Role of Mechanical Stress-induced Glutamate Signaling-associated Molecules in Cytodifferentiation of Periodontal Ligament Cells

Shinya Murakami,Chiharu Fujihara,Teruko Takano-Yamamoto,Satoru Yamada,Nobuhiro Ozaki,Harumi Kawaki,Nobuo Takeshita

doi:10.1074/jbc.m109.097303

Abstract

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.

Highlights

The ability of cells to sense and respond to physical stress is required for tissue homeostasis and normal development
To biologically evaluate whether the mechanical stress worked on human periodontal ligament (PDL) cells in this system, we examined the gene expression of C-FOS (FBJ murine osteosarcoma viral oncogene homolog), which is for hybridization
Determination of the intracellular Ca2ϩ influx showed that 100 ␮M exogenous glutamate apparently increased the fluorescence intensity compared with the control cells (Fig. 2B). These findings showed that exogenous glutamate induced an intracellular Ca2ϩ influx, followed by glutamate signaling in human PDL cells

Summary

EXPERIMENTAL PROCEDURES

Reagents—␣-Modified Eagle’s medium was obtained from Nikken Biomedical Laboratory (Kyoto, Japan). Human PDL cells were transferred to the chambers at a density of 1.5 ϫ 104 cell/ cm and cultured in ␣-modified Eagle’s medium with 10% FCS for 3 days. Human PDL cells stimulated by glutamate or mechanical stress counted with a hemocytometer after 0.5% trypan blue staining. After two concentrations of the cell lysates were measured using a BCA washes with PBS, the cells were sonicated in 2 ml of distilled protein assay kit (Pierce) according to the manufacturer’s water at 4 °C. Human PDL cell layers were washed twice with PBS and fixed with dehydrated ethanol for 10 min. B, human PDL cells were labeled with the Ca2ϩ fluorescent agent Fluo 3-AM and stimulated with 100 ␮M exogenous glutamate. C, human PDL cells were stimulated with glutamate (500 ␮M) for 40 min and lysed. 181850), that for mGluR5 was 505 bp (site location from 98 to 602 bp of accession number NM_001081414), and that for mGluR6 was 485 bp (site location from 58 to 542 bp of accession number NM_173372)

The DNA fragment for mouse

RESULTS

Functional Analysis of Glutamate Signaling in Human PDL

Release on the Cytodifferentiation and Mineralization of Human PDL

In Vivo Induction of the Gene

Findings

DISCUSSION