The role of p105 protein in NFκB activation in ANA-1 murine macrophages following stimulation with titanium particles

Alexander Soloviev, Edward M. Schwarz, Dmitry V. Kuprash, Sergei A. Nedospasov, J. Edward Puzas, Randy N. Rosier, Regis J. O'Keefe

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Macrophage activation by particulate debris from orthopaedic implants triggers an inflammatory response that ultimately leads to periprosthetic bone resorption and implant failure. TNFα has been identified as a critical cytokine involved in the response to debris particles but the mechanisms involved in activation of TNFα synthesis are unclear. The current study demonstrates rapid induction of TNFα following stimulation with titanium particles in the murine macrophage cell line, ANA-1. Electrophoretic mobility shift assays demonstrated NFκB DNA binding activity within 15 min of exposure to titanium particles, and experiments with an NFκB luciferase promoter confirmed the induction of NFκB mediated transcription by titanium particles. Furthermore, titanium particles induced a 2-fold induction in TNFα promoter activity, and mutation of the κB2a site, one of the four NFκB-binding sites in the TNFα promoter, resulted in decreased activation. Since NFκB is a critical regulator of inflammation and is involved in activation of the TNFα promoter, additional experiments were performed to determine the mechanism of NFκB activation by particles. NFκB activation was found to be dependent upon proteasome activity, since administration of MG 132, a proteasome inhibitor, blocked NFκB activation. However, IκBα is only slightly decreased following Ti treatment, in contrast to marked degradation following stimulation with LPS. Recently, another proteasome-dependent pathway of NFκB activation has been described involving degradation of p105, a precursor of p50 that binds to p65. p105 degradation occurred following titanium stimulation, suggesting that this recently described mechanism for NFκB activation is operant in ANA-1 cells following exposure to titanium particles. These findings demonstrate that activation of the NFκB signaling pathway is rapidly induced by titanium particles in ANA-1 cells and is associated with p105 degradation. TNFα induction appears to be mediated, at least in part, through NFκB binding to the κB2a site of the TNFα promoter.

Original languageEnglish
Pages (from-to)714-722
Number of pages9
JournalJournal of Orthopaedic Research
Volume20
Issue number4
DOIs
StatePublished - 2002

Keywords

  • NFκB
  • Osteolysis
  • Particles
  • TNFα
  • Wear debris

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