The type II collagen N-propeptide, PIIBNP, inhibits cell survival and bone resorption of osteoclasts via integrin-mediated signaling

Objective: Type IIB procollagen is characteristic of cartilage, comprising 50% of the extracellular matrix. The NH2-propeptide of type IIB collagen, PIIBNP, can kill tumor cells via binding to integrins alpha(V)beta(3) and alpha(V)beta(5). As osteoclasts rely on alpha(V)beta(3) integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function. Methods: We undertook in vitro and in vivo experiments to evaluate both osteoblast and osteoclast functions in the presence of recombinant PIIBNP. Adhesion of osteoclasts to PIIBNP was analyzed by staining of attached cells with crystal violet. PIIBNP-induced cell death was evaluated by counting Trypan Blue stained cells. The mechanism of cell death was evaluated by DNA fragmentation, TUNEL staining and western blotting to detect cleaved caspases. To determine the role of alpha(V)beta(3) integrin, osteoclasts were pretreated with alpha(V) or beta(3) integrin specific siRNA before the treatment with PIIBNP. To explore PIIBNP function in vivo, a lipopolysaccharide-induced mouse calvaria lysis model was employed. Results: Osteoclasts adhered to PIIBNP via an RGD-mediated mechanism. When osteoclasts were plated on extracellular matrix proteins, PIIBNP induced apoptosis of osteoclasts via caspase 3/8 activation. Osteoblasts and macrophages were not killed. Reduction of alpha(V) or beta(3) integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption. Conclusion: We conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the alpha(V)beta(3) integrins. Because of this property and the cell specificity, we propose that PIIBNP may play a role in vivo in protecting cartilage from osteoclast invasion and also could be a new therapeutic strategy for decreasing bone loss. (C) 2011 Elsevier Inc. All rights reserved.