Mechanical Stress-Induced Glutamate Signaling in 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 microarray analysis revealed that homer homolog1 (HOMER1) and glutamate receptor ionotropic NMDA3A (GRIN3A), which are related to glutamate signaling, were up-regulated by mechanical stress. In this study, we further investigated the expression and the function of glutamate signaling molecules in HPDL cells under mechanical stress. Methods: In vitro-maintained HPDL cells were constantly stretched and relaxed at 0.5 Hz with a 10% elongation by a stretch apparatus. Then, the mRNA expressions of various glutamate-signaling related genes were analyzed by RT-PCR or real-time PCR. The concentration of glutamate released from HPDL cells was measured by an enzyme-linked fluorometeric glutamate assay. HPDL cells were cultured in the mineralization-inducing medium in the presence of exogenous glutamate, and the alkaline phosphatase (ALPase) activities were measured. Results: RT-PCR and real-time PCR analyses revealed that the mRNA expressions of HOMER1 and several glutamate receptors (mGluR2-6, GRIA3, GRIN1, 2C, 2D, 3A, and 3B) were constantly expressed in HPDL cells, and mGluR2-6, GRIN3A and HOMER1 mRNA expressions were up-regulated by mechanical stress. Interestingly, HPDL cells spontaneously produced glutamate and the production was significantly increased by the mechanical stress or the induction of cytodifferentiation. Furthermore, the addition of exogenous glutamate enhanced the ALPase activities during the course of cytodifferentiation of HPDL cells. Conclusions: Present study demonstrates that glutamate signaling induced by mechanical stress positively regulates cytodifferentiation of HPDL cells and suggests that glutamate signaling may modulate the cellular functions of HPDL cells under mechanical stress in vivo.