The W9 peptide directly stimulates osteoblast differentiation via RANKL signaling

Abstract Objective A RANKL-binding peptide, WP9QY (W9), is known to inhibit mouse osteoclastogenesis by stimulating the production of autocrine factors such as bone morphogenetic proteins (BMPs) to induce osteoblast differentiation. In the present study, we investigated whether osteoblastic differentiation is mediated by RANKL signaling. Methods The effect of W9 on the differentiation of osteoclasts and osteoblasts was examined in mouse bone-marrow cultures, and in a mouse co-culture system consisting of primary osteoblasts derived from RANKL-deficient or wild-type (WT) newborn mouse calvariae, with WT-derived bone marrow mononuclear cells. Results The addition of the W9 peptide to the WT mouse bone-marrow culture simultaneously inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation, and stimulated alkaline phosphatase (ALP)-positive osteoblastic calcified nodule formation. RANKL-deficient osteoblasts exhibited weak ALP activity compared to WT osteoblasts. W9 treatment strongly inhibited TRAP-positive osteoclast formation, and stimulated ALP-positive osteoblast differentiation in co-cultures of WT-derived osteoblasts and bone-marrow cells, in the presence of bone-resorbing factors. In contrast, W9 exerted only a weak effect on ALP-positive osteoblast differentiation in co-cultures with RANKL-deficient osteoblasts, even in the presence of the W9 peptide, parathyroid hormone, and/or BMP-2. Conclusions The W9 peptide inhibited RANKL-mediated osteoclast formation in osteoblasts. It also directly stimulated osteoblast differentiation, both via RANKL signaling-mediated autocrine factors, and alternative mechanisms.