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

Abstract

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 αVβ3 and αVβ5. As osteoclasts rely on αVβ3 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 function 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 cell 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. 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 αV or β3 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 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.

Novel functions for Type II procollagen

“…PIIBNP did not induce cell death of primary bone marrow macrophages nor primary osteoblasts. Interestingly, the mechanism of cell death in osteoclasts is different from tumor cells, in that osteoclasts die via apoptosis rather than programmed necrosis (20). Ultimately the ability of PIIBNP to inhibit bone resorption by osteoclasts must be shown in vivo.…”

“…Adhesion assays performed with primary cells and cell lines of osteoclasts and osteoblasts yielded identical results. For example, undifferentiatied bone marrow macrophages, cells that do not express avb3 and avb5 integrins, did not adhere to PIIBNP, nor did osteoblasts, whereas osteoclasts successfully adhered to PIIBNP (20). In order to test the effect of PIIBNP on cell survival, osteoclasts and osteoblasts were cultured on various extracellular matrix molecules that have different integrin binding properties: type I collagen, fibronectin and vitronection-coated plates.…”

“…In fact, the function of these integrins is considered vital to the function of osteoclasts (19). Therefore, studies were undertaken to investigate the ability of PIIBNP to kill osteoclasts and thus potentially function in vivo to inhibit bone resorption (20). To investigate whether the RGDRGD sequence in PIIBNP was used to adhere to osteoclasts, we performed adhesion assays to recombinant proteins PIIBNP and mutPIIBNP.…”

“…In contrast, the calvaria of LPS + PIIBNP-treated animals were protected from bone erosion: that is, the resorption area, indicated by the red osteoclast-specific TRAP staining, was greatly reduced in the PIIBNP-treated animals ( Figure 3B). In order to quantify the changes in bone resorption, the number of osteoclasts per millimeter of bone and the percentage of eroded bone surface covered by osteoclasts were counted (20). The PIIBNP reduced osteoclasts number by 60%.…”

Alternative splicing of type II procollagen: IIB or not IIB?

“…There have been interesting reports that a recombinant IIB NH 2 -propeptide protein (encoded by human exons 3-8) has anti-angiogenic activity (76) and can also induce cell death of tumor cells and osteoclasts via an integrin-mediated mechanism (77, 78). Normal, healthy articular cartilage is avascular and resistant to tumor invasion.…”