Grp78 Is Critical for Amelogenin‐Induced Cell Migration in a Multipotent Clonal Human Periodontal Ligament Cell Line

Abstract

Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose‐regulated protein 78 (Grp78) as a new amelogenin‐binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1‐17, which possesses the most typical characteristics of PDLSCs. Confocal co‐localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1‐17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration‐associated genes in 1‐17 cells. Moreover, Grp78 overexpression enhanced rM180‐induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin‐induced migration in 1‐17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin‐induced periodontal tissue regeneration. J. Cell. Physiol. 231: 414–427, 2016. © 2015 Wiley Periodicals, Inc.

Leucine-Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast Requires Flotillin-1 Mediated Endocytosis

“…More recently, putative full‐length amelogenin (rM180)‐binding partners were identified in SaOS‐2 osteoblastic cells using mass spectrometry, and included chaperone molecules (HSP70 family proteins), cytoskeletal proteins (actin, vimentin, tubulin), actin‐binding proteins (gelsolin, tropomyosin), proton pump protein (ATPase), sialic acid‐binding Ig‐like lectins (Siglec‐10), endoplasmic reticulum (ER) protein (glucose‐regulated protein 78 [Grp78], calreticulin), mitochondrial membrane protein (prohibitin), and nuclear proteins (nucleophosmin and hnRNP A2/B1) (Fukuda et al, ). Some of these receptor candidates for full‐length amelogenin were also validated by co‐localization assays, including Lamp1 in murine dental follicle cells and OCCM‐30 cells (Zhang et al, ); LAMP1 and CD63 in human osteoblast (hFOP_1.19), murine pre‐osteoblast cells (MC3T3‐E1) and mouse ameloblast‐like LS8 cells (Shapiro et al, ); Grp78 in SaOS‐2 cells (Fukuda et al, ) and undifferentiated HPDL cell line (Toyoda et al, ). Previous studies have additionally demonstrated that exogenously added recombinant full‐length amelogenin (rM180) can rapidly move into the cell, through Lamp1 or CD63‐positive vesicles and subsequently localize to the perinuclear region in ameloblast (Shapiro et al, ), osteoblast (Shapiro et al, ; Fukuda et al, ), cementoblast, and dental follicle cells (Zhang et al, ).…”

“…Endocytosis of enamel matrix proteins has been considered as a crucial condition for amelogenin‐induced cellular responses. For example, reduction of cellular uptake of amelogenin as a result of Grp78/Bip knockdown affected amelogenin‐induced cell proliferation and cell migration in osteoblasts and HPDL cells, respectively (Fukuda et al, ; Toyoda et al, ). In the current study, we hypothesized that reduced expression of flotilin‐1 would significantly reduce LRAP cellular uptake leading to an impact on the biological properties reported for LRAP.…”

Microarray Analysis of the Effects of Amelogenin on U937 Monocytic Cells

“…In scatter plot analysis aimed at confirming the temporal changes in gene expression caused by amelogenin stimuli, our findings showed that unlike cytokines, amelogenin did not induce notable variations in gene expression; our findings also confirmed that the effects of amelogenin generally developed at a relatively early stage (4h) (Figure 1). We previously reported that the growth of osteoblasts and the migration of periodontal ligament stem cells were enhanced by amelogenin [14] [15]. In other studies, amelogenin was reported to enhance the differentiation of cementoblasts as well as that of periodontal ligament cells;…”

“…on the cell membrane of periodontal ligament stem cells served as a receptor for amelogenin [15]. GRP78 is a ubiquitous chaperone protein present mainly in the endoplasmic reticulum [26], but in undifferentiated stem cells, it is also expressed on the cell membrane [27] and has been reported to function as a receptor for Cript [28] and DMP1 [29].…”

“…By proteomic analysis focusing on amelogenin, which is a major protein in EMD, we identified a new group of amelogenin-associated molecules such as Grp78 in osteoblasts [14]. In addition, the association between amelogenin (rM180) and Grp78 has been reported to be involved in the cellular migratory capacity of periodontal ligament stem cells [15].…”