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Abstract: Osteogenesis associated with persistent inflammation or infection exists in a broad range of conditions including rheumatoid arthritis and traumatic bone fracture. The poor outcomes of these conditions will benefit from more effective treatments. Here we investigated the molecular mechanisms and tested NEMO-binding domain peptide as a new approach of circumventing TNF-α inhibition of osteoblast differentiation. Our results showed: TNF-α markedly decreased BMP-2-induced alkaline phosphatase activity in the multipotent myoblast C2C12 cells in a dose dependent manner; stepwise experiments demonstrated that BMP-2-induced Smad1 activity was abrogated by addition of exogenous TNF-α or overexpression of NF-κB, and it was significantly elevated by overexpression of IκBα, an inhibitor of NF-κB; Western blotting showed that TNF-α markedly decreased the amount of phospho-Smad1 in BMP-2-activated C2C12 cells, but it did not alter Smad1 mRNA abundance as measured by real-time PCR; addition of a functional cell-permeable NEMO-binding domain (NBD) peptide antagonized NF-κB activity and ameliorated TNF-α inhibition of osteoblast differentiation. Taken together, our study reveals for the first time that NF-κB activation inhibits osteoblast differentiation by attenuating Smad1 activity and application of NBD peptide ameliorates this inhibitory effect. This could lead to new therapeutic drugs that circumvent the inflammatory inhibition of osteogenesis for treatment of traumatic open fractures with infection, rheumatoid arthritis and other bone loss disorders. [Copyright &y& Elsevier]

Abstract: It has been reported that the 6CPCP9 motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the 6CPCP9 motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the 6CPCP9 tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the 6CPCP9 motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the 6CPCP9 motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1–13) may also influence the peptide conformation and contribute to the protein’s bioactivity. [Copyright &y& Elsevier]