Leucine-rich Amelogenin Peptide Promotes Chondrogenesis

Abstract

Purposes: Leucine-rich amelogenin peptide (LRAP) is an amelogenin splicing variant, and is suggested to be a signal peptide that promotes the maturation of osteoblasts as well as the proliferation and migration of periodontal ligament cells. Recently, amelogenin expression has been reported in cartilage in vivo. However, the involvement of LRAP in cartilage formation is unknown. The purpose of this study was to clarify the function of LRAP in mesenchymal cells during chondrogenic lineage differentiation. Methods: After removal of the limb buds of embryonic day 10 C57BL6/CrSlc mouse embryos, the cells were dissociated by trypsin/collagenase treatment and then applied to micromass culture. Recombinant porcine LRAP, which has 85% homology with mouse LRAP, was added to the medium at 0.1, 1, and 10 ng/ml. Alcian Blue staining was performed at 2, 4, and 6 days of culture, and the intensity was measured by the absorbance of the solubilized micromass cultures. To assess the effect of LRAP on cell proliferation, a BrdU assay was performed at 24 and 48 hours. Total RNA was extracted at 12, 24, and 48 hours, and subjected to semi-quantitative RT-PCR analysis of chondrogenic differentiation marker genes Sox9, collagen type II, and aggrecan. One-way analysis of variance (ANOVA) and two-way ANOVA were performed using Bonferroni's multiple comparison as a post-hoc test. Results: At 24 and 48 hours after addition of LRAP, the number of BrdU-positive cells was significantly increased in a concentration-dependent manner compared with that in the control. Alcian Blue-positive nodules appeared in micromass cultures after 2 days. In addition, at 2, 4 and 6 days of culture, the intensity of Alcian Blue staining was significantly increased by LRAP in a concentration-dependent manner compared with that in the control. Furthermore, Sox9 gene expression was significantly increased by LRAP after 24 and 48 hours of culture. Gene expression of collagen type II and aggrecan was also increased by LARP after 48 hours of culture compared with that in the control. Conclusion: These results suggest that LRAP promotes the proliferation of mesenchymal cells derived from the mouse limb bud, and increases chondrogenic marker gene expression-involved production of chondrogenic matrices as indicated by Alcian Blue staining in micromass cultures. Our study may provide useful information for further investigation of the application of amelogenin proteins in cartilage regeneration.