Amelogenin is one of the enamel matrices secreted by ameloblasts and is known to have at least 15 alternative mRNA splicing isoforms. The leucine rich amelogenin peptide (LRAP) is one of most abundant amelogenin isoforms and numerous studies have shown that amelogenin peptides including LRAP, are expressed in cartilage and bone. Lysosome-associated membrane protein-1 (LAMP-1) has been identified as amelogenin binding partner, however, the mechanism of action for LRAP in chondrogenesis or osteogenesis is still unclear. This study aimed to assay the effect of chemically synthesized LRAP (csLRAP) on chondrogenic or osteogenic differentiation with the involvement of LAMP-1. The csLRAP used in this study was generated by F-moc solid phase chemistry based on the mouse LRAP amino acid sequence and was added to the culture medium of the chondrogenic cells, ATDC5 and the osteoblast cells, MC3T3-E1. Both chondrogenic and osteoblast cells, in which an immunopositive reaction to LAMP-1 antibody was observed by immunocytochemistry, showed significant suppression in cell number in the presence of csLRAP at 10 g/ml compared to that in control, but this effect was rescued in the presence of LAMP-1 antibody. On the other hand, the intensity of alcian blue staining in chondrogenic cells and alizarin red staining in osteoblast cells was significantly increased in the presence of csLRAP at a dose of 10 g/ml after 4 weeks culture, and this effect was suppressed in the presence LAMP-1 antibody. Moreover, the chondrogenic or osteogenic differentiation marker genes including Sox9, Type II Collagen, Type X Collagen, Runx2, Alkaline Phosphatase, and Type I Collagen, were upregulated in the presence of csLRAP after one week of culture and were suppressed in the presence of LAMP-1 antibody. These results suggest that csLRAP could promote osteogenic and chondrogenic differentiation in vitro, and that LAMP-1 may be involved in the differentiation and proliferation of these cells.
Ameloblastoma is a locally invasive benign epithelial odontogenic tumor and its histopathological structures are similar to the enamel organ. Although various studies have investigated cell proliferation in ameloblastoma to elucidate the biological behavior and clinicopathological mechanisms, it remains poorly understood. The studies on the development of the enamel organ reports that FGF-9, -10, and TGF-β1 are strongly involved in dental epithelial cell differentiation and cell proliferation. In this study, we attempt to evaluate the effect of these growth factors on ameloblastoma cells. Both collagen-coated and normal plastic cell culture plate cell growth curves were steeper in the presence of growth supplement than in the absence of growth supplement. The presence of TGF-β1 at each dose (1 to 10 ng/ml), however, suppressed the number of cells cultured on the collagen-coated plate but made no significant difference on the normal plastic plate. The number of cells was increased in the presence of FGF-10 at 100 ng/ml, but not in the presence of FGF-9 after 48 h culture. These results suggest that FGF-10 and TGF-β1 play distinct roles in the cell proliferation of human ameloblastoma cells.
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