IADR Abstract Archives
Activin A reversely works on the osteoblastic differentiation in human pre-osteoblastic cells and periodontal ligament cells
Objectives: Activin A (ActA) that is a member of the TGF-β superfamily and is composed of a dimer of inhibinβa. ActA contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. However, the expression and function of ActA in human periodontal ligament cells (HPDLCs) and human pre-osteoblastic cells (Saos2) have not been reported. Thus, we aimed to analyze the biological effects of ActA on HPDLCs and Saos2, and compare the signal pathway between HPDLCs and Saos2 treated with ActA.
Methods: Saos2 was purchased from RIKEN (Saitama, Japan) and used HPDLCs that were isolated from a healthy third molar from a 23-year-old male. These cells were cultured with or without ActA in the presence or absence of CaCl2 that was used for inducing osteoblastic differentiation. The cells were then subjected to Alizarin red S staining and von Kossa staining. Gene expressions were evaluated by semi-quantitative or quantitative RT-PCR, and protein expressions were investigated by western blotting and immunocytochemical analyses. All procedures were performed in compliance with the Research Ethics Committee, Faculty of Dentistry, Kyushu University.
Results: Immunocytochemical analysis with an anti-ActA antibody revealed positive staining in HPDLCs and Saos2. Expression of Activin receptors (ALK4, IIa and IIb) was detected in HPDLCs and Saos2. ActA inhibited mineralization and expression of bone-related genes in HPDLCs, while it promoted those in Saos2. Western blotting data showed that ActA up-regulated the phosphorylation of Smad2/3 in HPDLCs, whereas promoted the phosphorylation of Smad1/5/8 in Saos2.
Conclusions: These results suggested that ActA inhibited osteoblastic differentiation of HPDLCs through phosphorylation of Smad2/3 pathway, while promoted osteoblastic differentiation of Saos2 through phosphorylation of Smad1/5/8 pathway. Thus, ActA may reversely work on these cells through activation of different Smad pathway.
Methods: Saos2 was purchased from RIKEN (Saitama, Japan) and used HPDLCs that were isolated from a healthy third molar from a 23-year-old male. These cells were cultured with or without ActA in the presence or absence of CaCl2 that was used for inducing osteoblastic differentiation. The cells were then subjected to Alizarin red S staining and von Kossa staining. Gene expressions were evaluated by semi-quantitative or quantitative RT-PCR, and protein expressions were investigated by western blotting and immunocytochemical analyses. All procedures were performed in compliance with the Research Ethics Committee, Faculty of Dentistry, Kyushu University.
Results: Immunocytochemical analysis with an anti-ActA antibody revealed positive staining in HPDLCs and Saos2. Expression of Activin receptors (ALK4, IIa and IIb) was detected in HPDLCs and Saos2. ActA inhibited mineralization and expression of bone-related genes in HPDLCs, while it promoted those in Saos2. Western blotting data showed that ActA up-regulated the phosphorylation of Smad2/3 in HPDLCs, whereas promoted the phosphorylation of Smad1/5/8 in Saos2.
Conclusions: These results suggested that ActA inhibited osteoblastic differentiation of HPDLCs through phosphorylation of Smad2/3 pathway, while promoted osteoblastic differentiation of Saos2 through phosphorylation of Smad1/5/8 pathway. Thus, ActA may reversely work on these cells through activation of different Smad pathway.