Glutamate Signaling Enhances Cytodifferentiation of Periodontal Ligament Cells

Objectives: In our previous study, we analyzed the influences of mechanical stress on the gene expression of human periodontal ligament (HPDL) cells in vitro. The DNA chip analysis showed that mechanical stress up-regulated the mRNA expression of glutamate signaling-associated genes in HPDL cells and increased the release of glutamate from HPDL cells. In this study, we investigated the roles of the glutamate signaling in the functions of HPDL cells. Methods: We stimulated in vitro-maintained HPDL cells with exogenous glutamate (100 µM). After 12 h, we extracted total RNA and performed real-time RT-PCR to analyze the gene expression of c-fos, RUNX2 and Alkaline phosphatase (ALP). We also cultured HPDL cells in the mineralization-inducing medium in the presence of exogenous glutamate, Riluzole (a inhibitor of the release of glutamate) or MK801 (an antagonist of NMDA glutamate receptors). Then, we measured ALP activities and stained the cells with alizarin red. Utilizing a stretch apparatus (Scholartec NS-350), we stretched HPDL cells with 110% elongation at 0.5 Hz for 48 h in the presence of Riluzole. Then, we extracted total RNA and performed real-time RT-PCR to analyze the gene expression of c-fos, RUNX2 and ALP. Results: The analysis of real-time PCR revealed that exogenous glutamate (100 µM) up-regulated the gene expression of c-fos, RUNX2 and ALP in HPDL cells. The exogenous glutamate enhanced ALP activities during the cytodifferentiation of HPDL cells. Riluzole and MK801 suppressed ALP activities and calcified nodule formation during the cytodifferentiation of HPDL cells. Riluzole down-regulated the expression of c-fos, RUNX2 and ALP under mechanical stress. Conclusion: The present study demonstrated that mechanical stress-induced glutamate signaling, at least via NMDA receptors, enhances the cytodifferentiation and calcified nodule formation of HPDL cells in vitro.