S100A4 inhibition by RNAi up-regulates osteoblast related genes in periodontal ligament cells

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Abstract

Periodontal ligament (PDL) is a thin fibrous connective tissue located between alveolar bone and cementum that remains unmineralized physiologically. It is thus thought that PDL cells possess mechanisms to inhibit mineralization. It has been demonstrated that S100A4, a member of the S100 calcium-binding protein family, is synthesized and secreted by PDL cells, and that it may act as an inhibitor of mineralization. However, the mechanisms of action of S100A4 in mineralization have not been thoroughly clarified. In the present study we investigated the effects of S100A4 inhibition by a short interfering RNA (siRNA) on the expression of osteoblast related genes by human PDL cells. Inhibition of S100A4 by siRNA resulted in increased expression of osteoblastic markers such as osteopontin and osteocalcin, and the osteoblast-specific transcription factors, Runx2/Cbfa1 and Osterix. These results indicate that S100A4 suppresses the expression of osteoblastic genes in PDL cells and may thus inhibit mineralization in the PDL.

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Materials and methods

Cell culture. Human PDL (hPDL) cells were obtained from healthy human teeth indicated for extraction for orthodontic treatment according to the report described by Somerman et al. [16]. Periodontal tissue was removed from the middle third of the root using a sterile scalpel. The tissue was rinsed five times with growth media (α-modified minimal essential medium, α-MEM; Gibco) and transferred to culture dishes.

Cultures were maintained in α-MEM supplemented with 10% fetal bovine serum (FBS;

Expression of S100A4 after siRNA transfection in hPDL cells

mRNA expression of S100A4 in cultured hPDL cells 72 h after siRNA transfection was detected by semi-quantitative RT-PCR methods. Non-sense siRNA was also transfected as a control. The results of the semi-quantitative RT-PCR analysis of S100A4 are shown in Fig. 1. S100A4 mRNA was expressed in cultured hPDL cells. The expression was down-regulated after siRNA transfection, while no change was observed after non-sense siRNA transfection.

Immunostaining of S100A4 in cultured hPDL cells with or

Discussion

PDL cells are known to be a heterogeneous cell population and are indispensable for the regeneration of periodontal tissues including the unmineralized PDL and two mineralized tissues, i.e., cementum and bone [18].

S100A4 was detected in cultured hPDL cells at the protein and mRNA levels. Duarte et al. [14] have suggested that a decrease in S100A4 expression may be associated with a terminal osteoblastic differentiation and/or the initiation of mineralized matrix formation. Other reports have

Acknowledgments

This study was supported by Japan Society for the Promotion of Science (JSPS-B-4656) and Grant-in-Aid for Scientific Research of the Ministry of Education, Science, Sports and Culture of Japan (13470459).

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