasts had already differentiated. Subsequently, BrdU-IR shifted in the apical direction to be localized around Hertwig's epithelial root sheath during days 5–30, never overlapping with concomitantly apically-shifted HSP-25-IR. On days 60–100, BrdU-immunoreactive cells were hardly recognizable in the dental pulp, where HSP-25-IR was exclusively localized in the odontoblast layer. Furthermore, the odontoblast- and ameloblast-lineage cells exhibited two steps in the expression of HSP-25 throughout the postnatal stages: first, dental epithelial and pulpal mesenchymal cells showed a weak IR for HSP-25 after the cessation of their proliferative activity, and subsequently Odontoblasts and ameloblasts consistently expressed an intense HSP-25-IR. Conclusion Odontoblast- and ameloblast-lineage cells acquire HSP-25-IR after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. The consistent expression of HSP-25-IR in the formative cells may be involved in the maintenance of their functional integrity.
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possible role of immunocompetent cells and the expression of heat shock protein 25 in the process of pulpal regeneration after tooth injury in rat molars
Journal of Electron Microscopy, 2003Co-Authors: Kuniko Nakakuraohshima, Junichi Watanabe, Shinichi Kenmotsu, Hayato OhshimaAbstract:Recent studies have established that heat shock proteins (HSPs) potentially play a role in immunosurveillance. The purpose of the present study was to clarify the relationship between the chronological changes of immunocompetent cells and the expression of HSP-25 in the process of pulpal regeneration after tooth injury in rat molars by immunocytochemistry for HSP-25 and class II major histocompatibility complex (MHC ) antigen. In untreated control teeth, intense HSP-25 immunoreactivity was found in the cell bodies of Odontoblasts. Both cavity preparation and tooth replantation caused the degeneration of the odontoblast layer to result in the loss of HSP-25 immunoreactions in the suffered dental pulp at the early stages after tooth injury. Numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their cell processes into the dentinal tubules at 12-24 h after cavity preparation and 3 days after tooth replantation. Newly differentiated odontoblast-like cells with HSP-25 immunoreactivity were arranged at the pulp-dentin border and the class II MHC-positive cells retreated towards the subodontoblastic layer by post-operative days 3-5 after tooth injury. Thus, the common cellular events occur during pulpal regeneration following two different experimental injuries. These findings indicate that the time course of changes in the expression of HSP-25 immunoreactivity reflects the degeneration/regeneration process of Odontoblasts and that the temporal appearance of the class II MHC-positive cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defence reactions during the pulpal regeneration process.
Guohua Yuan - One of the best experts on this subject based on the ideXlab platform.
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mdm2 promotes odontoblast like differentiation by ubiquitinating dlx3 and p53
Journal of Dental Research, 2020Co-Authors: H Zheng, Zhi Chen, Guobin Yang, Guohua YuanAbstract:Dentin is an important structural component of the tooth. Odontoblast differentiation is an essential biological process that guarantees normal dentin formation, which is precisely regulated by various proteins. Murine double minute 2 (Mdm2) is an E3 ubiquitin ligase, and it plays a pivotal role in the differentiation of different cell types, such as osteoblasts and myoblasts. However, whether Mdm2 plays a role in odontoblast differentiation remains unknown. Here, we investigated the spatiotemporal expression of Mdm2 by immunostaining and found that Mdm2 was highly expressed in the Odontoblasts and slightly in the dental papilla cells of mouse incisors and molars. Gene knockdown and overexpression experiments verified that Mdm2 promoted the odontoblast-like differentiation of mouse dental papilla cells (mDPCs). Intranuclear colocalization and physical interaction between Mdm2 and distal-less 3 (Dlx3), a transcription factor important for odontoblast differentiation, was found during the odontoblast-like differentiation of mDPCs by double immunofluorescence and immunoprecipitation. Mdm2 was proved to monoubiquitinate Dlx3, which enhanced the expression of Dlx3 target gene Dspp. In addition, p53, the canonical substrate of Mdm2, was validated to be also ubiquitinated but degraded by Mdm2 during the odontoblast-like differentiation of mDPCs. Gene knockdown experiments confirmed that p53 inhibited the odontoblast-like differentiation of mDPCs. p53 and Mdm2 double knockdown partially rescued the reduced odontoblast-like differentiation by knockdown of Mdm2 alone. Taken together, our study revealed that Mdm2 promoted the odontoblast-like differentiation of mDPCs by ubiquitinating both Dlx3 and p53. On one hand, the monoubiquitination of Dlx3 by Mdm2 led to upregulation of Dspp, which is a marker of the odontoblast differentiation. On the other hand, ubiquitination of p53 by Mdm2 resulted in its degradation, which eliminated the inhibitory effect of p53 on the odontoblast-like differentiation of mDPCs.
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sp1 promotes the odontoblastic differentiation of dental papilla cells
Development Growth & Differentiation, 2015Co-Authors: Jie Zhang, Guohua Yuan, Lu Zhang, Huan Liu, Heng Lin, Zhi ChenAbstract:Odontoblasts are a type of terminally differentiated and matrix-secreting cells that are responsible for dentinogenesis. The process of odontoblast differentiation is regulated by a variety of transcription factors. The transcription factor SP1 is known to play an essential regulatory role in cell proliferation and differentiation. The purpose of this study was to investigate the role of SP1 in odontoblastic differentiation. Immunohistochemistry verified that SP1 was specifically expressed in polarizing and secretory Odontoblasts in vivo. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence revealed that the expression of SP1 was significantly upregulated during odontoblastic differentiation of mDPC6T cells, a dental papilla cell line. Overexpression of SP1 significantly increased the expression of odontoblast-related genes, including DSPP, DMP1 and ALP, and promoted the formation of mineralized nodules. Meanwhile, knockdown of SP1 decreased the expression of these odontoblast-related genes and suppressed the formation of mineralized nodules. Our results demonstrate that SP1 promotes the odontoblastic differentiation and mineralization of dental papilla cells.
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klf4 promoted odontoblastic differentiation of mouse dental papilla cells via regulation of dmp1
Journal of Cellular Physiology, 2013Co-Authors: Guohua Yuan, Lu Zhang, Zhi ChenAbstract:Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of Odontoblasts. We have recently found that Kruppel-like factor 4 (Klf4) was expressed in the polarizing and elongating Odontoblasts, but the function of Klf4 in the differentiation of Odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of Odontoblasts by up-regulating some odontoblast-related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4 significantly up-regulated odontoblast-related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4 down-regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4—Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of Odontoblasts via the up-regulation of Dmp1. J. Cell. Physiol. 228: 2076–2085, 2013. © 2013 Wiley Periodicals, Inc.
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klf4 promoted odontoblastic differentiation of mouse dental papilla cells via regulation of dmp1
Journal of Cellular Physiology, 2013Co-Authors: Heng Lin, Guohua Yuan, Lu Zhang, Huan Liu, Qin Sun, Zhi ChenAbstract:Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of Odontoblasts. We have recently found that Kruppel-like factor 4 (Klf4) was expressed in the polarizing and elongating Odontoblasts, but the function of Klf4 in the differentiation of Odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of Odontoblasts by up-regulating some odontoblast-related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4 significantly up-regulated odontoblast-related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4 down-regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4-Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of Odontoblasts via the up-regulation of Dmp1.
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mir 145 and mir 143 regulate odontoblast differentiation through targeting klf4 and osx genes in a feedback loop
Journal of Biological Chemistry, 2013Co-Authors: Guohua Yuan, Lu Zhang, Shuo Chen, Zhi ChenAbstract:Dentin tissue is derived from mesenchymal cells induced into the odontoblast lineage. The differentiation of Odontoblasts is a complex process regulated by several transcriptional factor signaling transduction pathways. However, post-translational regulation of these factors during dentinogenesis remains unclear. To further explore the mechanisms, we investigated the role of microRNA (miRNA) during odontoblast differentiation. We profiled the miRNA expression pattern during mouse odontoblast differentiation using a microarray assay and identified that miR-145 and miR-143 were down-regulated during this process. In situ hybridization verified that the two miRNAs were gradually decreased during mouse odontoblast differentiation. Loss-of-function and gain-of-function experiments revealed that down-regulation of miR-145 and miR-143 could promote odontoblast differentiation and increased Dspp and Dmp1 expression in mouse primary dental pulp cells and vice versa. We found that miR-145 and miR-143 controlled odontoblast differentiation through several mechanisms. First, KLF4 and OSX bind to their motifs in Dspp and Dmp1 gene promoters and up-regulate their transcription thereby inducing odontoblast differentiation. The miR-145 binds to the 3′-UTRs of Klf4 and Osx genes, inhibiting their expression. Second, KLF4 repressed miR-143 transcription by binding to its motifs in miR-143 regulatory regions as detected by ChIP assay and dual luciferase reporter assay. Third, miR-143 regulates odontoblast differentiation in part through miR-145 pathway. Taken together, we for the first time showed that the miR-143 and miR-145 controlled odontoblast differentiation and dentin formation through KLF4 and OSX transcriptional factor signaling pathways.
