Abstract
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 μg/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 μg/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.
| Original language | English |
|---|---|
| Pages (from-to) | 51-60 |
| Number of pages | 10 |
| Journal | Journal of Hard Tissue Biology |
| Volume | 26 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2017 |
| Externally published | Yes |
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Keywords
- Chondrogenesis
- Leucine-rich amelogenin peptide (LRAP)
- Lysosome-associated membrane proteins (LAMPs)
- Osteogenesis
ASJC Scopus subject areas
- Medicine (miscellaneous)
- Biochemistry
- Biomaterials
- Orthopedics and Sports Medicine
- Dentistry(all)
- Cell Biology
Cite this
Effects of a chemically synthesized leucine-rich amelogenin peptide (csLRAP) on chondrogenic and osteogenic cells. / Matsuda, Yuko; Hatakeyama, Yuji; Nakashima, Kazuki; Kamogashira, Naoko; Hatakeyama, Junko; Tamaoki, Sachio; Sawa, Yoshihiko; Ishikawa, Hiroyuki.
In: Journal of Hard Tissue Biology, Vol. 26, No. 1, 2017, p. 51-60.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Effects of a chemically synthesized leucine-rich amelogenin peptide (csLRAP) on chondrogenic and osteogenic cells
AU - Matsuda, Yuko
AU - Hatakeyama, Yuji
AU - Nakashima, Kazuki
AU - Kamogashira, Naoko
AU - Hatakeyama, Junko
AU - Tamaoki, Sachio
AU - Sawa, Yoshihiko
AU - Ishikawa, Hiroyuki
PY - 2017
Y1 - 2017
N2 - 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 μg/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 μg/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.
AB - 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 μg/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 μg/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.
KW - Chondrogenesis
KW - Leucine-rich amelogenin peptide (LRAP)
KW - Lysosome-associated membrane proteins (LAMPs)
KW - Osteogenesis
UR - http://www.scopus.com/inward/record.url?scp=85009164472&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85009164472&partnerID=8YFLogxK
U2 - 10.2485/jhtb.26.51
DO - 10.2485/jhtb.26.51
M3 - Article
AN - SCOPUS:85009164472
VL - 26
SP - 51
EP - 60
JO - Journal of Hard Tissue Biology
JF - Journal of Hard Tissue Biology
SN - 1341-7649
IS - 1
ER -