NEMO Binding Domain (NBD) peptide inhibits the activity of osteoclasts

NF-κB is an essential inducible transcription factor that is rapidly and transiently elicited in response to proinflammatory cytokines in osteoclasts. Activation of NF-κB requires the complex of kinases (IKKα and IKKβ) and the regulatory protein NEMO (NF-κB essential modifier). NBD peptide is a small cell permeable peptide spanning the IKKβ and disrupts the association between NEMO and IKKβ. Consequently NBD peptide blocks NF-κB activation, hence becomes an attractive target for the development of inhibitors of bone resorption. How NBD peptide affects osteoclastic activity is yet to be found. The functional capacity of osteoclasts is measured by determining the volume of bone or dentine which they resorb. Objectives: To ascertain how NBD peptide inhibits the activity of osteoclasts in vitro. Methods: Wild type mice (C57BL/6) were used to isolate osteoblasts (new born) and bone marrow cells (7 week old). Pit formation assay was performed in the presence of NBD peptide. Quantitative analysis of resorbed area and volume was performed using Super Depth Surface Profile Measurement Microscope (Keyene Corporation, Osaka) interfaced to a computer with image analyzing software. Results: Statistical analysis revealed significant reduction in pit area (p<0.01) and pit volume (p<0.01) as well as the number of resorptive episodes (p<0.005) of osteoclasts in the presence of NBD peptide. NBD peptide reduces the maximum pit depths as well as average pit depths created by osteoclasts. Conclusion: This study clearly revealed that NBD peptide inhibits in vitro osteoclast function by reducing its resorbing activity.