Studies On NF-κB Signal Pathway And Mechanism Of Prion Peptide PrP106-126 On Mouse Macrophages

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal, neurodegenerative disorders that affect both animals and humans. These diseases include scrapie in goats and sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans. The"protein-only"hypothesis dictates that the central event in TSE diseases is the conversion of cellular prion protein (PrPC) into a pathological isoform, PrPSc. A number of aspects of the pathogenesis of TSEs remain to be elucidated. The cellular and molecular aspects of the neuropathology in prions suggest the possibility that the proinflammatory cytokines and iNOS could act as pathogenic mediators in this neurodegenerative disease. To investigate this possibility, we examined the effect of PrP106-126 induction on mRNA expression of iNOS and proinflammatory cytokines (IL-1β, TNF-α, IL-6), and the activation of nuclear factor-kappa B (NF-κB) in Ana-1 cells. We also analyzed the effect of the NF-κB inhibition on PrP-induced iNOS and cytokine gene expression. These results indicate that PrP106-126 stimulation of macrophages leads to the activation of transcription factor NF-κB, which in turn stimulates iNOS and inflammatory cytokine gene expression. Our study contributes to the understanding of the molecular mechanisms lying behind the interaction between PrP and macrophages which would provide a basis for the development of effective therapy to prion diseases.1. To determine if PrP106-126 activates mouse macrophages and to ascertain a possible signaling mechanism whereby the prion peptide exerts its effects, western blot and immunofluorescence analysis were performed. We demonstrated that stimulation for 24h with PrP106-126 induced NF-κB p65 subunit translocation and aggregation in nuclei of Ana-1 cells;that NF-κB activation and translocation decreased after incubation with NF-κB inhibitor for 1h. These results indicate that PrP106-126 induced NF-κB activation and nuclear translocation in Ana-1 cells.2. To investigate the role of NF-кB activation in relation to PrP106-126-induced iNOS and cytokine gene transcription, the mRNA levels were first determined by quantitative RT-PCR in PrP106-126-treated cells. We demonstrated that PrP peptide treatment up-regulated mRNA levels of both iNOS and proinflammatory cytokine IL-1β, TNF-α, IL-6 in Ana-1 cells. The mRNA levels of TNF-αand iNOS were significantly higher in treated cells compared to untreated control (P<0.05). The ScrPrP did not increase cytokines or iNOS mRNA levels.3. To confirm that the mRNA expression of iNOS and cytokines was induced via the NF-κB signaling pathway, Ana-1 cells were incubated with 12μM NF-κB inhibitor for 1h, and then treated with 25μM PrP106-126 for 24h. The quantitative analysis of the cytokines and iNOS mRNA levels was then performed. We demonstrated that a significant decrease of IL-1β, TNF-α, IL-6, and iNOS mRNAs in NF-κB inhibitor-treated cells compared to PrP-treated cells(P<0.05). The result indicate that NF-κB activation is involved in iNOS and inflammatory cytokine gene expression in PrP-induced mouse macrophage cells, and provide basic data for further elucidating the possible role of transcription factors in the interaction between PrP and immune cells.