Depletion of histone demethylase KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of stem cells from apical papilla.

Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), is evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A can regulate the cell proliferation and osteo/dentinogenic differentiation of MSCs. It is not known whether KDM2A is involved in the other cell lineages differentiation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can enhance adipogenic and chondrogenic differentiation potentials in human stem cells from apical papilla (SCAPs). We found that the stemness-related genes, SOX2, and the embryonic stem cell master transcription factor, NANOG were significantly increased after silence of KDM2A in SCAPs. Moreover, we found that knock-down of the KDM2A co-factor, BCOR also up-regulated the mRNA levels of SOX2 and NANOG. Furthermore, Chromatin immunoprecipitation assays demonstrate that silence of KDM2A increased the histone H3 Lysine 4 (H3K4) trimethylation in the SOX2 and NANOG locus and regulates its expression. In conclusion, our results suggested that depletion of KDM2A enhanced the adipogenic and chondrogenic differentiation potentials of SCAPs by up-regulated SOX2 and NANOG, BCOR also involved in this regulation as co-factor, and provided useful information to understand the molecular mechanism underlying directed differentiation in MSCs.

The role of inducible nitric oxide synthase and haem oxygenase 1 in growth and development of dental tissue'.

In this study, the activity of the antioxidant enzyme network was assessed spectrophotometrically in samples of dental pulp and dental papilla taken from third-molar gem extracts. The production of nitric oxide by the conversion of l-(2,3,4,5)-[3H] arginine to l-(3H) citrulline, the activity of haem oxygenase 1 (HO-1) through bilirubin synthesis and the expression of inducible nitric oxide synthase (iNOS), HO-1 proteins and messenger RNA by Western blot and reverse-transcribed polymerase chain reaction were also tested. The objective of this study was to evaluate the role of two proteins, iNOS and HO-1, which are upregulated by a condition of oxidative stress present during dental tissue differentiation and development. This is fundamental for guaranteeing proper homeostasis favouring a physiological tissue growth. The results revealed an over-expression of iNOS and HO-1 in the papilla, compared with that in the pulp, mediated by the nuclear factor kappa B transcription factor activated by the reactive oxygen species that acts as scavengers for the superoxide radicals. HO-1, a metabolically active enzyme in the papilla, but not in the pulp, seems to inhibit the iNOS enzyme by a crosstalk between the two proteins. We suggest that the probable mechanism through which this happens is the interaction of HO-1 with haem, a cofactor dimer indispensible for iNOS, and the subsequent suppression of its metabolic activity.

Expression of protein kinases C betaI, betaII, and VEGF during the differentiation of enamel epithelium in tooth development.

Protein kinase C (PKC) is an important molecule involved in various cell function, and mediates induced secretion of vascular endothelial growth factor (VEGF). It is hypothesized that PKC and VEGF may be associated with tooth development. Using the laser microdissection method and real-time reverse-transcription-polymerase chain-reaction (RT-PCR), we investigated the expression of PKC betaI and betaII, VEGF, and amelogenin (used as a marker of differentiation to ameloblasts) in the inner and outer enamel epithelia, stellate reticulum, and dental papilla in each stage of the dental germ. We found that the expression levels of PKC betaI and betaII were increased in the inner enamel epithelium during the early bell stage. In addition, the increased expression levels of PKC betaI and betaII were accompanied by increased VEGF expression. These results indicate that PKC betaI, betaII, and VEGF are closely associated with the differentiation of the inner enamel epithelium to ameloblasts.

Enamelysin (matrix metalloproteinase-20): localization in the developing tooth and effects of pH and calcium on amelogenin hydrolysis.

The formation of dental enamel is a precisely regulated and dynamic developmental process. The forming enamel starts as a soft, protein-rich tissue and ends as a hard tissue that is over 95% mineral by weight. Intact amelogenin and its proteolytic cleavage products are the most abundant proteins present within the developing enamel. Proteinases are also present within the enamel matrix and are thought to help regulate enamel development and to expedite the removal of proteins prior to enamel maturation. Recently, a novel matrix metalloproteinase named enamelysin was cloned from the porcine enamel organ. Enamelysin transcripts have previously been observed in the enamel organ and dental papillae of the developing tooth. Here, we show that the sources of the enamelysin transcripts are the ameloblasts of the enamel organ and the odontoblasts of the dental papilla. Furthermore, we show that enamelysin is present within the forming enamel and that it is transported in secretory vesicles prior to its secretion from the ameloblasts. We also characterize the ability of recombinant enamelysin (rMMP-20) to degrade amelogenin under conditions of various pHs and calcium ion concentrations. Enamelysin displayed the greatest activity at neutral pH (7.2) and high calcium ion concentration (10 mM). During the initial stages of enamel formation, the enamel matrix maintains a neutral pH of between 7.0 and 7.4. Thus, enamelysin may play a role in enamel and dentin formation by cleaving proteins that are also present during these initial developmental stages.

A morphological, tracer and cytochemical study of the role of the papillary layer of the rat-incisor enamel organ during enamel maturation.

Thin-section, freeze-fracture, tracer and cytochemical methods showed that papillary cells were characterized by the presence of numerous mitochondria and microvilli. The cytoplasms contained Golgi apparatus, multivesicular bodies (MVB), tubular structures and coated vesicles. In freeze-fracture replicas, some tubular structures and vesicles were continuous with the cell surfaces. Intravenously-injected horseradish peroxidase (HRP) was rapidly incorporated into the papillary cells via coated vesicles and tubular structures. Horseradish peroxidase-labelled coated vesicles and tubular structures then fused with endocytic vacuoles and MVB. These endocytic vacuoles and MVB showed HRP-labelling of various degrees. Acid-phosphatase activity was demonstrated in the Golgi apparatus, endocytic vacuoles and MVB of papillary cells. Enzymic activity sometimes took place in tubular structures but never occurred in coated vesicles. These results indicate that absorptive and digestive activities of papillary cells are closely related to loss of organic materials from the enamel matrix during enamel maturation.

Expression and activity of matrix metalloproteinase-2 (MMP-2) in the development of rat first molar tooth germ.

Tooth germ development is associated with morphological and biochemical changes of the dental papilla and enamel organ. Enzymes with gelatinolytic activities were studied by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography in tooth germ of newborn to 15-day-old rats. Three major bands with gelatinolytic activity were detected at all periods and characterized as the latent and active forms of MMP-2 using their molecular weight and activity dependent on Zn++ and Ca++ ions as criteria. Expression and activity of MMP-2 increased progressively from 0 to 15 days after birth. Mechanical separation of the tooth germ from 10-day-old rats showed that the gelatinolytic activity was localized mainly in the dental papilla and not the dental organ. These data indicate that the expression and activity of MMP-2 varies during the development and maturation of rat first molar tooth germ.

Epiregulin can promote proliferation of stem cells from the dental apical papilla via MEK/Erk and JNK signalling pathways.

OBJECTIVES: Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but their molecular mechanisms of differentiation and proliferation remain unclear; this situation has restricted use of MSCs to a limited number of applications. A previous study of ours found a member of the epidermal growth factor family, epiregulin (EREG), to be involved in regulation of MSC differentiation. In the present study, we have used human dental stem cells from the apical papilla (SCAPs) to investigate the role of EREG on proliferation of MSCs. MATERIALS AND METHODS: SCAPs were isolated from apical papillae of immature third molars. Retroviral short hairpin RNA (shRNA) was used to silence EREG gene expression, and human recombinant EREG protein was used to stimulate SCAPs. SCAP proliferation was examined using tetrazolium dye colorimetric assay/cell growth curve. Western blotting was performed to detect expressions of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2), mitogen-activated protein kinases 1 and 2 (MEK1/2), protein kinase B (Akt), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). RESULTS: Depletion of EREG with shRNA inhibited SCAP proliferation and repressed phosphorylation of Erk1/2 and JNK. Human recombinant EREG protein promoted cell proliferation and enhanced Erk1/2, MEK and JNK phosphorylation in SCAPs. Furthermore, blocking MEK/Erk signalling with specific Erk1/2 inhibitor PD98059, or JNK signalling with specific inhibitor SP600125, abolished effects of EREG on cell proliferation. CONCLUSION: These findings indicate that EREG could enhance cell proliferation in dental tissue-derived MSCs by activating MEK/Erk and JNK signalling pathways.

The regulatory role of a disintegrin and metalloproteinase 28 on the biologic property of human periodontal ligament stem cells.

BACKGROUND: A disintegrin and metalloproteinase 28 (ADAM28) is considered to be the possible virulence gene for congenital hypoplasia of tooth root. Periodontal ligament stem cells (PDLSCs) are regarded as playing crucial roles in the developing process of the periodontium and are generally used in dental regenerative medicine. This study evaluates the influence of ADAM28 on the biologic property of human PDLSCs (HPDLSCs) and to postulate the possible mechanism of this influence. METHODS: HPDLSCs were acquired by immunomagnetic bead selection and identified by immunofluorescence detection. After ADAM28 eukaryotic plasmid and antisense oligodeoxynucleotides (AS-ODNs) were constructed and respectively transfected into HPDLSCs by a transfection reagent, the expression differences of ADAM28 among various groups were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide and cell-cycle assays were used to test the proliferation activity of HPDLSCs. Annexin V-fluorescein isothiocyanate/propidium iodide analysis was performed to detect the apoptotic level. Cell differentiation was tested by measuring the alkaline phosphatase level. Immunocytochemistry and Western blotting were carried out to determine the effects of ADAM28 AS-ODNs on HPDLSCs expressing cementum attachment protein (CAP), osteopontin, and osteocalcin. RESULTS: The ADAM28 eukaryotic plasmid group showed the highest expression level in HPDLSCs, whereas the AS-ODN group displayed the lowest. Furthermore, the overexpression of ADAM28 enhanced the proliferation of HPDLSCs and inhibited the specific differentiation of HPDLSCs, whereas the inhibition of ADAM28 produced the opposite effects and induced apoptosis. ADAM28 AS-ODNs were able to significantly inhibit CAP expression, and ADAM28 had a positive correlation with CAP. CONCLUSION: Our findings demonstrated that ADAM28 was able to effectively manipulate the proliferation, apoptosis, and differentiation of HPDLSCs.

Immunohistochemical expression of matrix metalloproteinases 1, 2, and 9 in odontogenic myxoma and dental germ papilla.

The aim of this study was to compare the immunohistochemical expression of matrix metalloproteinases (MMPs) 1, 2, and 9 in odontogenic myxomas and dental germ papillae. Twelve cases of odontogenic myxoma and eight tooth germ specimens were selected for analysis of the immunohistochemical expression and the pattern of distribution of MMPs 1, 2, and 9 in extracellular matrix (ECM), as well as of the number of MMP-positive cells. MMP-2 was expressed only in the ECM of myxomas (p<0.05). No significant difference was observed between ECM immunoreactivity for MMP-9 in myxomas and dental papillae (p>0.05). MMP-1 immunoreactivity was detected in most myxoma cases at a proportion similar to that observed in dental papillae (p>0.05). A significant difference was observed in the number of immunoreactive cells in myxomas (p<0.05), MMP-1 being present at higher proportions than MMPs 2 and 9. There was a gradient in the expression of MMPs in the ECM and in neoplastic cells of odontogenic myxomas, with higher immunoreactivity to MMP-1 and lower immunoreactivity to MMP-9. Taken together, our results suggest the existence of a coordinated mechanism between MMPs 1, 2, and 9 that aimed at the efficient degradation of extracellular matrix in odontogenic myxomas.

Essential role of ADAM28 in regulating the proliferation and differentiation of human dental papilla mesenchymal cells (hDPMCs).

Dental papilla mesenchymal cells (DPMCs) have been supposed to possess the relatively independent and critical role for tooth development and morphogenesis. Here, we characterized the role of ADAM28, a member of a disintegrin and metalloproteinase (ADAM) family, in the regulative mechanisms of odontogenic capability of hDPMCs. Immunofluorescence staining showed the ubiquitous expression of ADAM28 in multiple human dental mesenchymal and epithelial cells. After confirming the effect of eukaryotic expression plasmid containing ADAM28 coding region and ADAM28 antisense oligodeoxynucleotide (AS-ODN), we respectively transfected them into hDPMCs and observed the biological markers for proliferation and differentiation. Overexpression of ADAM28 favored the proliferation and lineage-specific differentiation of hDPMCs, while blockage of ADAM28 exerted the opposite effects and induced apoptosis. These results identified an unrecognized hypothesis that ADAM28 may function as positive regulator of growth and differentiation of hDPMCs and act as an important molecule mediating reciprocal epithelial-mesenchymal signaling during tooth organ development.

Characterization of protein kinases involved in dentinogenesis.

Protein phosphorylation and dephosphorylation control many different cell functions as well as responses to internal and external signals. It has also been shown that highly phosphorylated acidic proteins have an important role in matrix mediated biomineralization, perhaps functioning as nucleators for crystal formation. Dentine phosphoprotein (DPP) is one of such proteins which is exclusively synthesized by the odontoblast cells and therefore a likely candidate to play a significant role in normal and abnormal dentine biomineralization. These studies are directed at characterizing the protein kinases involved in dentinogenesis and in particular the enzyme(s) responsible for DPP phosphorylation. In this report we present data which indicate that there are several different types of kinases in the odontoblast-enriched dental papilla mesenchyme (DPM), some of which can phosphorylate DPP, such as casein kinase I and II. However, a different DPP-kinase activity was identified. This enzyme(s) appears to be different from other reported kinases, and it is the only kinase that can phosphorylate both phosphorylated DPP and enzymatically dephosphorylated DPP.

Widespread expression of tartrate-resistant acid phosphatase (Acp 5) in the mouse embryo.

Tartrate-resistant acid phosphatase (TRAP, Acp 5) is considered to be a marker of the osteoclast and studies using 'knockout' mice have demonstrated that TRAP is critical for normal development of the skeleton. To investigate the distribution of TRAP in the mammalian embryo, cryostat sections of 18 d murine fetuses were examined by in situ hybridisation, immunohistochemistry and histochemical reactions in situ. Abundant expression of TRAP mRNA was observed in the skin and epithelial surfaces of the tongue, oropharynx and gastrointestinal tract including the colon, as well as the thymus, ossifying skeleton and dental papillae. TRAP protein was identified at the same sites, but the level of expression in the different tissues did not always correlate with apparent enzyme activity. The findings indicate that abundant TRAP expression is not confined to osteoclasts in bone, but occurs in diverse tissues harbouring cells of bone marrow origin, including dendritic cells and other cells belonging to the osteoclast/macrophage lineage.