Selective Knockdown of the Long Variant of Cellular FLICE Inhibitory Protein Augments Death Receptor-mediated Caspase-8 Activation and Apoptosis

Death receptors trigger apoptosis by activating the apical cysteine proteases caspase-8 and -10 within a death-inducing signaling complex (DISC). c-FLIP (cellular FLICE inhibitory protein) is an enzymatically inactive relative of caspase-8 and -10 that binds to the DISC. Two major c-FLIP variants result from alternative mRNA splicing: a short, 26-kDa protein (c-FLIPS) and a long, 55-kDa form (c-FLIPL). The role of c-FLIPS as an inhibitor of death receptor-mediated apoptosis is well established; however, the function of c-FLIPL remains controversial. Although overexpression of transfected c-FLIPL inhibits apoptosis, ectopic expression at lower levels supports caspase-8 activation and cell death. Simultaneous ablation of both c-FLIP variants augments death receptor-mediated apoptosis, but the impact of selective depletion of c-FLIPL on caspase-8 activation and subsequent apoptosis is not well defined. To investigate this, we developed small interfering RNAs that specifically knock down expression of c-FLIPL in several cancer cell lines and studied their effect on apoptosis initiation by Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand). Knockdown of c-FLIPL augmented DISC recruitment, activation, processing, and release of caspase-8, thereby enhancing effector-caspase stimulation and apoptosis. Thus, endogenous c-FLIPL functions primarily as an inhibitor of death receptor-mediated apoptosis.