The Role of the Nfkb 1 Precursor Protein P105 in Murine Macrophages Activation after Stimulation with Ti Wear Debris

Introduction: Osteolysis following joint arthroplasty is believed to be mediated by implant-derived wear debris. Although it has been established that particles stimulate cytokine production, the molecular mechanisms involved in this process are unclear. Recently, TNFα has emerged as a central mediator of aseptic loosening, thus understanding the pathways leading to TNFα induction are very important for clinical translation. One of the well-known mediators of TNFα induction is the transcription factor NFκB. In the current study, we examined the mechanism involved in NFκB stimulation by titanium particles. Our findings suggest that the processing of the NFkB1 precursor protein p105 is more critically involved than the activation of the classic pathway mediated by IκBα degradation. Methods: The ANA-1 murine macrophage cell line was kindly provided by Dr.G.Cox. (NCI, Frederick, MD). Cells were grown on DMEM+10% FBS (GIBCO). Ti particles (Alpha AESAR, MA) were used at a concentration 5x10 particles/ml. Conditioned medium was collected after 18h of treatment (if not specified) and analyzed by ELISA for cytokine concentration. mRNA was isolated after specified times with QIAGEN kit and 10 μg was analyzed by Northern blotting. Cytoplasmic and nuclear cell extracts were analyzed for the presence of IκBα protein and NFκB binding activity respectively. The pNFκB-luc construct was obtained from Stratagen. The cRel, p65, p50 and mutated IkBα constructs were in pCMX-vector and p105 was in pcDNA3.1 Cells were transfected by the DEAE-dextran method. Results: Stimulation of murine macrophage ANA-1 cells with Ti particles resulted in stimulation of TNFα mRNA within 30 minutes and a dose and time-dependent release of TNFα (up to 20-fold) in culture. NFκB binding was rapidly induced (15 minutes) and peaked at one hour after stimulation with Ti particles, as shown by using gel shift assays. A supershift analysis of these samples displayed the presence of p50, p65 and cRel proteins in the NFκB DNA binding complexes. In contrast, gel mobility shift assays using AP-1, C/EBP, CREB, Ets, NFAT, and SRE binding oligonucleotides demonstrated no alteration. Collectively, these finding suggest that NFκB is an early mediator of the monocyte response to particles. To further analyze this NFκB activation, ANA-1 cells were transfected with the pNFκB-luc reporter construct and stimulated with Ti for 8 hr. This resulted in a 2-4 fold increase in luciferase activity compared with untreated controls. To examine transcriptional regulation, we co-transfected ANA-1 cells with pNFκB-luc reporter and pCMX-p50, -65, -cRel and – IκBα expression constructs. Consistent with the current paradigm, we found that p65 and cRel by themselves significantly (2-fold) increased luciferase activity while p50 had no effect and mutant IκBα completely blocked the basal level of luciferase activity. Stimulation of the co-transfected cells with Ti particles revealed similar results, but interestingly overexpression of p50 inhibited NFκB-luciferase activation in all samples. Since p50 homodimers have no transactivation potential, these latter findings suggest that Ti stimulation is dependent upon increasing the pool of p65 and cRel, and the subsequent heterodimer formation with p50. Pretreatment of ANA-1 cells with the specific proteosome inhibitor MG132 eliminated this inducible NFκB binding suggesting the requirement of a degradative process in NFκB induction. However, Western blotting for IκBα (the natural inhibitor of NFκB whose proteolysis is typically required for its nuclear translocation) did not reveal any significant changes in IκBα levels after Ti stimulation. In contrast, LPS and showed rapid (15 min) degradation of IκBα, followed by resynthesis and return to control levels by one hour. Furthermore, while cycloheximide blocked the resynthesis of ΙκΒ protein in the LPS samples, both control and Ti treated cultures had an identical slow rate of ΙκΒ catabolism in the presence of cycloheximide. In related experiments, ΙκΒα was immunoprecipitated (100% recovery) and assessed for tyrosine phosphorylation. Tyrosine phosphorylation of ΙκΒ results in activation of NFκB in the absence of ΙκΒ degradation. While a number of proteins are tyrosine phosphorylated, we did not observe any evidence of ΙκΒ phosphorylation following Ti treatment. Thus, NFκB activation by titanium particles appears to be independent both degradative and non-degradative ΙκΒ-mediated mechanisms. Recently, p105 proteosomal degradation has been shown to induce NFκB independent of ΙκΒ, through mechanism involving the release of sequestered p65. Western blot was performed using a polyclonal antibody that detects p105, p50, and other p105 catabolic products (fig.1). p105 levels declined precipitously within 15 minutes with a parallel increase in p105 degradation products. However, p50 levels remained constant, consistent with prior studies examining the pattern of p105 degradation.