Inflammation-associated proteinase functions are key determinants of inflammatory stromal tissues deconstruction. As a specialized inflammatory pathological process, dental internal resorption (IR) includes both soft and hard tissues deconstruction within the dentin-pulp complex, which has been one of the main reasons for inflammatory tooth loss. Mechanisms of inflammatory matrix degradation and tissue resorption in IR are largely unclear. In this study, we used a combination of Cre-loxP reporter, flow cytometry, cell transplantation, and enzyme activities assay to mechanistically investigate the role of regenerative cells, odontoblasts (ODs), in inflammatory mineral resorption and matrices degradation. We report that inflamed ODs have strong capabilities of matrix degradation and tissue resorption. Traditionally, ODs are regarded as hard-tissue regenerative cells; however, our data unexpectedly present ODs as a crucial population that participates in IR-associated tissue deconstruction. Specifically, we uncovered that nuclear factor-kappa b (NF-κB) signaling orchestrated Tumor necrosis factor α (TNF-α)-induced matrix metalloproteinases (Mmps) and Cathepsin K (Ctsk) functions in ODs to enhance matrix degradation and tissue resorption. Furthermore, TNF-α increases Rankl/Opg ratio in ODs via NF-κB signaling by impairing Opg expression but increasing Rankl level, which utterly makes ODs cell line 17IIA11 (A11) become Trap⁺ and Ctsk⁺ multinucleated cells to perform resorptive actions. Blocking of NF-κB signaling significantly rescues matrix degradation and resorptive functions of inflamed ODs via repressing vital inflammatory proteinases Mmps and Ctsk. Utterly, via utilizing NF-κB specific small molecule inhibitors we satisfactorily attenuated inflammatory ODs-associated human dental IR in vivo. Our data reveal the underlying mechanisms of inflammatory matrix degradation and resorption via proteinase activities in IR-related pathological conditions.
Humans ; Matrix Metalloproteinases/metabolism* ; Minerals/metabolism* ; NF-kappa B/metabolism* ; Odontoblasts/metabolism* ; Osteoclasts/metabolism* ; RANK Ligand/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

Multiple signaling pathways are involved in the regulation of cell proliferation and differentiation in odontogenesis and dental tissue renewal, but the details of these mechanisms remain unknown. Here, we investigated the expression patterns of a transcription factor, Krüppel-like factor 6 (KLF6), during the development of murine tooth germ and its function in odontoblastic differentiation. KLF6 was almost ubiquitously expressed in odontoblasts at various stages, and it was co-expressed with P21 (to varying degrees) in mouse dental germ. To determine the function of Klf6, overexpression and knockdown experiments were performed in a mouse dental papilla cell line (iMDP-3). Klf6 functioned as a promoter of odontoblastic differentiation and inhibited the proliferation and cell cycle progression of iMDP-3 through p21 upregulation. Dual-luciferase reporter assay and chromatin immunoprecipitation showed that Klf6 directly activates p21 transcription. Additionally, the in vivo study showed that KLF6 and P21 were also co-expressed in odontoblasts around the reparative dentin. In conclusion, Klf6 regulates the transcriptional activity of p21, thus promoting the cell proliferation to odontoblastic differentiation transition in vitro. This study provides a theoretical basis for odontoblast differentiation and the formation of reparative dentine regeneration.
Animals ; Cell Differentiation/physiology* ; Cell Proliferation ; Mice ; Odontoblasts/metabolism* ; Odontogenesis ; Tooth Germ

OBJECTIVE: To investigate the role of autophagy in lipopolysaccharide (LPS)-induced apoptosis of murine odontoblasts. METHODS: Murine odontoblasts (mDPC-23 cells) were treated with 5 μg/mL LPS for 6, 12 and 24 h, and the changes in cell viability was examined using CCK8 kit and cell apoptosis was detected by TUNEL staining. The changes in the protein levels of LC3, Beclin1, Atg5, AKT, p-AKT, mTOR and p-mTOR were detected using Western blotting. The effect of 3-MA treatment for 24 h on LPS-induced apoptosis of mDPC-23 cells was evaluated by detecting the expressions of apoptosis-related proteins caspase-3 and Bax using Western blotting. RESULTS: Stimulation with LPS for 6 and 12 h did not cause significant changes in the proliferation or apoptosis of mDPC-23 cells, but LPS treatment for 24 h significantly suppressed cell proliferation ( CONCLUSIONS LPS stimulation induces autophagy to promote apoptosis of mDPC-23 cells, and suppression of autophagy attenuates LPS-induced apoptosis. Autophagy may play an important role in the injury of inflamed pulp tissues.
Animals ; Apoptosis ; Autophagy ; Lipopolysaccharides/pharmacology* ; Mice ; Odontoblasts/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction

OBJECTIVE: To verify the effect of the mutant gene vps4b on the expression of tooth development-related proteins, dentin sialophosphoprotein (DSPP) and collagenⅠ (COL-Ⅰ). METHODS: Paraffin tissue sections of the first molar tooth germ were obtained from the heads of fetal mice at the embryonic stages of 13.5, 14.5, and 16.5 days and from the mandibles of larvae aged 2.5 and 7 days after birth. The immunohistochemical method was used to detect the expression and location of DSPP and COL-Ⅰ in wild-type mouse and vps4b knockout mouse. RESULTS: DSPP and COL-Ⅰ were not found in the bud and cap stages of wild-type mouse molar germ. In the bell stage, DSPP was positively expressed in the inner enamel epithelium and dental papilla, whereas COL-Ⅰ was strongly expressed in the dental papilla and dental follicle. During the secretory and mineralized periods, DSPP and COL-Ⅰ were intensely observed in ameloblasts, odontoblasts, and dental follicles, but COL-Ⅰ was also expressed in the dental papilla. After vps4b gene knockout, DSPP was not expressed in the dental papilla of the bell stage and in the dental papilla and dental follicle of the secretory phase. The expression position of COL-Ⅰ in the bell and mineralization phase was consistent with that in the wild-type mice. Moreover, the expression of COL-Ⅰ in the dental papilla changed in the secretory stage. CONCLUSIONS Gene vps4b plays a significant role in the development of tooth germ. The expression of DSPP and COL-Ⅰ may be controlled by gene vps4b and regulates the development of tooth dentin and cementum together with vps4b.
ATPases Associated with Diverse Cellular Activities ; genetics ; Animals ; Collagen ; metabolism ; Endosomal Sorting Complexes Required for Transport ; genetics ; Extracellular Matrix Proteins ; metabolism ; Mice ; Mice, Knockout ; Molar ; Odontoblasts ; Phosphoproteins ; metabolism ; Sialoglycoproteins ; metabolism ; Tooth Germ

OBJECTIVETo detect the expression of D-E-A-D-box polypeptide 41 (DDX41) in human dental pulp tissues and cells.

METHODSThe mRNA and protein expressions of DDX41 in human dental pulp cells were detected by RT-PCR and immunocytochemistry, and the expression of DDX41 in human dental pulp tissues was investigated by immunohistochemistry.

RESULTSStrong expressions of DDX41 mRNA and protein were detected in dental pulp cells. In dental pulp tissues, DDX41 was expressed in the cytoplasm and nucleus of odontoblasts.

CONCLUSIONDDX41/STING-dependent TBK1-IRF3-IFN-β signaling pathway may play a role in innate immune responses of the dental pulp to caries and pulpitis.

Cell Nucleus ; metabolism ; Cells, Cultured ; Cytoplasm ; metabolism ; DEAD-box RNA Helicases ; metabolism ; Dental Pulp ; metabolism ; Humans ; Immunohistochemistry ; Odontoblasts ; metabolism ; RNA, Messenger ; Signal Transduction

OBJECTIVETo invesitgate the expression patterns of amelogenin and enamelin in the developing tooth germs.

METHODSMandible sections of postnatal day 1, 3, 7 and 14 mouse were prepared, immunohistochemical analysis and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detect the expression patterns of amelogenin and enamelin in mandibular first molars.

RESULTSAmelogenin was observed in the cytoplasm of secretory ameloblasts and the whole enamel matrix layer. It was also transiently expressed in the odontoblasts of postnatal day 1 molars. Enamelin proteins were observed in the enamel layer deposited by secretory ameloblasts, especially intense beneath the ameloblast process and dentino-enamel junction. The mRNA levels of both amelogenin and enamelin were highest on postnatal day 7 (the ratio to glyceraldehyde phosphate dehydrogenase of amelogenin and enamelin: 0.813 ± 0.085 and 0.799 ± 0.064, respectively, P < 0.05).

CONCLUSIONSAmelogenin and enamelin were enamel matrix proteins predominately expressed by secretory ameloblasts. The temporal-spatial expression patterns of amelogenin and enamelin indicate the important roles they played in amelogenesis and biomineralization.

Ameloblasts ; metabolism ; Amelogenesis ; Amelogenin ; genetics ; metabolism ; Animals ; Dental Enamel ; metabolism ; Dental Enamel Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Molar ; metabolism ; Odontoblasts ; metabolism ; RNA, Messenger ; metabolism ; Time Factors ; Tooth Germ ; growth & development ; metabolism

Epithelial-mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA. Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development. Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses. Robust differences in gene expression,as well as genes not previously associated with root formation,were identified. Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction (RT-PCR) supported our findings. These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family. In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.
Animals ; Dental Cementum ; cytology ; metabolism ; Epithelial-Mesenchymal Transition ; physiology ; Gene Expression Regulation, Developmental ; Laser Capture Microdissection ; Mice ; Mice, Inbred Strains ; Odontoblasts ; metabolism ; Oligonucleotide Array Sequence Analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Tooth Germ ; metabolism ; Tooth Root ; growth & development

OBJECTIVETo investigate the regulation effects of upstream stimulatory factor 1 (USF1) on osteopontin expression in odontoblasts.

METHODSOdontoblast MDPC-23 was cultured and stably transfected with PCMV-USF1 or A-USF plasmids. Total RNA was extracted and osteopontin expression examined by semi-quantitative RT-PCR. Gray value of osteopontin was measured and statistic analysis performed.

RESULTSClones of stable PCMV-USF1 and A-USF plasmids transfection were obtained. Compared with the control, osteopontin was upregulated in PCMV-USF1 transfection group, and downregulated in A-USF transfection group.

CONCLUSIONSUpstream stimulatory factor 1 could regulate the osteopontin expression in odontoblasts, which could be blocked partly by A-USF.

Cell Line, Tumor ; Humans ; Odontoblasts ; metabolism ; Osteopontin ; genetics ; metabolism ; Plasmids ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Upstream Stimulatory Factors ; genetics

OBJECTIVETo examine the expression and subcellular localization of transcription factor USF1 in odontoblasts and investigate whether nuclear translocation occurs under stimuli.

METHODSOdontoblasts MDPC-23 were cultured on coverslips and divided into 2 groups. Group 1 received no stimuli, and group 2 was stimulated by nicotine with various concentrations respectively for 1h. Then the mountings of odontoblasts were prepared and immunocytochemical staining was performed with specific USF1 antibody via SABC method. Hela cells were used as positive control.

RESULTSThe staining was positive in the cytoplasm of odontoblasts in group 1, but in the nuclei of Hela cells and in 100 mg/L nicotine-stimulated odontoblasts in group 2.

CONCLUSIONSThere exists USF1 protein in odontoblasts, which locates in the cytoplasm and could translocate into nuclei under the stimulation of nicotine.

Cells, Cultured ; HeLa Cells ; Humans ; Nicotine ; pharmacology ; Odontoblasts ; drug effects ; metabolism ; Protein Sorting Signals ; Protein Transport ; drug effects ; Upstream Stimulatory Factors ; metabolism

OBJECTIVETo investigate the effect of adenovirus expressing human bone morphogenetic protein-7 (hBMP-7) on proliferation and differentiation of human dental pulp cells.

METHODSThe replication-deficient adenoviral vector encoding hBMP-7 gene was constructed by using homologous recombinant modality. The efficiency of transfection was evaluated by fluorescent microscopy and flow cytometry. The expression of hBMP-7 protein in adenovirus-infected dental pulp cells was determined by Western blot. The proliferation of cells was tested by MTT method, the activity of alkaline phosphatase was assayed, von Kossa staining was used to detect mineralized nodule formation, and the expression of DSPPmRNA in cells was detected using semi-quantitative RT-PCR.

RESULTSGreen fluorescent protein was visible under fluorescent microscopy. Higher transfection efficiency (91.1 +/- 1.0)% could be obtained at MOI of 75. Western blot from dental pulp cells infected with Ad-hBMP-7 for 48h detected protein expression of a hBMP-7 gene. The activity of alkaline phosphatase in cells was significantly higher than those of the control groups (P < 0.05). The cells infected with Ad-hBMP-7 had the ability of mineralization. DSPP mRNA expression of cells was in a time- and dose- dependent manner.

CONCLUSIONSAd-hBMP-7 can induce human pulp cells into odontoblasts, but has no obvious effect on their proliferation.

Adenoviridae ; genetics ; Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Dental Pulp ; cytology ; Humans ; Odontoblasts ; cytology ; Transfection