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 3

WP9QY blocks RANKL-induced signaling.

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WP9QY blocks RANKL-induced signaling. (A) RAW 264.7 cells treated with s...
(A) RAW 264.7 cells treated with sRANKL with or without WP9QY were stained with anti-p65 antibody (green) and nuclear stain (blue). WP9QY prevented RANKL-induced nuclear accumulation of p65. (B) RAW 264.7 cells were treated with sRANKL with or without WP9QY (10 μM or 25 μM) or OPG. EMSA was performed using a consensus NF-κB oligonucleotide probe. WP9QY prevented the RANKL-induced activation of NF-κB. (C) Nonadherent bone marrow cells differentiated with M-CSF were treated for 48 hours with 100 ng/ml RANKL alone or with WP9QY (10 μM or 25 μM) or OPG. NFATc1 and c-Fos were detected by Western blotting. WP9QY inhibited RANKL-induced NFATc1 and c-Fos expression, albeit less effectively than OPG. (D) RAW 264.7 cells were treated with M-CSF (20 ng/ml for 5 minutes) or sRANKL, with or without 50 μM WP9QY as indicated. Phosphorylated and total Erk1/2 (upper panels) or phosphorylated and total glycogen synthase kinase 3α/β (GSK3α/β; lower panels) were assayed. WP9QY did not affect M-CSF–induced activation of Erk or Akt. (E) RAW 264.7 cells were treated with sRANKL with or without WP9QY (5–50 μM), the control Y6N peptide, or OPG as indicated. Erk, JNK, and Akt activation were monitored as described in Methods. Upper panels: phosphorylated and total Erk and phosphorylated and total c-Jun. The control peptide inhibited RANKL-induced activation of Erk1/2 and JNK significantly less than WP9QY. Lower panels: phosphorylated GSK3α/β. Protein loading was monitored by Ponceau S staining before blocking the membrane. WP9QY inhibited the RANKL-induced activation of Akt.