Grp78 is Essential for Amelogenin-Induced Cell Migration in PDLSCs

Objectives: Amelogenin, the major component of enamel matrix proteins, is a potential bioactive molecule for periodontal regeneration, although the downstream target molecules and/or signaling are still unknown. Recently, we performed proteomics analysis to elucidate amelogenin-interaction networks. We identified Grp78 as a new amelogenin binding protein, and the biological interaction of amelogenin with this molecule enhanced cell proliferation in osteoblastic cells (Fukuda et al, 2013). Since specific migration and proliferation of periodontal ligament stem/progenitor cells (PDLSCs) cells play key role in successful periodontal regeneration, in this study, we, therefore, evaluated the biological interaction between amelogenin and Grp78, and its effect on cellular responses in PDLSCs.
Methods: An established multipotent clonal human PDLSC, cell line 1-17 was used. GST pull down assay and confocal co-localization ware performed to validate the binding of recombinant amelogenin (rM180:10mg/mL) and endogenous Grp78. Grp78 was overexpressed or knock-downed in 1-17 cells for further biological evaluation experiments including gene expression profiles (microarray, pathway, and heatmap analysis), cell motility (wound healing and boyden chamber assay), cell viability (WST-8, Ki-67 positive staining), Rho signal transductions (Rho family activation), and morphological changes (confocal microscopy).
Results: Internalization of rM180 via binding to the cell surface Grp78 was observed in 1-17 cells. Microarray analysis demonstrated that rM180 activates TGF-b pathway, and Grp78 altered the expression of several cell migration related genes. Overexpression of Grp78 enhanced rM180-induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Binding of rM180 with Grp78 promoted lamellipodia formation, and the simultanous activation of Rac1 was observed.
Conclusions: The results suggested that Grp78 was essential for enhancing amelogenin-induced cell migration in PDLSCs. The Rac1 activation and lamellipodia formation are critical steps for amelogenin-induced cell migration.