Pimrumpai Rochanakit Sindhavajiva1, Panunn Sastravaha2, Mansuang Arksornnukit3, Prasit Pavasant4. 1. Graduate Program in Prosthodontics, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Mineralized Tissue Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. 2. Department of Surgery, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. 3. Department of Prosthodontics, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. 4. Mineralized Tissue Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: prasit215@gmail.com.
Abstract
OBJECTIVE: Purinergic 2X7 receptor (P2X7R) activation modulates in vitro mineralization by primary rat and human osteoblasts. However, the detailed mechanism of how P2X7R activation affects primary human osteoblasts remains unclear. The aim of this study was to investigate the effect of P2X7R activation on human mandibular-derived osteoblast (hMOB) differentiation. DESIGN: Primary human osteoblasts were obtained from non-pathologic mandibular bone from healthy patients. The hMOBs were cultured in osteogenic medium with or without 0.5-5μM 2'(3')-O-(4-benzoyl) benzoyl-ATP (BzATP), a selective P2X7R agonist. The mRNA expression of osteogenic differentiation markers and WNT-signaling molecules was investigated by quantitative real time polymerase chain reaction. In vitro mineral deposition was determined by Alizarin Red S staining. Transfection of small interfering RNA was performed to confirm the effect of P2X7R activation. WNT/β-catenin signaling was detected by immunofluorescence staining for β-catenin. RESULTS: BzATP inhibited osteogenic medium-induced RUNX2 and OSX mRNA expression in hMOBs. Moreover, BzATP significantly retarded in vitro mineralization. These findings indicated that BzATP/P2X7R activation inhibited hMOB differentiation. Interestingly, reduced WNT3A mRNA expression and blockage of osteogenic medium-induced β-catenin nuclear translocation were also found. These data suggested that WNT signaling might be a target of P2X7R-regulated osteogenic differentiation. Furthermore, when recombinant human WNT3A was added to the BzATP-treated group, it rescued the reduced RUNX2 and OSX expression, and in vitro mineralization. CONCLUSION: Our results demonstrate that P2X7R activation by BzATP inhibits hMOB differentiation. This inhibitory effect was associated with inhibition of the WNT/β-catenin signaling pathway.
OBJECTIVE: Purinergic 2X7 receptor (P2X7R) activation modulates in vitro mineralization by primary rat and human osteoblasts. However, the detailed mechanism of how P2X7R activation affects primary human osteoblasts remains unclear. The aim of this study was to investigate the effect of P2X7R activation on human mandibular-derived osteoblast (hMOB) differentiation. DESIGN: Primary human osteoblasts were obtained from non-pathologic mandibular bone from healthy patients. The hMOBs were cultured in osteogenic medium with or without 0.5-5μM 2'(3')-O-(4-benzoyl) benzoyl-ATP (BzATP), a selective P2X7R agonist. The mRNA expression of osteogenic differentiation markers and WNT-signaling molecules was investigated by quantitative real time polymerase chain reaction. In vitro mineral deposition was determined by Alizarin Red S staining. Transfection of small interfering RNA was performed to confirm the effect of P2X7R activation. WNT/β-catenin signaling was detected by immunofluorescence staining for β-catenin. RESULTS:BzATP inhibited osteogenic medium-induced RUNX2 and OSX mRNA expression in hMOBs. Moreover, BzATP significantly retarded in vitro mineralization. These findings indicated that BzATP/P2X7R activation inhibited hMOB differentiation. Interestingly, reduced WNT3A mRNA expression and blockage of osteogenic medium-induced β-catenin nuclear translocation were also found. These data suggested that WNT signaling might be a target of P2X7R-regulated osteogenic differentiation. Furthermore, when recombinant humanWNT3A was added to the BzATP-treated group, it rescued the reduced RUNX2 and OSX expression, and in vitro mineralization. CONCLUSION: Our results demonstrate that P2X7R activation by BzATP inhibits hMOB differentiation. This inhibitory effect was associated with inhibition of the WNT/β-catenin signaling pathway.