IADR Abstract Archives
A Stimulatory Mechanism of RANKL-Binding Peptide on Osteoblast Differentiation
Objectives: Cell to cell communication in biological systems comprises a large variety of interactions, one of which is a switching on system which is caused by the clustering of molecules on the plasma membrane. We herein describe the possible mechanism regulating osteoblast differentiation by the receptor activator of NF-κB ligand (RANKL)-binding peptides.
Methods: The histidine(His)-tagged RANKL with biotinylated Ni-nitrilotriacetic acid on the tamavidin-2-LPI-2D-crystals-coated surface was used to mimic the RANKL-expressed plasma membrane of osteoblasts, and a high-speed atomic force microscope (hsAFM) was used to observe the molecular events on the coated surface. Primary osteoblasts isolated from mice calvariae were used for the in vitro study. In both the acellular and cellular systems, the RANKL-binding peptide, OP3-4, and osteoprotegerin (OPG) were used. OPG-Fc and IgM were also used in the cell culture system. The alkaline phosphatase (ALP) activity, one of the osteoblast differentiation markers, were measured using a spectral photometer. ANOVA and the Tukey post hoc test were used for multiple comparisons.
Results: In the osteoblast culture, OP3-4 (100 μM) stimulated the ALP activity significantly more than the vehicle control (p<0.05), but not OPG (20 nM). The hsAFM was used to clarify the reason for this difference and the clustering of the RANKL molecules on the RANKL-coated surface in the presence of OP3-4 (100 μM) was observed, but not in the presence of OPG (20 nM). An ALP assay was again performed to clarify the biological significance of clustered RANKL on the simulated plasma membrane using IgM, which induces the pentamer-clustering of Fc-conjugated protein. OPG-Fc (20 nM) did not stimulate the ALP activity, but it significantly stimulated the ALP activity in the presence of IgM compared to OPG-Fc (20 nM) alone (p<0.05).
Conclusions: A reagent, which induces the clustering of RANKL molecules on the osteoblast surface could thus be a new stimulator of osteoblast differentiation.
Methods: The histidine(His)-tagged RANKL with biotinylated Ni-nitrilotriacetic acid on the tamavidin-2-LPI-2D-crystals-coated surface was used to mimic the RANKL-expressed plasma membrane of osteoblasts, and a high-speed atomic force microscope (hsAFM) was used to observe the molecular events on the coated surface. Primary osteoblasts isolated from mice calvariae were used for the in vitro study. In both the acellular and cellular systems, the RANKL-binding peptide, OP3-4, and osteoprotegerin (OPG) were used. OPG-Fc and IgM were also used in the cell culture system. The alkaline phosphatase (ALP) activity, one of the osteoblast differentiation markers, were measured using a spectral photometer. ANOVA and the Tukey post hoc test were used for multiple comparisons.
Results: In the osteoblast culture, OP3-4 (100 μM) stimulated the ALP activity significantly more than the vehicle control (p<0.05), but not OPG (20 nM). The hsAFM was used to clarify the reason for this difference and the clustering of the RANKL molecules on the RANKL-coated surface in the presence of OP3-4 (100 μM) was observed, but not in the presence of OPG (20 nM). An ALP assay was again performed to clarify the biological significance of clustered RANKL on the simulated plasma membrane using IgM, which induces the pentamer-clustering of Fc-conjugated protein. OPG-Fc (20 nM) did not stimulate the ALP activity, but it significantly stimulated the ALP activity in the presence of IgM compared to OPG-Fc (20 nM) alone (p<0.05).
Conclusions: A reagent, which induces the clustering of RANKL molecules on the osteoblast surface could thus be a new stimulator of osteoblast differentiation.
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