A role for the calcium-sensing receptor in the expression of inflammatory mediators in LPS-treated human dental pulp cells.

The aim of this study is to investigate the role of calcium-sensing receptor (CaSR) in the expression of inflammatory mediators of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). The expression profile of CaSR in LPS-simulated hDPCs was detected using immunofluorescence, real time quantitative PCR (RT-qPCR), and Western blot analyses. Then, its regulatory effects on the expression of specific inflammatory mediators such as interleukin (IL)-1ß, IL-6, cyclooxygenase 2 (COX2)-derived prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and IL-10 were determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA). LPS significantly downregulated the gene expression of CaSR, but upregulated its protein expression level in hDPCs. Treatments by CaSR agonist R568 or its antagonist Calhex231, and their combinations with protein kinase B (AKT) inhibitor LY294002 showed obvious effects on the expression of selected inflammatory mediators in a time-dependent manner. Meanwhile, an opposite direction was found between the action of R568 and Calhex231, as well as the expression of the pro- (IL-1ß, IL-6, COX2-derived PGE2, and TNF-α) and anti-inflammatory (IL-10) mediators. The results provide the first evidence that CaSR-phosphatidylinositol-3 kinase (PI3K)-AKT-signaling pathway is involved in the release of inflammatory mediators in LPS-treated hDPCs, suggesting that the activation or blockade of CaSR may provide a novel therapeutic strategy for the treatment of pulp inflammatory diseases.

CaSR as a Therapeutic Target and Tool in Human Dental Pulp: A Concise Review and Novel Hypothesis.

PURPOSE: To review the essential characteristics of calcium sensing receptor (CaSR) and explore the hypothesis that elevated extracellular calcium ions (Ca2+) may affect the odontogenic/osteogenic differentiation and mineralisation of human dental pulp cells (hDPCs) through the CaSR signal. MATERIALS AND METHODS: Based on a literature search of databases using different combinations of the key words and our previous researches, we gleaned the following important viewpoints. RESULTS: The Ca2+ released from pulp capping materials plays an essential role in maintaining the viability and function of human dental pulp, and elevated extracellular Ca2+ concentrations can promote the odontogenic/osteogenic differentiation and mineralisation of hDPCs. Ca2+ is the primary physiological ligand of the CaSR, which has been reported to be widely expressed in a broad range of cells, including various osteoblast-like cell lines, osteoprogenitor cells, and mature osteoblasts. hDPCs consist of different subpopulations and have been shown to share phenotypical features with osteoblasts. Thus, we speculated that hDPCs also express CaSR and respond to extracellular Ca2+ via this receptor. Calcimimetics are indirect allosteric regulators of CaSR function and can increase the receptor's sensitivity to ambient Ca2+. CONCLUSION: The local use of calcimimetics and calcium-based pulp capping materials could create an option for promoting the Ca2+ influx of hDPCs from the extracellular space via the CaSR. Such elevated Ca2+ concentrations could enhance the odontogenic/osteogenic differentiation and mineralisation of hDPCs and eventually improve the success rate of direct pulp capping treatments in patients suffering from accidental dental pulp exposure.

Identification of a Calcium-sensing Receptor in Human Dental Pulp Cells That Regulates Mineral Trioxide Aggregate-induced Mineralization.

INTRODUCTION: The purpose of this study was to verify the expression of the calcium-sensing receptor (CaSR) and its role in mineral trioxide aggregate (MTA)-induced odontoblastic differentiation and mineralization in human dental pulp cells (hDPCs). METHODS: The expression of CaSR in human dental pulp tissue and hDPCs was detected using immunohistochemical and immunofluorescent assays. Then, hDPCs were cultured in specific medium supplemented with defined concentrations of MTA dilute alone or in combination with calcimimetic R-568 (a positive allosteric modulator of CaSR [Tocris Bioscience, Bristol, UK]), and cell viability was monitored by Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) analysis. Alkaline phosphatase activity, alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot were used to investigate the gene/protein expression of odontoblastic-associated markers and CaSR in medium supplemented with different combinations of diluted MTA, R-568, and calcilytic Calhex 231 (a negative allosteric modulator of CaSR [Sigma-Aldrich, St Louis, MO]). RESULTS: CaSR was slightly expressed in the central pulp tissue, whereas it was strongly expressed in the odontoblast layer, plasma membrane, and cytoplasm of hDPCs. Cell Counting Kit-8 assay indicated maximum cell viability in cultures treated with 1:8 diluted MTA additives. Compared with undifferentiated controls, the cells at the early stage of odontoblastic differentiation exhibited lower CaSR protein expression. The combination of 1:8 diluted MTA with 0.1 and 1.0 µmol/L R-568 led to significantly increased cell vitality but decreased alkaline phosphatase activity and mineralized deposit formation, and this negative effect could be attenuated by 1.0 µmol/L Calhex 231 supplementation. Quantitative polymerase chain reaction results showed a significant up-regulation of RUNX2, DSPP, DMP-1, and OCN gene expression in the 1 µmol/L R-568-treated hDPCs. Western blot analysis indicated that the treatment by MTA and R-568 alone or their combination gave no clear trend on the protein levels of CaSR and dentin sialophosphoprotein, whereas Calhex 231 can increase their expressions. In addition, the up-regulation of Akt phosphorylation was observed in R-568- and Calhex 231-treated hDPCs. CONCLUSIONS: Our data indicated that CaSR is expressed in human dental pulp and hDPCs and that it can negatively or positively regulate MTA-induced mineralization of hDPCs via the phosphoinositide 3-kinase/Akt pathway in a ligand-dependent manner, suggesting a therapeutic target for modulating reparative dentin formation.

Enterococcus faecalis lipoteichoic acid regulates macrophages autophagy via PI3K/Akt/mTOR pathway.

Enterococcus faecalis (E. faecalis) infection is considered an important etiological factor for the development of persistent apical periodontitis (PAP), but the exact mechanisms of autophagy between E. faecalis and immune cells remain unknown. In this study, we elucidated how E. faecalis lipoteichoic acid (LTA) is associated with macrophages autophagy. We found that E. faecalis LTA apparently activated macrophage autophagy with significant increase of autophagosomes and autophagy relative protein. Meanwhile, we noticed significantly decreasing expression of p-Akt and p-mTOR. However, these effect were absent in macrophages knockdown of Beclin1. In summary, these findings suggested E. faecalis LTA may increased macrophages autophagy via inhibiting PI3K/Akt/mTOR pathway and this process was Beclin1 dependent.

The Starvation Resistance and Biofilm Formation of Enterococcus faecalis in Coexistence with Candida albicans, Streptococcus gordonii, Actinomyces viscosus, or Lactobacillus acidophilus.

INTRODUCTION: Enterococcus faecalis is the most frequently detected species in root canal-treated teeth, and it is able to survive under starvation conditions. However, persistent periapical disease is often caused by multispecies. The aim of this study was to explore the survival of E. faecalis in starvation conditions and biofilm formation with the 4 common pathogenic species. METHODS: A dual-species model of Candida albicans, Streptococcus gordonii, Actinomyces viscosus, or Lactobacillus acidophilus in combination with E. faecalis was established and allowed to grow in phosphate-buffered saline for the examination of starvation survival. Cefuroxime sodium and vancomycin at a concentration of 100 mg/L were added into brain-heart infusion plate agar to count the 2 bacteria separately in the dual species. Scanning electron microscopy was used to observe the dual species and multiple species on the root canal dentin of bovine teeth for 48 hours. A confocal laser scanning microscope was used to show the 4 groups of dual-species biofilms on substrates with glass bottoms for 48 hours. RESULTS: E. faecalis was more resistant to starvation in coexistence with C. albicans, S. gordonii, A. viscosus, or L. acidophilus, and S. gordonii was completely inhibited in coexistence with E. faecalis. The dual-species biofilm showed that E. faecalis formed thicker and denser biofilms on the root canal dentin and glass slides in coexistence with S. gordonii and A. viscosus than C. albicans and L. acidophilus. CONCLUSIONS: The multispecies community is conducive to the resistance to starvation of E. faecalis and biofilm formation in root canals.