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

ObjectiveType 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 αVβ3 and αVβ5. As osteoclasts rely on αVβ3 integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function.MethodsWe 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 αVβ3 integrin, osteoclasts were pretreated with αV or β3 integrin specific siRNA before the treatment with PIIBNP. To explore PIIBNP function in vivo, a lipopolysaccharide-induced mouse calvaria lysis model was employed.ResultsOsteoclasts 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 αV or β3 integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption.ConclusionWe conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the αVβ3 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.

► PIIBNP adheres to osteoclasts via the RGD domain. ► PIIBNP induced cell death of osteoclasts, but not macrophages or osteoblasts. ► PIIBNP induced apoptosis of osteoclasts via αV and β3 integrins. ► PIIBNP inhibited bone resorption in vivo and decreased osteoclast number.