A TNF receptor loop peptide mimic blocks RANK ligand–induced signaling, bone resorption, and bone loss
Kazuhiro Aoki, Hiroaki Saito, Cecile Itzstein, Masaji Ishiguro, Tatsuya Shibata, Roland Blanque, Anower Hussain Mian, Mariko Takahashi, Yoshifumi Suzuki, Masako Yoshimatsu, Akira Yamaguchi, Pierre Deprez, Patrick Mollat, Ramachandran Murali, Keiichi Ohya, William C. Horne, Roland Baron
Kazuhiro Aoki, Hiroaki Saito, Cecile Itzstein, Masaji Ishiguro, Tatsuya Shibata, Roland Blanque, Anower Hussain Mian, Mariko Takahashi, Yoshifumi Suzuki, Masako Yoshimatsu, Akira Yamaguchi, Pierre Deprez, Patrick Mollat, Ramachandran Murali, Keiichi Ohya, William C. Horne, Roland Baron
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Research Article Bone biology

A TNF receptor loop peptide mimic blocks RANK ligand–induced signaling, bone resorption, and bone loss

Abstract

Activating receptor activator of NF-κB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-α–induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF–induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-α promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF.

Authors

Kazuhiro Aoki, Hiroaki Saito, Cecile Itzstein, Masaji Ishiguro, Tatsuya Shibata, Roland Blanque, Anower Hussain Mian, Mariko Takahashi, Yoshifumi Suzuki, Masako Yoshimatsu, Akira Yamaguchi, Pierre Deprez, Patrick Mollat, Ramachandran Murali, Keiichi Ohya, William C. Horne, Roland Baron

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Figure 4

WP9QY inhibits sRANKL-induced osteoclastogenesis and bone resorption in vitro.

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WP9QY inhibits sRANKL-induced osteoclastogenesis and bone resorption in ...
(A) Murine bone marrow cells from CD-1 mice were cultured in the presence of M-CSF (25 ng/ml) and sRANKL (10 ng/ml). Increasing amounts of WP9QY were included as indicated. After 4 days, the cells were fixed and stained for TRAP. Ctr, control. (B) Murine bone marrow cells from CD-1 mice were cultured in the presence of M-CSF (25 ng/ml) and increasing concentrations of sRANKL (10, 30, or 100 ng/ml, as indicated). Increasing amounts of WP9QY were included as indicated. After 4 days, the cells were fixed and stained for TRAP, and the TRAP+ cells were counted. Increasing concentrations of WP9QY progressively inhibited the sRANKL-induced osteoclast formation at all concentrations of sRANKL. Progressively more WP9QY was required to achieve the same degree of inhibition as the concentration of sRANKL increased. The values are the mean ± SD of triplicate determinations and are representative of 3 independent experiments. (C) Murine bone marrow cells from CD-1 mice were cultured for 4 days in the presence of M-CSF (25 ng/ml) and sRANKL (10 ng/ml) with increasing amounts of WP9QY (filled circles) or the control Y6N peptide (open circles) or 30 ng/ml OPG. The cells were fixed and stained for TRAP and the TRAP+ multinucleated cells counted.