scholarly journals HDAC6 Regulates the Fusion of Autophagosome and Lysosome to Involve in Odontoblast Differentiation

2020 ◽  
Vol 8 ◽  
Author(s):  
Yunyan Zhan ◽  
Haisheng Wang ◽  
Lu Zhang ◽  
Fei Pei ◽  
Zhi Chen
Keyword(s):  
Tooth Development ◽  
Expression Patterns ◽  
Autophagic Flux ◽  
Differentiation Capacity ◽  
Odontoblast Differentiation ◽  
Dental Papilla ◽  
Odontoblastic Differentiation ◽  
Dental Papilla Cells

Odontoblast differentiation is an important process during tooth development in which pre-odontoblasts undergo elongation, polarization, and finally become mature secretory odontoblasts. Many factors have been found to regulate the process, and our previous studies demonstrated that autophagy plays an important role in tooth development and promotes odontoblastic differentiation in an inflammatory environment. However, it remains unclear how autophagy is modulated during odontoblast differentiation. In this study, we found that HDAC6 was involved in odontoblast differentiation. The odontoblastic differentiation capacity of human dental papilla cells was impaired upon HDAC6 inhibition. Moreover, we found that HDAC6 and autophagy exhibited similar expression patterns during odontoblast differentiation both in vivo and in vitro; the expression of HDAC6 and the autophagy related proteins ATG5 and LC3 increased as differentiation progressed. Upon knockdown of HDAC6, LC3 puncta were increased in cytoplasm and the autophagy substrate P62 was also increased, suggesting that autophagic flux was affected in human dental papilla cells. Next, we determined the mechanism during odontoblastic differentiation and found that the HDAC6 substrate acetylated-Tubulin was up-regulated when HDAC6 was knocked down, and LAMP2, LC3, and P62 protein levels were increased; however, the levels of ATG5 and Beclin1 showed no obvious change. Autophagosomes accumulated while the number of autolysosomes was decreased as determined by mRFP-GFP-LC3 plasmid labeling. This suggested that the fusion between autophagosomes and lysosomes was blocked, thus affecting the autophagic process during odontoblast differentiation. In conclusion, HDAC6 regulates the fusion of autophagosomes and lysosomes during odontoblast differentiation. When HDAC6 is inhibited, autophagosomes can't fuse with lysosomes, autophagy activity is decreased, and it leads to down-regulation of odontoblastic differentiation capacity. This provides a new perspective on the role of autophagy in odontoblast differentiation.

scholarly journals RORα Regulates Odontoblastic Differentiation and Mediates the Pro-Odontogenic Effect of Melatonin on Dental Papilla Cells

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1098
Author(s):  
Jun Kang ◽  
Haoling Chen ◽  
Fuping Zhang ◽  
Tong Yan ◽  
Wenguo Fan ◽  
...  
Keyword(s):  
Tooth Development ◽  
Critical Role ◽  
Orphan Receptor ◽  
Dental Papilla ◽  
Odontoblastic Differentiation ◽  
Alp Activity ◽  
Matrix Mineralisation ◽  
Dental Papilla Cells

Dental papilla cells (DPCs), precursors of odontoblasts, are considered promising seed cells for tissue engineering. Emerging evidence suggests that melatonin promotes odontoblastic differentiation of DPCs and affects tooth development, although the precise mechanisms remain unknown. Retinoid acid receptor-related orphan receptor α (RORα) is a nuclear receptor for melatonin that plays a critical role in cell differentiation and embryonic development. This study aimed to explore the role of RORα in odontoblastic differentiation and determine whether melatonin exerts its pro-odontogenic effect via RORα. Herein, we observed that RORα was expressed in DPCs and was significantly increased during odontoblastic differentiation in vitro and in vivo. The overexpression of RORα upregulated the expression of odontogenic markers, alkaline phosphatase (ALP) activity and mineralized nodules formation (p < 0.05). In contrast, odontoblastic differentiation of DPCs was suppressed by RORα knockdown. Moreover, we found that melatonin elevated the expression of odontogenic markers, which was accompanied by the upregulation of RORα (p < 0.001). Utilising small interfering RNA, we further demonstrated that RORα inhibition attenuated melatonin-induced odontogenic gene expression, ALP activity and matrix mineralisation (p < 0.01). Collectively, these results provide the first evidence that RORα can promote odontoblastic differentiation of DPCs and mediate the pro-odontogenic effect of melatonin.

scholarly journals IPO7 Promotes Odontoblastic Differentiation of Mouse Dental Papilla Cells by Selectively Importing Odontogenic Transcription Factors

2020 ◽  
Author(s):  
Yue Zhang ◽  
Hao Zhang ◽  
Guohua Yuan ◽  
Guobin Yang
Keyword(s):  
Transcription Factors ◽  
Biological Process ◽  
Expression Patterns ◽  
Nucleocytoplasmic Transport ◽  
Immunoprecipitation Assay ◽  
Odontoblast Differentiation ◽  
Dental Papilla ◽  
Odontoblastic Differentiation ◽  
Dentin Formation ◽  
Dental Papilla Cells

Abstract Background: An important biological process for dentin formation and mineralization is odontoblast differentiation, which is precisely governed by a series of transcription factors (TFs). Importin 7 (IPO7) is a member of the Karyopherin β-superfamily mediating nucleocytoplasmic transport of proteins. In this study, we aimed to study the mechanisms by which IPO7 participates in odontoblastic differentiation. Methods: The expression patterns of IPO7 was investigated by immunofluorescence staining. Besides, mouse dental papilla cells (mDPCs) were extracted and cultured. After silencing Ipo7 in mDPCs, odontoblastic differentiation and the effect on odontogenic TFs were evaluated. Using nuclear and cytoplasmic extraction and co-immunoprecipitation assay, we aimed to confirm that IPO7 imports some odontogenic transcriptions factors to promote odontoblastic differentiation.Results: We found that IPO7 was increasingly expressed from the pre-odontoblasts to mature odontoblasts from PN2 to PN9. IPO7 enhanced odontoblast differentiation in mDPCs and imported essential TFs for odontoblastic differentiation, such as Distal-less homeobox 3 (DLX3), Osterix (OSX), Krüppel-like factor 4 (KLF4) and P-SMAD1/5 except for RUNX2. Besides, only RUNX2 showed no obvious interaction with IPO7.Conclusion: In conclusion, our data demonstrated that IPO7 enhances odontoblast differentiation in mDPCs by selectively importing odontogenic TFs.

WWP2 Promotes Odontoblastic Differentiation by Monoubiquitinating KLF5

2020 ◽  
pp. 002203452097086
Author(s):  
J. Fu ◽  
H. Zheng ◽  
Y. Xue ◽  
R. Jin ◽  
G. Yang ◽  
...  
Keyword(s):  
Tooth Development ◽  
Marker Genes ◽  
Alizarin Red ◽  
Odontoblast Differentiation ◽  
Dental Papilla ◽  
Odontoblastic Differentiation ◽  
C Terminus ◽  
Elevated Expression ◽  
Dentin Formation ◽  
Dental Papilla Cells

WW domain-containing E3 Ub-protein ligase 2 (WWP2) belongs to the homologous to E6AP C-terminus (HECT) E3 ligase family. It has been explored to regulate osteogenic differentiation, chondrogenesis, and palatogenesis. Odontoblasts are terminally differentiated mesenchymal cells, which contribute to dentin formation in tooth development. However, it remained unknown whether WWP2 participated in odontoblast differentiation. In this study, WWP2 was found to be expressed in mouse dental papilla cells (mDPCs), odontoblasts, and odontoblastic-induced mDPCs by immunohistochemistry and Western blotting. Besides, WWP2 expression was decreased in the cytoplasm but increased in the nuclei of differentiation-induced mDPCs. When Wwp2 was knocked down, the elevated expression of odontoblast marker genes ( Dmp1 and Dspp) in mDPCs induced by differentiation medium was suppressed. Meanwhile, a decrease of alkaline phosphatase (ALP) activity was observed by ALP staining, and reduced formation of mineralized matrix nodules was demonstrated by Alizarin Red S staining. Overexpression of WWP2 presented opposite results to knockdown experiments, suggesting that WWP2 promoted odontoblastic differentiation of mDPCs. Further investigation found that WWP2 was coexpressed and interacted with KLF5 in the nuclei, leading to ubiquitination of KLF5. The PPPSY (PY2) motif of KLF5 was essential for its physical binding with WWP2. Also, cysteine 838 (Cys838) of WWP2 was the active site for ubiquitination of KLF5, which did not lead to proteolysis of KLF5. Then, KLF5 was confirmed to be monoubiquitinated and transactivated by WWP2, which promoted the expression of KLF5 downstream genes Dmp1 and Dspp. Deletion of the PY2 motif of KLF5 or mutation of Cys838 of WWP2 reduced the upregulation of Dmp1 and Dspp. Besides, lysine (K) residues K31, K52, K83, and K265 of KLF5 were verified to be crucial to WWP2-mediated KLF5 transactivation. Taken together, WWP2 promoted odontoblastic differentiation by monoubiquitinating KLF5.

Zeb1 Promotes Odontoblast Differentiation in a Stage-Dependent Manner

2021 ◽  
pp. 002203452098224
Author(s):  
Y. Xiao ◽  
Y.X. Lin ◽  
Y. Cui ◽  
Q. Zhang ◽  
F. Pei ◽  
...  
Keyword(s):  
Late Stage ◽  
Tooth Development ◽  
Early Stage ◽  
Rna Seq ◽  
Cis Elements ◽  
Odontoblast Differentiation ◽  
Odontoblastic Differentiation ◽  
Accessible Chromatin

A comprehensive study of odontoblastic differentiation is essential to understand the process of tooth development and to achieve the ability of tooth regeneration in the future. Zinc finger E-box-binding homeobox 1 ( Zeb1) is a transcription factor expressed in various neural crest–derived tissues, including the mesenchyme of the tooth germ. However, its role in odontoblastic differentiation remains unknown. In this study, we found the expression of Zeb1 gradually increased during odontoblast differentiation in vivo, as well as during induced differentiation of cultured primary murine dental papilla cells (mDPCs) in vitro. In addition, the differentiation of mDPCs was repressed in Zeb1-silenced cells. We used RNA sequencing (RNA -seq) to identify the transcriptome-wide targets of Zeb1 and used assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to explore the direct targets of Zeb1 in both the early stage (embryonic day 16.5; E16.5) and the late stage (postnatal day 0; PN0) of tooth development. We identified the motifs of transcription factors enriched in Zeb1-dependent accessible chromatin regions and observed that only in the early stage of mDPCs could Zeb1 significantly change the accessibility of chromatin regions. In vivo and in vitro experiments confirmed that silencing of Zeb1 at E16.5 inhibited dentinogenesis. Analysis of RNA-seq and ATAC-seq resulted in the identification of Runx2, a gene directly regulated by Zeb1 during early odontoblast differentiation. Zeb1 enhances the expression of Runx2 by binding to its cis-elements, and ZEB1 interacts with RUNX2. In the late stage of tooth development, we found that ZEB1 could directly bind to and increase the enhancer activity of an element upstream of Dspp and promote dentinogenesis. In this study, for the first time, we revealed that ZEB1 promoted odontoblast differentiation in the early stage by altering chromatin accessibility of cis-elements near genes such as Runx2, while in the late stage, it directly enhanced Dspp transcription, thereby performing a dual role.

Changes in the distribution of tenascin during tooth development

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 289-296 ◽  
Author(s):  
I. Thesleff ◽  
E. Mackie ◽  
S. Vainio ◽  
R. Chiquet-Ehrismann
Keyword(s):  
Tooth Development ◽  
Polyclonal Antibodies ◽  
Staining Intensity ◽  
Mesenchymal Cells ◽  
Hard Tissue ◽  
Connective Tissues ◽  
Odontoblast Differentiation ◽  
Dental Papilla ◽  
Extracellular Matrix Molecule ◽  
Dental Papilla Cells

Tenascin is an extracellular matrix molecule that was earlier shown to be enriched in embryonic mesenchyme surrounding the budding epithelium in various organs including the tooth. In the present study tenascin was localized by immunohistology throughout the course of tooth development in the mouse and rat using polyclonal antibodies against chick tenascin. The results indicate that tenascin is expressed by the lineage of dental mesenchymal cells throughout tooth ontogeny. The intensity of staining with tenascin antibodies in the dental papilla mesenchyme was temporarily reduced at cap stage when the tooth grows rapidly and undergoes extensive morphogenetic changes. During the bell stage of morphogenesis, the staining intensity increased and tenascin was accumulated in the dental pulp even after completion of crown development and eruption. Tenascin was present in the dental basement membrane at the time of odontoblast differentiation. The dental papilla cells ceased to express tenascin upon differentiation into odontoblasts and tenascin was completely absent from dentin. It can be speculated that the remarkable expression of tenascin in the dental mesenchymal cells as compared to other connective tissues is associated with their capacity to differentiate into hard-tissue-forming cells.

Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth

Development ◽  
1993 ◽  
Vol 117 (2) ◽  
pp. 461-470 ◽  
Author(s):  
A.K. Jowett ◽  
S. Vainio ◽  
M.W. Ferguson ◽  
P.T. Sharpe ◽  
I. Thesleff
Keyword(s):  
Gene Expression ◽  
Tooth Development ◽  
Homeobox Gene ◽  
Oral Epithelium ◽  
Tooth Formation ◽  
Dental Papilla ◽  
Tissue Interactions ◽  
Dental Epithelium

Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth. By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation. msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.

scholarly journals C-Jun N-terminal kinase (JNK) pathway activation is essential for dental papilla cells polarization

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0233944
Author(s):  
Jiao Luo ◽  
Xiujun Tan ◽  
Ling Ye ◽  
Chenglin Wang
Keyword(s):  
Cell Function ◽  
Tooth Development ◽  
Tooth Germ ◽  
Cell Polarization ◽  
Positive Role ◽  
Jnk Pathway ◽  
Jnk Signaling ◽  
Dental Papilla ◽  
Dental Papilla Cells

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.

The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 269-285 ◽  
Author(s):  
A. Mackenzie ◽  
G.L. Leeming ◽  
A.K. Jowett ◽  
M.W. Ferguson ◽  
P.T. Sharpe
Keyword(s):  
Neural Crest ◽  
Tooth Development ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Branchial Arch ◽  
Developing Brain ◽  
Stage Of Development ◽  
Tooth Germs

Hox 7.1 is a murine homeobox-containing gene expressed in a range of neural-crest-derived tissues and areas of putative epithelial-mesenchymal interactions during embryogenesis. We have examined the expression of Hox 7.1 during craniofacial development in the mouse embryo between days 8 and 16 of development. Whereas facial expression at day 10 of gestation is broadly localised in the neural-crest-derived mesenchyme of the medial nasal, lateral nasal, maxillary and mandibular processes, by day 12 expression is restricted to the mesenchyme immediately surrounding the developing tooth germs in the maxillary and mandibular processes. Hox 7.1 expression in the mesenchyme of the dental papilla and follicle is maximal at the cap stage of development and progressively declines in the bell stage prior to differentiation of odontoblasts and ameloblasts. Hox 7.1 expression in tooth germs is independent of overall embryonic stage of development but is dependent on stage of development of the individual tooth. Similar patterns of transient Hox 7.1 expression can also be detected in tooth germs in vitro in organ cultures of day 11 first branchial arch explants cultured for up to 7 days. Hox 7.1 is also expressed early in development (days 10/11) in the epithelium of the developing anterior pituitary (Rathke's pouch), the connective tissue capsule and meninges of the developing brain, and specific regions of neuroepithelium in the developing brain.

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