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

Macrophage activation by particulate debris from orthopaedic implants triggers an inflammatory response that ultimately leads to periprosthetic bone resorption and implant failure. TNFalpha has been identified as a critical cytokine involved in the response to debris particles but the mechanisms involved in activation of TNFalpha synthesis are unclear. The current study demonstrates rapid induction or TNFalpha following stimulation with titanium particles in the murine macrophage cell line. ANA-1. Electrophoretic mobility shift assays demonstrated NFkappaB DNA binding activity within 15 min of exposure to titanium particles, and experiments with an NFkappaB luciferase promoter confirmed the induction of NFkappaB mediated transcription by titanium particles. Furthermore, titanium particles induced a 2-fold induction in TNFalpha promoter activity, and mutation of the kappaB2a site, one of the four NFkappaB-binding sites in the TNFalpha promoter, resulted in decreased activation. Since NFtB is a critical regulator of inflammation and is involved in activation of the TNFalpha promoter, additional experiments were performed to determine the mechanism of NFkappaB activation by particles. NFKB activation was found to be dependent upon proteasome activity, since administration of MG 132, a proteasome inhibitor, blocked NFkappaB activation. However, IkappaBalpha is only slightly decreased following Ti treatment, in contrast to marked degradation following stimulation with LPS. Recently, another proteasome-dependent pathway of NFkappaB 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 NFKB activation is operant in ANA-1 cells following exposure to titanium particles. These findings demonstrate that activation of the NFkappaB signaling pathway is rapidly induced by titanium particles in ANA-1 cells and is associated with p105 degradation. TNFalpha induction appears to be mediated, at least in part, through NFkappaB binding to the kappaB2a site of the TNFalpha promoter.