Regulation of Cytodifferentiation of Periodontal Ligament Cells by SFRP4

Objectives: We have been conducting large-scale gene expression analysis of human periodontal ligament (PDL) in attempt to understand the molecular and genetic basis of PDL. In this study, we utilized oligo DNA chip that contains 30,000 genes including known function, confirmed, and predicted genes in order to identify genes that play crucial roles in PDL functions. Methods: To identify genes showing differential expression during the process of cytodifferentiation of PDL cells, we isolated RNA from PDL cells cultured in α-MEM supplemented with 10%FCS, 10mM β-glycerophosphate and 50mg/ml ascorbic acid (mineralization-inducing media) and analyzed the gene expression by oligo DNA chip. We constructed the SFRP4 (secreted frizzled-related protein 4) expressing vector and transfected MPDL22 (mouse periodontal ligament cell clone) cells. The transfectant was cultured in the mineralization-inducing media and then, the alkaline phosphatase (ALP) activity was measured according to the procedure of Bessay et al. Calcified nodule formation was detected by Alizarin Red S staining. We then suppressed endogenous SFRP4 gene in MPDL22 cells by RNA interference (RNAi). Results: We detected 103 up-regulated genes in the cytodifferentiation process of PDL cells by oligo DNA chip. Among these genes, SFRP4 showed the highest induction ratio of expression. Over-expression of SFRP4 in MPDL22 cells resulted in suppression of the ALP activity and calcified nodule formation in the process of the cytodifferentiation. On the other hand, RNAi of SFRP4 showed enhancement of the ALP activity and calcified nodule formation. Conclusion: Present results suggest that SFRP4 identified by oligo DNA chip is one of the negative regulators for the cytodifferentiation of PDL cells. This work was supported in part by a Grant-in-Aid from the Japan Society for the Promotion of Science (No.15390646, No.17390560 and No.17390561) and was a part of the 21st Century COE program.