Periodontal ligament (PDL) plays crucial roles in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In attempt to understand the molecular and genetic basis of PDL functions, we investigated the expression profile of active genes in human periodontal ligament obtained by collecting sequences with 3′-directed cDNA library, which faithfully represents composition of the mRNA population. We succeeded to obtain a total of 1752 cDNA sequences by sequencing randomly selected clones and a total of 1318 different species was identified as gene signatures (GS) by their sequence identity. The resulting expression profile showed that collagen types I and III were the most abundant genes and osteogenesis-relating genes, such as osteonectin and periostin were highly expressed. In the gene expression profile of human PDL, we found a novel gene which was highly expressed in PDL, but not in other tissue-cDNA libraries. We cloned a full-length cDNA of the gene and identified that it codes a novel protein, which is a new member of class I of small leucine-rich repeat proteoglycan (SLRP) family. We designated it periodontal ligament associated protein-1 (PLAP-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. In situ mRNA hybridization analysis using mouse periodontium revealed that PLAP-1 was expressed only in PDL tissues. Over-expression of PLAP-1 in PDL-derived clone cells interfered with both naturally and bone morphogenetic protein 2 (BMP-2)-induced mineralization of the PDL cells. On the other hand, knockdown of PLAP-1 transcript levels by RNA interference enhanced BMP-2-induced differentiation of PDL cells. Furthermore, co-immunoprecipitation assays showed a direct interaction between PLAP-1 and BMP-2 in vitro. These results suggest that PLAP-1 plays a specific role(s) in the periodontal ligament as a negative regulator of cytodifferentiation and mineralization, probably by regulating BMP-2 activity to prevent the periodontal ligament from developing non-physiological mineralization, such as ankylosis.
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