CHIP inhibits odontoblast differentiation through promoting DLX3 polyubiquitylation and degradation.

Dentin is the major hard tissue of teeth formed by differentiated odontoblasts. How odontoblast differentiation is regulated remains enigmatic. Here, we report that the E3 ubiquitin ligase CHIP is highly expressed in undifferentiated dental mesenchymal cells and downregulated after differentiation of odontoblasts. Ectopic expression of CHIP inhibits odontoblastic differentiation of mouse dental papilla cells, whereas knockdown of endogenous CHIP has opposite effects. Chip (Stub1) knockout mice display increased formation of dentin and enhanced expression of odontoblast differentiation markers. Mechanistically, CHIP interacts with and induces K63 polyubiquitylation of the transcription factor DLX3, leading to its proteasomal degradation. Knockdown of DLX3 reverses the enhanced odontoblastic differentiation caused by knockdown of CHIP. These results suggest that CHIP inhibits odontoblast differentiation by targeting its tooth-specific substrate DLX3. Furthermore, our results indicate that CHIP competes with another E3 ubiquitin ligase, MDM2, that promotes odontoblast differentiation by monoubiquitylating DLX3. Our findings suggest that the two E3 ubiquitin ligases CHIP and MDM2 reciprocally regulate DLX3 activity by catalyzing distinct types of ubiquitylation, and reveal an important mechanism by which differentiation of odontoblasts is delicately regulated by divergent post-translational modifications.

piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

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.

Image fusion-based low-dose CBCT enhancement method for visualizing miniscrew insertion in the infrazygomatic crest.

Digital dental technology covers oral cone-beam computed tomography (CBCT) image processing and low-dose CBCT dental applications. A low-dose CBCT image enhancement method based on image fusion is proposed to address the need for subzygomatic small screw insertion. Specifically, firstly, a sharpening correction module is proposed, where the CBCT image is sharpened to compensate for the loss of details in the underexposed/over-exposed region. Secondly, a visibility restoration module based on type II fuzzy sets is designed, and a contrast enhancement module using curve transformation is designed. In addition to this, we propose a perceptual fusion module that fuses visibility and contrast of oral CBCT images. As a result, the problems of overexposure/underexposure, low visibility, and low contrast that occur in oral CBCT images can be effectively addressed with consistent interpretability. The proposed algorithm was analyzed in comparison experiments with a variety of algorithms, as well as ablation experiments. After analysis, compared with advanced enhancement algorithms, this algorithm achieved excellent results in low-dose CBCT enhancement and effective observation of subzygomatic small screw implantation. Compared with the best performing method, the evaluation metric is 0.07-2 higher on both datasets. The project can be found at: https://github.com/sunpeipei2024/low-dose-CBCT .

A WWP2-PTEN-KLF5 signaling axis regulates odontoblast differentiation and dentinogenesis in mice.

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

IPO7 Promotes Odontoblastic Differentiation and Inhibits Osteoblastic Differentiation Through Regulation of RUNX2 Expression and Translocation.

RUNX2, an important transcriptional factor for both odontoblastic and osteoblastic differentiation, is upregulated during osteoblastic differentiation, but downregulated during late odontoblastic differentiation. However, the specific mechanism of the different RUNX2 expression in bone and dentin remains largely unknown. Importin 7 (IPO7), a member of the karyopherin ß-superfamily, mediates nucleocytoplasmic transport of proteins. In this study, we found that IPO7 was increasingly expressed from pre-odontoblasts to mature odontoblasts. IPO7 expression was increased with odontoblastic differentiation of mouse dental papilla cells (mDPCs) and knockdown of IPO7-inhibited cell differentiation. While in MC3T3-E1 cells, IPO7 was decreased during osteoblastic differentiation and knockdown of IPO7-promoted cell differentiation. In mPDCs, IPO7 was able to bind with some odontoblastic transcription factors, and imported them into the nucleus, but not with RUNX2. Furthermore, IPO7 inhibited the total RUNX2 expression by promoting HDAC6 nuclear localization during odontoblastic differentiation. However, in MC3T3-E1 cells, IPO7 inhibited the nuclear distribution of RUNX2 but did not affect the total protein level of RUNX2. In conclusion, we found that IPO7 promotes odontoblastic differentiation and inhibits osteoblastic differentiation through regulating RUNX2 expression and translocation differently.

Effects of transforming growth factor-ß1 on odontoblastic differentiation in dental papilla cells is determined by IPO7 expression level.

Transforming growth factor ß1 (TGF-ß1) is one of the broad-spectrum growth-promoting factors that participate in tooth development. The influence of TGF-ß1 on the odontoblastic differentiation is still controvercy. Mouse primary dental papilla cells (mDPCs) as well as an immortalized mouse dental papilla cell line (mDPC6Ts) were treated with exogenous TGF-ß1 during odontoblastic differentiation. RT-qPCR, Western blot, alizarin red staining and ALP staining were carried out to investigate the influence of TGF-ß1 on odontoblastic differentiation. IPO7, important for SMAD complex translocation was also detected in mDPCs and mDPC6Ts in response to TGF-ß1. After silencing IPO7 by transfection, the translocation process of P-SMAD2 was investigated by nuclear and cytoplasmic extraction as well as co-immunoprecipitation assay. The odontogenic markers, mineralization and IPO7 expression were significantly up-regulated in TGF-ß1-treated mDPCs while down-regulated in mDPC6Ts. The total level of P-SMAD2 was not influenced by IPO7 in mDPCs, however, IPO7 could bind to P-SMAD2 and affect the nuclear-cytoplasm-shuttling of P-SMAD2. Our data demonstrated that TGF-ß1 plays opposite roles in odontoblast differentiation in mDPCs and immortalized mouse dental papilla cell line (mDPC6Ts), which is determined by IPO7.

Quaking promotes the odontoblastic differentiation of human dental pulp stem cells.

Odontoblastic differentiation of human dental pulp stem cells (hDPSCs) is essential for the formation of reparative dentin after dental caries or injury. Our previous studies have demonstrated that krüppel-like factor 4 (KLF4) is a critical transcription factor that promotes the odontoblastic differentiation of hDPSCs. Analysis of the microRNA binding sites within the 3'-UTR of KLF4 revealed that QKI, an RNA-binding protein, shared the most microRNAs with KLF4, presumably served as a "competent endogenous RNA (ceRNA)" with KLF4. Thus, we hypothesized QKI could also promote odontoblastic differentiation. In this study, we found QKI was up-regulated during mouse odontoblast differentiation in vivo and hDPSCs odontoblastic differentiation in vitro. Overexpression or knockdown of QKI in hDPSCs led to the increase or decrease of odontoblast marker genes' expressions, indicating its positive role in odontoblastic differentiation. We further validated that QKI served as a key ceRNA of KLF4 via interaction of the shared miRNAs in hDPSCs. Last, we found that, as an RNA binding protein, QKI protein could bind to, and stabilize dentin sialophosphoprotein (DSPP) mRNA, resulting in the augmented accumulation of DSP protein. Taken together, our study indicates that QKI promotes the odontoblastic differentiation of hDPSCs by acting as a ceRNA of KLF4 and as a binding protein of DSPP mRNA to stabilize its level. These two mechanisms of QKI will together positively regulate the downstream pathways and hence potentiate odontoblastic differentiation.

The non-canonical BMP and Wnt/ß-catenin signaling pathways orchestrate early tooth development.

BMP and Wnt signaling pathways play a crucial role in organogenesis, including tooth development. Despite extensive studies, the exact functions, as well as if and how these two pathways act coordinately in regulating early tooth development, remain elusive. In this study, we dissected regulatory functions of BMP and Wnt pathways in early tooth development using a transgenic noggin (Nog) overexpression model (K14Cre;pNog). It exhibits early arrested tooth development, accompanied by reduced cell proliferation and loss of odontogenic fate marker Pitx2 expression in the dental epithelium. We demonstrated that overexpression of Nog disrupted BMP non-canonical activity, which led to a dramatic reduction of cell proliferation rate but did not affect Pitx2 expression. We further identified a novel function of Nog by inhibiting Wnt/ß-catenin signaling, causing loss of Pitx2 expression. Co-immunoprecipitation and TOPflash assays revealed direct binding of Nog to Wnts to functionally prevent Wnt/ß-catenin signaling. In situ PLA and immunohistochemistry on Nog mutants confirmed in vivo interaction between endogenous Nog and Wnts and modulation of Wnt signaling by Nog in tooth germs. Genetic rescue experiments presented evidence that both BMP and Wnt signaling pathways contribute to cell proliferation regulation in the dental epithelium, with Wnt signaling also controlling the odontogenic fate. Reactivation of both BMP and Wnt signaling pathways, but not of only one of them, rescued tooth developmental defects in K14Cre;pNog mice, in which Wnt signaling can be substituted by transgenic activation of Pitx2. Our results reveal the orchestration of non-canonical BMP and Wnt/ß-catenin signaling pathways in the regulation of early tooth development.

TGF-ß signaling inhibits canonical BMP signaling pathway during palate development.

During early palate development, gene expression and regulation exhibit heterogeneity along the anterior-posterior axis. Transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling pathways play essential roles in secondary palatal formation but the exact relationship between the TGF-ß and BMP pathways in palate development remains unknown. Here, we demonstrate that, during early secondary palate development, phospho-(p)Smad1/5/8 is highly expressed in the anterior palate but relatively lowly expressed in the posterior palate. Conversely, pSmad2/3 has a lower expression level in the anterior palate than in the posterior palate. With the BRE-Gal reporter, we found that the canonical BMP signaling pathway was not activated in the anterior palate but exhibited a moderate level in the posterior palate. Co-immunoprecipitation revealed that Smad4 bound to pSmad1/5/8 only in the posterior palate and not in the anterior palate. Knocking-out of Tgfbr2 (Wnt1-Cre;Tgfbr2 f/f;BRE) in the palatal mesenchyme enhanced canonical BMP activity in the posterior palate but not in the anterior palate, because of decreased pSmad2/3. pSmad1/5/8-Smad4 complexes were found to be dramatically increased in posterior palatal mesenchymal cells at embryonic day 13.5 cultured in the presence of SB525334. Proximity ligation assay also showed pSmad1/5/8-Smad4 complexes were increased in the posterior palate of Wnt1-Cre;Tgfbr2 f/f mice. Therefore, the reduction of pSmad2/3 level in the palatal mesenchyme of Wnt1-Cre;Tgfbr2 f/f;BRE mice contributes primarily to the increase of pSmad1/5/8-Smad4 complexes leading to enhanced canonical BMP activity in the posterior palate. Moreover, during early development, canonical BMP signaling operates in the posterior palate but is completely absent in the anterior palate. Canonical TGF-ß signaling suppresses canonical BMP signaling activity in the posterior palate by competing limited Smad4.

An atypical canonical bone morphogenetic protein (BMP) signaling pathway regulates Msh homeobox 1 (Msx1) expression during odontogenesis.

Bone morphogenetic protein (BMP) signaling plays an essential role in early tooth development, evidenced by disruption of BMP signaling leading to an early arrested tooth development. Despite being a central mediator of BMP canonical signaling pathway, inactivation of Smad4 in dental mesenchyme does not result in early developmental defects. In the current study, we investigated the mechanism of receptor-activated Smads (R-Smads) and Smad4 in the regulation of the odontogenic gene Msx1 expression in the dental mesenchyme. We showed that the canonical BMP signaling is not operating in the early developing tooth, as assessed by failed activation of the BRE-Gal transgenic allele and the absence of phospho-(p)Smad1/5/8-Smad4 complexes. The absence of pSmad1/5/8-Smad4 complex appeared to be the consequence of saturation of Smad4 by pSmad2/3 in the dental mesenchyme as knockdown of Smad2/3 or overexpression of Smad4 led to the formation of pSmad1/5/8-Smad4 complexes and activation of canonical BMP signaling in dental mesenchymal cells. We showed that Smad1/5 but not Smad4 are required for BMP-induced expression of Msx1 in dental mesenchymal cells. We further presented evidence that in the absence of Smad4, BMPs are still able to induce pSmad1/5/8 nuclear translocation and their binding to the Msx1 promoter directly in dental mesenchymal cells. Our results demonstrate the functional operation of an atypical canonical BMP signaling (Smad4-independent and Smad1/5/8-dependent) pathway in the dental mesenchyme during early odontogenesis, which may have general implication in the development of other organs.

SP1 promotes the odontoblastic differentiation of dental papilla cells.

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.

miR-145 and miR-143 regulate odontoblast differentiation through targeting Klf4 and Osx genes in a feedback loop.

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.

The distribution and ultrastructure of the forming blood capillaries and the effect of apoptosis on vascularization in mouse embryonic molar mesenchyme.

Vascularization is essential for organ and tissue development. Teeth develop through interactions between epithelium and mesenchyme. The developing capillaries in the enamel organ, the dental epithelial structure, occur simultaneously by mechanisms of vasculogenesis and angiogenesis at the onset of dentinogenesis. The vascular neoformation in the dental mesenchyme has been reported to start from the cap stage. However, the mechanisms of vascularization in the dental mesenchyme remain unknown. In the hope of understanding the mechanisms of the formation of dental mesenchymal vasculature, mouse lower molar germs from embryonic day (E) 13.5 to E16.5 were processed for immunostaining of CD31 and CD34, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and transmission electron microscopy (TEM). In addition, the role of apoptosis for the vascularization in dental mesenchyme was examined by in vitro culture of E14.0 lower molars in the presence of the apoptosis inhibitor (z-VAD-fmk) and a subsequent subrenal culture. Our results showed that CD31- and CD34-positive cells progressively entered the central part of the dental papilla from the peridental mesenchyme. For TEM, angioblasts, young capillaries with thick endothelium and endothelial cells containing vacuoles were observed in peripheral dental mesenchyme, suggesting vasculogenesis was taking place. The presence of lateral sprouting, cytoplasmic filopodia and transluminal bridges in the dental papilla suggested angiogenesis was also occurring. Inhibition of apoptosis delayed the angiogenic vascularization of the dental papilla. Therefore, these data demonstrated that molar mesenchyme is progressively vascularized by mechanisms of both vasculogenesis and angiogenesis and apoptosis partially contributes to the vascularization of the dental papilla.

Application of Oxygen Saturation Test after Replantation of Avulsed Immature Permanent Teeth: A Prospective Observational Study.

INTRODUCTION: The evaluation of pulp status is crucial for avulsed immature permanent teeth after replantation. In addition to commonly used clinical and radiographic examinations providing clinical evidence, the oxygen saturation test may offer valuable assistance. The aim of this study was to analyze the efficacy of a pulse oximeter in evaluating pulp status in avulsed and replanted immature permanent teeth. METHODS: A prospective observational study was performed including 51 avulsed and replanted immature permanent teeth. Routine clinical and radiographic examinations were performed and used as the basis for the diagnosis of pulp status during the 1-year follow-up period. Meanwhile, the oxygen saturation values of these teeth were recorded using a modified pulse oximeter at each visit. RESULTS: Seven teeth completed pulp revascularization (success group), whereas 44 teeth failed to revascularize (failure group). Abnormal clinical and/or radiographic manifestations in the failure group were observed at an average period of 42.7 days, which was too late because a high incidence of inflammatory root resorption (43.18%) had occurred. For oxygen saturation tests, teeth in the success group showed an immediate postreplantation oxygen value of 70.71 ± 3.35, then an upward trend starting from the 2-week postreplantation visit, and a significantly increased final value of 81.86 ± 2.34 at the 1-year visit. In contrast, no increase trend was found for teeth in the failure group because abnormal clinical and/or radiographic manifestations emerged. CONCLUSIONS: The oxygen saturation test is a reliable diagnostic method to evaluate pulp status of avulsed teeth as early as 2 weeks after replantation.

KLF4 promoted odontoblastic differentiation of mouse dental papilla cells via regulation of DMP1.

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 Krüppel-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.

Autophagy appears during the development of the mouse lower first molar.

Odontogenesis consists of a series of consecutive tooth morphogenesis stages, in which apoptosis is involved to eliminate the unnecessary cells. Autophagy, a lysosome or endosome-mediated self-degradation process, is indicated to participate in embryogenesis and tissue morphogenesis associated with apoptosis. This study hypothesized that autophagy may be involved and associated with apoptosis in odontogenesis. The transcripts of autophagy-related genes (Atg5, Atg7, and Atg12) were positively detected in tooth germs at embryonic day (E) 14.5 and postnatal day (P) 5.5 by quantitative real-time PCR. The protein expression of Atg5-Atg12 conjugate and lipidation of LC3 (microtubule-associated protein 1 light chain 3, autophagic marker) were revealed in the developing tooth germs by western blot. Meanwhile, LC3 was immunolocalized in the enamel organ and dental papilla at embryonic stages (E13.5-E18.5), especially stage E14.5 cervical loop and the PEK that facing the mesenchyme. At postnatal stages (P1.5-P15.5), besides the dental epithelium cells, LC3 was detected in the differentiating and differentiated odontoblasts, dental follicle cells, and Hertwig's epithelium root sheath cells. Moreover, double-immunofluorescence analysis revealed the partial colocalization of LC3 and TUNEL signal in the E14.5 PEK that facing the mesenchyme, the E16.5 stratum intermedium and outer enamel epithelium, the P5.5 stratum intermedium and stellate reticulum. Nevertheless, LC3 was also found in non-apoptotic cells. Furthermore, the transmission electron microscopic images revealed the presence of autophagy, as well as the partial colocalization of autophagic vacuoles and apoptotic nuclei during tooth development. Our findings imply the developmental appearance of autophagy and its partial colocalization with apoptosis during odontogenesis.

MicroRNA-338-3p promotes differentiation of mDPC6T into odontoblast-like cells by targeting Runx2.

Odontoblasts are terminally differentiated cells that play a vital role in dentinogenesis. The differentiation of odontoblasts is regulated by a variety of genetic and epigenetic mechanisms. Our previous microRNA microarray studies verified that miR-338-3p was up-regulated during odontoblast differentiation. The purpose of this study was to determine the function of miR-338-3p during odontoblast differentiation. The upregulation of miR-338-3p expression during odontoblast differentiation was validated by qRT-PCR. Odontoblast differentiation was enhanced after over-expression of miR-338-3p, while a loss of function approach using a miR-338-3p inhibitor impaired odontoblast differentiation. Bioinformatic analysis identified Runx2 as a potential target of miR-338-3p. Overexpression of miR-338-3p caused a decreased in the expression of Runx2 at both mRNA and protein levels, while Runx2 expression increased after treatment with miR-338-3p inhibitors. Furthermore, the activity of a luciferase reporter plasmid containing the 3'-UTR of Runx2 was significantly suppressed by ectopic expression of miR-338-3p. These results suggested that miR-338-3p promotes odontoblast differentiation through targeting Runx2.

Reactive Oxygen Species-Induced Inhibition of Odontoblastic Differentiation of Mouse Dental Papilla Cells Is Mediated by Downregulation of Importin 7.

Tooth dentin is a crucial tooth structure. The biological process of odontoblast differentiation is essential for formation of normal dentin. Accumulation of reactive oxygen species (ROS) leads to oxidative stress, which can influence the differentiation of several cells. As a member of the importin-ß superfamily, importin 7 (IPO7) is essential for nucleocytoplasmic transport and plays an important role in the processes of odontoblast differentiation and oxidative stress. Nevertheless, the association between ROS, IPO7, and odontoblast differentiation in mouse dental papilla cells (mDPCs) and the underlying mechanisms remain to be elucidated. In this study, we confirmed that ROS suppressed odontoblastic differentiation of mDPCs as well as the expression and nucleocytoplasmic shuttle of IPO7 in cells, while overexpression of IPO7 can rescue these effects. ROS resulted in increased phosphorylation of p38 and cytoplasmic aggregation of phosphorylated p38 (p-p38), which was able to be reversed by overexpression of IPO7. p-p38 interacted with IPO7 in mDPCs without hydrogen peroxide (H2O2) treatment, but in the presence of H2O2, the interaction between p-p38 and IPO7 was significantly decreased. Inhibition of IPO7 increased the expression level and nuclear translocation of p53, which are mediated by cytoplasmic aggregation of p-p38. In conclusion, ROS inhibited odontoblastic differentiation of mDPCs, which is mediated by downregulation and damaged nucleocytoplasmic shuttle of IPO7.

IPO7 promotes lipopolysaccharide-induced inflammatory responses in human dental pulp cells via p38 MAPK and NF-κB signaling pathways.

Pulpitis is a chronic inflammatory process that greatly affects the physical, mental health and life quality of patients. Human dental pulp cells (hDPCs) are essential components of dental pulp tissue and play a significant role in pulpitis. Lipopolysaccharide (LPS) is an initiator of pulpitis and can induce the production of inflammatory cytokines in hDPCs by activating p38 MAPK and NF-κB signaling pathways. Importin7 (IPO7), a member of the importin-ß family, is widely expressed in many tissues. Previous studies have shown that IPO7 mediated nuclear translocation of p-p38 after stimulation, and IPO7 homologous protein IPO8 participated in human dental pulp inflammation. This research aims to investigate whether IPO7 is involved in pulpitis and explore its underlying mechanisms. In the current study, we found the expression of IPO7 was increased in pulpitis tissue. In vitro, hDPCs treated with LPS to mimic the inflammatory environment, the expression of IPO7 was increased. Knockdown of IPO7 significantly inhibited the production of inflammatory cytokines and suppressed the p38 MAPK and NF-κB signaling pathways. Activating the p38 MAPK and NF-κB signaling pathways by the p38 activator and p65 activator reversed the inflammatory responses. IPO7 interacted with p-p38 under LPS stimulation in hDPCs. In addition, the increased binding between IPO7 and p-p38 is associated with the decreased binding ability of IPO7 to Sirt2. In conclusion, we found that IPO7 was highly expressed in pulpitis and played a vital role in modulating human dental pulp inflammation.

AAV6-Mediated Gene Therapy Prevents Developmental Dentin Defects in a Dentinogenesis Imperfecta Type â ¢ Mouse Model.

Dentin is a major type of hard tissue of teeth and plays essential roles for normal tooth function. Odontoblasts are responsible for dentin formation. Mutations or deficiency in various genes affect the differentiation of odontoblasts, leading to irreversible dentin developmental defects in animals and humans. Whether such dentin defects can be reversed by gene therapy for odontoblasts remains unknown. In this study, we compare the infection efficiencies of six commonly used adeno-associated virus (AAV) serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVDJ) in cultured mouse odontoblast-like cells (OLCs). We show that AAV6 serotype infects OLCs with the highest efficiency among the six AAVs. Two cellular receptors, which are able to recognize AAV6, AAV receptor (AAVR), and epidermal growth factor receptor (EGFR), are strongly expressed in the odontoblast layer of mouse teeth. After local administration to mouse molars, AAV6 infects the odontoblast layer with high efficiency. Furthermore, AAV6-Mdm2 was successfully delivered to teeth and prevents the defects in odontoblast differentiation and dentin formation in Mdm2 conditional knockout mice (a mouse model of dentinogenesis imperfecta type Ⅲ). These results suggest that AAV6 can serve as a reliable and efficient vehicle for gene delivery to odontoblasts through local injection. In addition, human OLCs were also successfully infected by AAV6 with high efficiency, and both AAVR and EGFR are strongly expressed in the odontoblast layer of extracted human developing teeth. These findings suggest that AAV6-mediated gene therapy through local injection may be a promising treatment approach for hereditary dentin disorders in humans.