Methods: Human-cementoblast-like (HCEM) cells were cultured and treated with recombinant human full-length amelogenin (rh174) in the presence or absence of anti-CD63 antibody. The cell proliferation was evaluated by use of BrdU and MTS method. In addition, the activity of extracellular signal regulated kinases (ERK)1/2 was measured by means of ELISA. Real-time-PCR was performed with light-Cycler system to examine the mRNA levels of alkaline-phosphatase (ALP), osteocalcin (OCN), receptor-activator-for-nuclear factor-kb-ligand (RANKL), and osteoprotegerin (OPG). In addition, Alizalin red staining were used to examine the effects of rh174 on the differentiation of HCEM cells.
Results: Proliferation of HCEM cells was enhanced significantly by the treatment with rh174 and inhibied by the addition of anti-CD63 blocking antibody. In addition, the activity of phospho/ERK1/2 signaling of HCEM cells was enhanced significantly by the treatment with rh174, and inhibied by the addition of anti-CD63 blocking antibody. The mRNA expression of ALP, OCN and OPG was up-regulated significantly by the treatment with rh174, whereas gene expression of RANKL was down-regulated significantly by the treatment with rh174. In addition, calcified deposit of HCEM cells, defected by Alizalin red staining, increaced by treatment with rh174.
Conclusions: It is shown that CD63 may be a receptor of rh174 in HCEM cells, and CD63/rh174 interaction activates the signaling pathway of ERK1/2, resulting in the induction of both proliferation and differentiation of HCEM cells. It is thus highly anticipated that amelogenin may contribute to the application to the regeneration of cementum in terms of the enhancement of HCEM proliferation and differentiation.