IADR Abstract Archives
Recombinant-human Osteopontin Induced Osteogenic Differentiation via Calcium-binding Protein-ALK1 Signaling Pathway
Objectives: Osteopontin (OPN) plays an important role in bone remodeling. We recently showed that recombinant human osteopontin (rhOPN) from Nicotiana benthamiana induced osteogenic-differentiation by human periodontal ligament stem cells (HPDLSCs), however, the detailed mechanism of this induction remains unclear. The aim of this study was to determine the molecular mechanism by which rhOPN generates this osteogenic induction.
Methods: Five HPDLSCs lines were isolated from third molars extracted from five different donors. Cells were seeded on OPN-coated surfaces with or without various inhibitors. Reverse-transcription polymerase chain reaction, Western analysis, and in vitro calcification as judged by alizarin red staining were performed to confirm the osteogenic differentiation. In silico analysis was also carried out to examine the ligand-receptor interaction.
Results: RhOPN could induce osterix (OSX) and dentin matrix protein 1 (DMP-1) expression as well as in vitro calcification. Adding an ALK-1 inhibitor abolished the expression of OSX and DMP-1, in cells seeded on rhOPN. The molecular docking data from in silico analysis suggested the interaction between ALK-1 and the OPN calcium-binding domain (CaBD-OPN). The CaBD-OPN was generated and showed the ability to induce OSX and DMP-1 expression and in vitro calcification similar to the effect of full length rhOPN. The effect of CaBD-OPN was significantly abolished by ALK-1 inhibitor (p<0.05). Western blot analysis revealed an increase in phospho-Smad-1 protein in HPDLSCs seeded on CaBD-OPN, which was inhibited by the ALK1 inhibitor.
Conclusions: This study reveals the potential of the CaBD as the functional osteogenic-induction domain of OPN and the interaction between CaBD-ALK-1 promoted osteogenic differentiation. These findings indicate the potential role of this plant-produced recombinant OPN on bone induction. The possible application of this plant-OPN, focusing on the calcium binding domain, is to coat this domain on a scaffold to enhance bone regeneration in patients with bone defects.
Methods: Five HPDLSCs lines were isolated from third molars extracted from five different donors. Cells were seeded on OPN-coated surfaces with or without various inhibitors. Reverse-transcription polymerase chain reaction, Western analysis, and in vitro calcification as judged by alizarin red staining were performed to confirm the osteogenic differentiation. In silico analysis was also carried out to examine the ligand-receptor interaction.
Results: RhOPN could induce osterix (OSX) and dentin matrix protein 1 (DMP-1) expression as well as in vitro calcification. Adding an ALK-1 inhibitor abolished the expression of OSX and DMP-1, in cells seeded on rhOPN. The molecular docking data from in silico analysis suggested the interaction between ALK-1 and the OPN calcium-binding domain (CaBD-OPN). The CaBD-OPN was generated and showed the ability to induce OSX and DMP-1 expression and in vitro calcification similar to the effect of full length rhOPN. The effect of CaBD-OPN was significantly abolished by ALK-1 inhibitor (p<0.05). Western blot analysis revealed an increase in phospho-Smad-1 protein in HPDLSCs seeded on CaBD-OPN, which was inhibited by the ALK1 inhibitor.
Conclusions: This study reveals the potential of the CaBD as the functional osteogenic-induction domain of OPN and the interaction between CaBD-ALK-1 promoted osteogenic differentiation. These findings indicate the potential role of this plant-produced recombinant OPN on bone induction. The possible application of this plant-OPN, focusing on the calcium binding domain, is to coat this domain on a scaffold to enhance bone regeneration in patients with bone defects.
