title: Inhibition of the PI3K-Akt signaling pathway reduces tumor necrosis factor-alpha production in response to titanium particles in vitro.
abstract: BACKGROUND
  Wear debris contributes to implant loosening after total joint arthroplasty, and few advances have been made in our ability to inhibit the biological response to wear particles. Bacterial endotoxins augment the effects of wear particles in vitro and in vivo. The cytokine, tumor necrosis factor-alpha (TNF-alpha), is produced by macrophages in response to bacterial endotoxins and wear particles, and it increases osteoclast activity resulting in bone resorption and implant loosening. The phosphoinositol-3-kinase (PI3K)-Akt intracellular signal transduction pathway contributes to cytokine production in response to soluble endotoxin. We investigated the role of the PI3K-Akt pathway in the production of TNF-alpha in response to wear particles with adherent endotoxin and so-called endotoxin-free wear particles.
  
  
  METHODS
  Cultured RAW264.7 murine macrophages were incubated with titanium particles with adherent endotoxin or with endotoxin-free titanium particles in the presence and absence of specific inhibitors of PI3K (LY294002) or Akt (SH-5). Akt activation was assessed with use of Western blot. TNF-alpha production was measured with use of enzyme-linked immunosorbent assay. Cytotoxicity was determined by measuring lactic dehydrogenase release.
  
  
  RESULTS
  Titanium particles with adherent endotoxin increased Akt activation, whereas endotoxin-free titanium particles did not. The PI3K inhibitor reduced TNF-alpha production by 70% in response to titanium with adherent endotoxin without increasing cytotoxicity. Similarly, the Akt inhibitor reduced TNF-alpha production by 83% in response to titanium particles with adherent endotoxin without increasing cytotoxicity. High concentrations of endotoxin-free titanium particles resulted in a small delayed increase in TNF-alpha production that was completely blocked by the PI3K inhibitor.
  
  
  CONCLUSIONS
  Inhibition of the PI3K-Akt pathway reduces macrophage TNF-alpha production in response to titanium particles with adherent endotoxin and endotoxin-free particles in vitro.
authors.affiliation: Department of Orthopaedics, Case Western Reserve University/University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106, USA. mvsmith23@gmail.com
authors.forename: Matthew V
authors.initials: MV
authors.lastname: Smith
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lee
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Islam
authors.forename: Jacqueline L
authors.initials: JL
authors.lastname: Rohrer
authors.forename: Victor M
authors.initials: VM
authors.lastname: Goldberg
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Beidelschies
authors.forename: Edward M
authors.initials: EM
authors.lastname: Greenfield
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D019736:Prostheses and Implants; D011475:Prosthesis Failure; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014025:Titanium / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D002867:Chromones; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D014025:Titanium; D014409:Tumor Necrosis Factor-alpha; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one