A novel single-base deletion of the RUNX Family Transcription Factor 2 gene associated with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a rare, autosomal dominant hereditary disorder characterized by skeletal malformations and dental abnormalities. The purpose of this study was to explore the functional role of a novel mutation in the pathogenesis of CCD. Genomic DNA was extracted from peripheral blood mononuclear cells collected from family members of a Chinese patient with CCD. An analysis of their RUNX Family Transcription Factor 2 (RUNX2) gene sequences was performed by PCR amplification and Sanger sequencing. The function of the mutant RUNX2 was studied by bioinformatics, real-time PCR, western blotting, and subcellular localization analysis. Sanger sequencing identified a novel single-base deletion (NM_001024630.4:c.132delG;NP_001019801.3: Val45Trpfs* 99) in the RUNX2 gene present in the Chinese patient with CCD. In vitro, functional studies showed altered protein localization and increased expression of mutant RUNX2 mRNA and mutant Runt-related transcription factor 2 (RUNX2). Luciferase reporter assay demonstrated that the novel RUNX2 mutations significantly increased the transactivation activity of RUNX2 on the osteocalcin gene promoter. In conclusion, we identified a patient with sporadic CCD carrying a novel deletion/frameshift mutation of the RUNX2 gene and performed screening and functional analyses to determine the cause of the CCD phenotype. This study provides new insights into the pathogenesis of CCD.3.

Glycogen synthase kinase-3ß (GSK-3ß) deficiency inactivates the NLRP3 inflammasome-mediated cell pyroptosis in LPS-treated periodontal ligament cells (PDLCs).

Bacterial infection caused cell pyroptosis and gingival inflammation contributes to periodontitis progression, and lipopolysaccharide (LPS) is the main infectious agent of gram-negative bacteria, which is reported to be closely associated with gingival inflammation and periodontitis. In this study, the primary human periodontal ligament cells (PDLCs) were isolated, cultured, and exposed to LPS treatment, and the results suggested that LPS suppressed cell viability and promoted pro-inflammatory cytokines' (IL-1ß, IL-18, IL-6, and TNF-α) generation and secretion in the PDLCs and its supernatants in a time- and concentration-dependent manner. Also, we noticed that LPS upregulated NLRP3, Gasdermin D, and cleaved caspase-1 to trigger pyroptotic cell death in the PDLCs. Further experiments identified that glycogen synthase kinase-3ß (GSK-3ß) was upregulated by LPS treatment, and inhibition of GSK-3ß by its inhibitor (GSKI) or GSK-3ß downregulation vectors was effective to restore normal cellular functions in LPS-treated PDLCs. Mechanistically, blockage of GSK-3ß restrained NLRP3-meidated cell pyroptosis and inflammation, resulting in the recovery of cell viability and inhibition of cell death in PDLCs treated with LPS, which further ameliorated periodontitis progression. Finally, we collected the serum from periodontitis patients and healthy volunteers, and the clinical data supported that those pro-inflammatory cytokines were also upregulated in patients' serum but not in the healthy participants. Taken together, we concluded that targeting the GSK-3ß/NLRP3 pathway mediated cell pyroptosis was effective to attenuate LPS-induced cell death and inflammation in PDLCs, and this study firstly investigated this issue, which broadened our knowledge in this field.

Dental pulp stem cells from human teeth with deep caries displayed an enhanced angiogenesis potential in vitro.

BACKGROUND/PURPOSE: Dental pulp stem cells can be isolated from human teeth with deep caries (cDPSCs), but their biological characteristics are still unclear. The aim of this study was to investigate the angiogenic potential of cDPSCs and compare them to dental pulp stem cells from human normal teeth (nDPSCs). MATERIALS AND METHODS: Cells were isolated from human pulp tissue of normal and infected teeth with deep caries. Basic mesenchymal stem cell (MSC) characterization was conducted. Colony forming units and proliferation ability were evaluated in nDPSCs and cDPSCs. Expression of VEGF in both tissues and cells was examined by immunohistochemical staining. After stimulating nDPSCs and cDPSCs with an angiogenic medium, angiogenic markers were evaluated by qRT-PCR and western blotting. Finally, tube formation assays were used to evaluate the in vitro angiogenesis potential of both cell populations. RESULTS: Both nDPSCs and cDPSCs possessed typical MSC characteristics. cDPSCs had enhanced colony formation and proliferation capacities than nDPSCs did. The expression of VEGF was higher in pulp tissue from teeth with deep caries and cDPSCs than in normal tissue and nDPSCs. When both cell types were grown in vitro under angiogenic conditions, cDPSCs expressed a higher level of angiogenic markers and showed a stronger angiogenesis potential than nDPSCs did. CONCLUSION: cDPSCs maintained MSC traits and presented a higher angiogenesis potential than nDPSCs.

Effects of vascular endothelial growth factor and insulin growth factor­1 on proliferation, migration, osteogenesis and vascularization of human carious dental pulp stem cells.

The present study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and insulin­like growth factor­1 (IGF­1) on the proliferation, migration and differentiation of human carious dental pulp stem cells (hCDPSCs), and to elucidate the underlying mechanism(s). Cell counting kit­8 assay was used to detect the effect of different concentrations of IGF­1 and VEGF on the proliferation of hCDPSCs. Transwell assay was used to detect the migratory ability of the hCDPSCs. Alizarin red and alkaline phosphatase (ALP) staining were used to detect the osteogenic ability of hCDPSCs, whereas the angiogenic ability of the hCDPSCs was tested by tube formation assay. Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting were used to detect the expression levels of associated genes and proteins. IGF­1 (100 ng/ml) or VEGF (25 ng/ml) alone were revealed to be able to promote proliferation and migration of hCDPSCs; however, the combined use of IGF­1 and VEGF enhanced this effect when compared with the use of either agent in isolation. Alizarin red and ALP staining revealed that the use of either VEGF or IGF­1 alone did not result in any significant effects, whereas their use in combination promoted the osteogenic differentiation of hCDPSCs. In addition, the RT­qPCR and western blotting analyses revealed that the expression levels of Runt­related transcription factor 2 (RUNX2), bone sialoprotein (BSP) and ALP were increased upon combined treatment of the cells with VEGF and IGF­1. The expression levels of VEGF and plateletderived growth factor (PDGF) in hCDPSCs were enhanced upon treatment with either VEGF or IGF­1 in isolation, with greater effects observed when VEGF and IGF­1 were added in combination, indicating that VEGF and IGF­1 may exert a synergistic role in these events. Further experiments revealed that the combination of VEGF and IGF­1 led to an activation of the AKT signaling pathway. The proliferation and angiogenesis of hCDPSCs were also shown to be more effective compared with treatment with either VEGF or IGF­1 in isolation. Taken together, the present study has demonstrated that the combined use of VEGF and IGF­1 leads to an increase in the proliferation, migration, osteogenesis and angiogenesis of hCDPSCs and, furthermore, these signaling molecules may mediate their effects via activation of the AKT signaling pathway.