Identification of N-methyl-D-aspartate receptor subunit in human periodontal ligament fibroblasts: potential role in regulating differentiation.
BACKGROUND
Periodontal ligament fibroblasts (PDLFs), which can be differentiated into osteoblasts, are crucial cells for the regeneration of the periodontal tissue. Although N-methyl-D-aspartate (NMDA) receptors were reported to be involved in bone formation by affecting osteoblasts, the existence and function of NMDA receptors in PDLFs have not been confirmed. The purpose of this study was to examine the expression of NMDA receptors and their role in human PDLFs.


METHODS
Human PDLFs were cultured and evaluated to identify the subunits of NMDA receptors (NR) by reverse transcription-polymerase chain reaction, Western blot analysis, and immunocytochemistry. Then, the cells were assigned to four different groups: a control media group, a control media with NMDA receptor antagonist group, a differentiation media group, and a differentiation media with NMDA receptor antagonist group. Cell proliferation assay, alkaline phosphatase (ALP) activity analysis, and mineralization assay were performed to determine whether NMDA receptors affected the function of PDLFs.


RESULTS
NR1, NR2B, and NR2D were detected in human PDLFs. There was no statistically significant difference in proliferation among the groups. However, the NMDA receptor antagonist-treated group showed a significant reduction in ALP activity (P <0.05). Moreover, the NMDA receptor antagonist-supplemented group presented no mineralization.


CONCLUSIONS
This study revealed the existence of NMDA receptors in human PDLFs and specified their subunits. Moreover, NMDA receptors had a significant influence on the differentiation and mineralization of human PDLFs but did not affect their proliferation. These results suggest that NMDA receptors may play an important role in the differentiation and mineral tissue formation of human PDLFs.
Yu
Ji-Hee
JH
Department of Oral Anatomy, Brain Korea 21 Program, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea.
Lee
Seung-Pyo
SP
Kim
Tae-Il
TI
Jang
Jun-Hyeog
JH
D015153:Blotting, Western; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D010513:Periodontal Ligament / Q000737:chemistry* / Q000166:cytology; D021122:Protein Subunits / Q000032:analysis; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C409466:NR1 NMDA receptor; C121001:NR2B NMDA receptor; C464436:NR2D NMDA receptor; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate