title: 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 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.
  
  
  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 α(V)β(3) integrin, osteoclasts were pretreated with α(V) or β(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 α(V) or β(3) integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption.
  
  
  CONCLUSIONS
  We conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the α(V)β(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.
authors.affiliation: Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Shinya
authors.initials: S
authors.lastname: Hayashi
authors.forename: Zhepeng
authors.initials: Z
authors.lastname: Wang
authors.forename: Jennifer
authors.initials: J
authors.lastname: Bryan
authors.forename: Chikashi
authors.initials: C
authors.lastname: Kobayashi
authors.forename: Roberta
authors.initials: R
authors.lastname: Faccio
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Sandell
mesh_terms: D000818:Animals; D001862:Bone Resorption / Q000378:metabolism / Q000473:pathology*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000494:pharmacology*; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D002452:Cell Count; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D024042:Collagen Type I / Q000494:pharmacology; D024043:Collagen Type II / Q000737:chemistry / Q000494:pharmacology*; D005353:Fibronectins / Q000494:pharmacology; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000473:pathology; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry; D010006:Osteoblasts / Q000187:drug effects / Q000473:pathology; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D019096:Vitronectin / Q000494:pharmacology
chemical_list: D002135:Calcium-Binding Proteins; D024042:Collagen Type I; D024043:Collagen Type II; D005353:Fibronectins; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D034741:RNA, Small Interfering; D019096:Vitronectin; C040072:chondrocalcin; C047981:arginyl-glycyl-aspartic acid