title: Fibroblasts express RANKL and support osteoclastogenesis in a COX-2-dependent manner after stimulation with titanium particles.
abstract: Synovial fibroblasts are possible mediators of osteolysis. Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Fibroblasts pretreated with titanium or PGE2 stimulated osteoclast formation, showing the functional importance of RANKL induction. Synovial fibroblasts and their activation pathways are potential targets to prevent osteolysis.
  
  
  BACKGROUND
  Bone loss adjacent to the implant is a major cause of joint arthroplasty failure. Although the cellular and molecular response to microscopic wear debris particles is recognized as causative, little is known concerning role of synovial fibroblasts in these events.
  
  
  METHODS
  Murine embryonic fibroblasts and knee synovial fibroblasts in culture stimulated with titanium particles were examined by FACS, real time RT-PCR, Northern blot, and Western blot for expressions of vascular cell adhesion molecule (VCAM)1, RANKL, cyclooxygenase (COX)-1, and COX-2, and the four prostaglandin E2 (PGE2) receptor isoforms. Experiments were performed in the presence and absence of COX inhibitors, protein kinase A (PKA) and protein kinase C (PKC) inhibitors, and various EP receptor agonists. Osteoclast formation was examined in co-cultures of pretreated glutaraldehyde-fixed fibroblasts and primary murine spleen cells treated with macrophage-colony stimulating factor (M-CSF) for 7-days.
  
  
  RESULTS
  TNF-alpha stimulated VCAM1 expression, consistent with a synovial fibroblast phenotype. Titanium particles stimulated RANKL gene and protein expressions in fibroblasts in a dose-dependent manner. Gene expression was increased 5-fold by 4 h, and protein levels reached a maximum after 48 h. Within 1 h, titanium particles also induced COX-2 mRNA and protein levels, whereas both indomethacin and celecoxib blocked the stimulation of RANKL, suggesting a COX-2-mediated event. Furthermore, PGE2 induced RANKL gene and protein expression and rescued RANKL expression in titanium-treated cultures containing COX-2 inhibitors. Fibroblast cultures pretreated with either PGE2 or titanium particles enhanced osteoclast formation, indicating the functional importance of RANKL induction. EP4 was the most abundant PGE2 receptor isoform, EP1 and EP2 were expressed at low levels, and EP3 was absent. The EP1 selective agonist iloprost and the EP2 selective agonist butaprost minimally stimulated RANKL. In contrast, the EP2 and EP4 agonist misoprostol induced RANKL to a magnitude similar to PGE2. Finally, PKA antagonism strongly repressed RANKL stimulation by PGE2.
  
  
  CONCLUSIONS
  Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Thus, the synovial fibroblast is important mediator of osteolysis and target for therapeutic strategies.
authors.affiliation: Center for Musculoskeletal Research University of Rochester, School of Medicine and Dentistry, Rochester, New York, USA.
authors.forename: Xiaochao
authors.initials: X
authors.lastname: Wei
authors.forename: Xinping
authors.initials: X
authors.lastname: Zhang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zuscik
authors.forename: M Hicham
authors.initials: MH
authors.lastname: Drissi
authors.forename: Edward M
authors.initials: EM
authors.lastname: Schwarz
authors.forename: Regis J
authors.initials: RJ
authors.lastname: O'Keefe
mesh_terms: D000818:Animals; D001862:Bone Resorption; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D015870:Gene Expression; D017746:Joint Capsule / Q000166:cytology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000378:metabolism; D058309:Receptors, Prostaglandin E, EP4 Subtype; D012886:Skull / Q000166:cytology; D014025:Titanium / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008562:Membrane Glycoproteins; C545651:Ptger4 protein, mouse; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D014025:Titanium; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone