Signalling pathways activated by TRAIL : implications for apoptosis
thesisposted on 15.12.2014, 10:42 authored by Nicholas Harper
TRAIL (TNF-Related Apoptosis-Inducing Ligand) and its receptors represent a relatively new and poorly characterised subset of the TNF family. The transcription factor NF-kappaB is an important cellular survival pathway and its potential activation by TRAIL was assessed. TRAIL activates NF-kappaB only in TRAIL resistant cells whereas activation was only observed in sensitive cells in the presence of a caspase inhibitor suggesting a caspase-sensitive component. NF-kappaB activation could regulate TRAIL cytotoxicity as its inhibition in resistant cells led to them becoming sensitive to TRAIL while activation in sensitive cells made these cells refractory to TRAIL. The native TRAIL DISC (Death-Inducing Signalling Complex) was characterised and found to contain FADD (Fas-Associated Death Domain Protein) and the inhibitor caspases-8 and -10. FADD- and caspase-8-deficient cells were refractory to TRAIL-induced apoptosis indicating an obligatory role for these components in TRAIL signalling. DISCs were formed irrespective of the TRAIL sensitivity of the cell. Consistent with TRAIL activating NF-kappaB, RIP (Receptor Interacting Protein), a protein required for TNF-mediated NF-kappaB activation, was present within the DISC. Analysis of the native TNF-R1 signalling complex revealed the presence of proteins required for NF-kappaB activation, RIP and TRAF2 (TNF-Associated Factor 2) however the apoptotic mediators, namely FADD and caspase-8 were absent despite being required for apoptosis induction. Finally, others have reported that protein kinase C (PKC) activation can protect cells from death receptor apoptosis. PMA (Phorbol Myristate Acetate)-mediated PKC activation protected HeLa cells from TRAIL-induced apoptosis. DISC analysis revealed that PKC activation modulated apoptosis by inhibiting FADD recruitment.