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

ABSTRACT: Type IIB procollagen is characteristic of cartilage, comprising 50% of the extracellular matrix. The NH(2)-propeptide of type IIB collagen, PIIBNP, can kill tumor cells via binding to integrins ?(V)?(3) and ?(V)?(5). As osteoclasts rely on ?(V)?(3) integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function.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 ?(V)?(3) integrin, osteoclasts were pretreated with ?(V) or ?(3) integrin specific siRNA before the treatment with PIIBNP. To explore PIIBNP function in vivo, a lipopolysaccharide-induced mouse calvaria lysis model was employed.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 ?(V) or ?(3) integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption.We conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the ?(V)?(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.

SUBMITTER: Hayashi S

PROVIDER: S-EPMC3166963 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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

Hayashi Shinya S Wang Zhepeng Z Bryan Jennifer J Kobayashi Chikashi C Faccio Roberta R Sandell Linda J LJ

Bone 20110625 4

<h4>Objective</h4>Type IIB procollagen is characteristic of cartilage, comprising 50% of the extracellular matrix. The NH(2)-propeptide of type IIB collagen, PIIBNP, can kill tumor cells via binding to integrins α(V)β(3) and α(V)β(5). As osteoclasts rely on α(V)β(3) integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function.<h4>Methods</h4>We undertook in vitro and in vivo experiments to evaluate both osteoblast and osteoclast functions in the ...[more]

PMID: 21708300

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Project description:BACKGROUND:Osteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. The osteoclast resorption processes required to degrade subchondral bone and cartilage-the remodeling of which is important in the osteoarthritic disease process-have not been previously described, although cathepsin K has been indicated to participate. In this study we profile osteoclast-mediated degradation of bovine knee joint compartments in a novel in vitro model using biomarkers of extracellular matrix (ECM) degradation to assess the potential of osteoclast-derived resorption processes to degrade different knee joint compartments. METHODS:Mature human osteoclasts were cultured on ECMs isolated from bovine knees-articular cartilage, cortical bone, and osteochondral junction ECM (a subchondral bone-calcified cartilage mixture)-in the presence of inhibitors: the cystein protease inhibitor E-64, the matrix metalloproteinase (MMP) inhibitor GM6001, or the vacuolar-type H+-ATPase (V-ATPase) inhibitor diphyllin. Biomarkers of bone (calcium and C-terminal type I collagen (CTX-I)) and cartilage (C2M) degradation were measured in the culture supernatants. Cultures without osteoclasts were used as background samples. Background-subtracted biomarker levels were normalized to the vehicle condition and were analyzed using analysis of variance with Tukey or Dunnett's T3 post hoc test, as applicable. RESULTS:Osteochondral CTX-I release was inhibited by E-64 (19% of vehicle, p = 0.0008), GM6001 (51% of vehicle, p = 0.013), and E-64/GM6001 combined (4% of vehicle, p = 0.0007)-similarly to bone CTX-I release. Diphyllin also inhibited osteochondral CTX-I release (48% of vehicle, p = 0.014), albeit less than on bone (4% of vehicle, p <?0.0001). Osteochondral C2M release was only inhibited by E-64 (49% of vehicle, p = 0.07) and GM6001 (14% of vehicle, p = 0.006), with complete abrogation when combined (0% of vehicle, p = 0.004). Cartilage C2M release was non-significantly inhibited by E-64 (69% of vehicle, p = 0.98) and was completely abrogated by GM6001 (0% of vehicle, p = 0.16). CONCLUSIONS:Our study supports that osteoclasts can resorb non-calcified and calcified cartilage independently of acidification. We demonstrated both MMP-mediated and cysteine protease-mediated resorption of calcified cartilage. Osteoclast functionality was highly dependent on the resorbed substrate, as different ECMs required different osteoclast processes for degradation. Our novel culture system has potential to facilitate drug and biomarker development aimed at rheumatic diseases, e.g. osteoarthritis, where pathological osteoclast processes in specific joint compartments may contribute to the disease process.

Dataset Information

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

Publications

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

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