ADAMTSL-4 Regulated Assembly of Oxytalan Fiber during Periodontal Ligament Development

Objectives: ADAMTSL-4 is a novel extracellular matrix member designated from the ADAMTSL family that expressed in perichondrocyte, and seems to modulate protease activity of ADAMTS family. Interestingly, ADAMTSL-4 was also expressed in the periodontal ligament. In this study, we investigate if ADAMTSL-4 has a functional role for the formation of periodontal ligament. Methods: Both gene expression and protein distribution of ADAMTSL-4 were examined by in situ hybridization or immunohistochemistry during tooth germ development. Immunostaining patterning of ADAMTSL-4 was compared with fuchsin staining, if it associated with oxytalan fiber formation. Expression of ADAMTSL-4 was also examined by using immortalized mice dental follicle cells (MDFE6-EGFP). To investigate function of ADAMTSL-4, MG63, osteosarcoma cell line, was transduced with retrovirus carrying ADAMTSL-4 whether they form the periodontal ligament-like structure. Results: Results showed that ADAMTSL-4 mRNA and protein were found to express in dental follicle at bell or late bell stage of tooth germ, and became deposited as oxytalan fiber-like microfibrils in the periodontal ligament at late stage of root formation. The distribution of ADAMTSL-4-positive fiber was almost identical to the fuchsin staining positive oxytalan fiber, suggesting that ADAMTSL-4 is a novel component of the oxytalan fiber. MDFE6-EGFP found to form the ADAMTSL-4 positive oxytalan fiber-like structure upon implantation into immunodeficiency mice. Overexpression of ADAMTSL-4 demonstrated the formation of oxytalan fiber-like microfibrils, and they were partially colocalized with fibrillin-1. Immunoflorescence study using anti-ADAMTSL-4 and anti-fibrillin-1 antibody showed that they were partially colocalized in the periodontal ligament. Conclusion: Our results suggest that ADAMTSL-4 is a novel extracellular matrix component that involves oxytalan fiber assembly, and play an important role in formation of the periodontal ligament.