YB-1 is important for amelogenin induced periodontal regeneration

Objective: Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, and it is used in periodontal surgery to stimulate regeneration of periodontal tissues. However, the exact molecular mechanisms of periodontal regeneration remain largely unknown. In this study, we investigated the proteins bound to amelogenin, the major (>95%) component of EMD, to elucidate the molecules involved in the periodontal tissue regeneration. Methods: We first made GST-fusion amerogenin using the fetal mouse cDNA, to analyze the proteins bound to amelogenin by GST pull down assay. Purified GST fusion amelogenin immobilized on glutathione was incubated with the lysates of periodontal tissue cells. After extensive washing, bound proteins ware resolved by SDS-PAGE, and analyzed by silver staining or Western blotting. Thio-His fusion protein was also made to confirm the mutual interaction with amelogenin. To investigate the role of amelogenin on cell proliferation, we purified recombinant amelogenin. The siRNA was also transfected to knock-down the endogenous amelogenin binding protein. The effect of amelogenin and its binding protein on cell growth rates were analyzed by MTT assay using the SaOS-2 osteoblastic cells. Results: Silver staining analysis showed several proteins bound to amelogenin. Some proteins bound to amelogenin ware ubiquitous, others ware cell specific. YB-1, human Y-box protein, was identified as one of the amelogenin binding proteins by the Western blot analysis. MTT assay revealed that knock-down of YB-1 suppressed cell proliferation, but the stimulation with amelogenin restored the cell growth. Conclusions: We identified new amelogenin binding protein, YB-1 (human Y-box binding protein-1). YB-1 has multiple functions but was initially identified as a transcription factor. YB-1 protein is abundant and expressed ubiquitously in human cells, functioning in cell proliferation. Our data suggest that YB-1 regulates cell growth rates, and involved in regeneration of periodontal tissues by binding to amelogenin.