Date of Graduation
2005
Document Type
Dissertation/Thesis
Abstract
The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. Here, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by overexpression of dominant-negative mutants of PI3K (Δp85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis induced by FasL, indicating an unexpected pro-apoptotic role of PI3K/Akt in Fas signaling process. Electron spin resonance (ESR) studies also show that FasL treatment induces rapid generation of reactive oxygen species (ROS) and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox-sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas receptor expression is downregulated but FLICE-inhibitory protein (FLIP) expression is unaffected. We also investigate the role of TNF-α and NF-κB in FasL-mediated cell death in macrophages. Gene transfection studies using NF-κB-dependent reporter plasmid showed that FasL did indeed activate NF-κB promoter activity. Gel shift studies revealed that FasL mobilized the p50/p65 heterodimeric form of NF-κB. Inhibition of NF-κB by specific NF-κB inhibitor, caffeic acid phenylethyl ester, or by dominant expression of the NF-κB inhibitory subunit IκB caused an increase in FasL-induced apoptosis and a reduction in TNF-α expression. However, neutralization of TNF-α by specific anti-TNF-α antibody had no protective effective effect on FasL-induced apoptosis. These results indicate that FasL-mediated cell death in macrophages is regulated through NF-κB and is independent of TNF-α activation. The wild-type FLIPL and various N-terminal deletion plasmids were generated and used to study the role of FLIP in Fas-mediated NF-κB activation and apoptosis. NF-κB is activated by FLIPL and its deletion mutants except Δ7. Also, FLIPL and its deletion mutants Δ2-Δ6 (but not Δ7) inhibit FasL-induced apoptosis. We also tested whether our truncated mutants are capable of binding to TRAF2. We performed immunoprecipitation experiments and found that both DED1 and DED2 domains of FLIP are required for TRAF2 recruitment. These results are in good agreement with our apoptosis and NF-κB activation data.
Recommended Citation
Lu, Bin, "Roles of cellular FLICE-inhibitory protein (c-FLIP) and PI3K /Akt in Fas (CD95)-induced NF-κB activation and apoptosis through death effector domains." (2005). Graduate Theses, Dissertations, and Problem Reports. 9328.
https://researchrepository.wvu.edu/etd/9328