Porcine enamel extracts have been used to treat periodontal defects, but the mechanisms of action are evasive. Furthermore, enamel extracts are heterogeneous and may mask the perceived therapeutic effects of single constituent proteins/peptides. Here, we investigated the underlying mechanisms of amelogenin.
Method:
1) pCMV6-amelogenin was transfected into human PDL stem/progenitor cells. Cell seeded TCP scaffolds were implanted in vivo. The cells were transfected with b-catenin reporter and signaling activation was measured by luciferase activity. Recombinant amelogenin was encapsulated collagen gel and implanted into b-catenin reporter mice. The samples were subjected to LacZ staining to demonstrate activation of b-catenin signaling in vivo. 2) T7 phage library was constructed using mouse osteoblast cDNA. Recombinant full-length amelogenin was used as bait for bio-panning. Upon high-throughput screening, high affinity phage colonies were sequenced and confirmed by far-western and ELISA. Angiogenic effect of amelogenin and the identified protein was analyzed by metatarsal angiogenesis assay.
Result:
1) PDL stem/progenitor cells overexpressing amelogenin showed enhanced mineralized matrix formation. After implanted in vivo in TCP scaffold, the transfected PDL stem/progenitor cells formed cellular and acellular cementum-like structures with Sharpe fibers integration insertion. 2) Amelogenin functioned as a signaling molecule by activating b-catenin signaling in PDL stem/progenitor cells as demonstrated by b-catenin nuclear translocalization and reporter transactivation both in vitro and in vivo. Amelogenin induced PDL stem/progenitor cell differentiation in a b-catenin dependent manner. 3) Using osteoblast cDNA, a phage library was constructed. High-throughput screening identified TIMP2 as an amelogenin interaction protein. Amelogenin attenuated the inhibitory activity of TIMP2 on MMPs. Metatarsal angiogenesis assay demonstrated that amelogenin promotes angiogenesis through TIMP2 attenuation.
Conclusion:
Amelogenin induces PDL cell differentiation through activation of b-catenin signaling and promotes angiogenesis by suppressing the activity of TIMP-2, an anti-angiogenesis factor. Our study also presents a potential mechanism regulating tooth development.
Acknowledgements
NIH/NIDCR R01DE023112.