Abstract
Tumor Necrosis Factor α (TNFα) induces both the apoptotic pathway and anti-apoptotic factors. Incubation of human dermal fibroblasts with TAPF (TNF Apoptosis Protection Fraction) protects them from apoptosis induced by the subsequent addition of TNF and cycloheximide (CHX). TAPF does not protect against apoptosis induced by CHX in combination with either TRAIL (TNF related apoptosis inducing ligand) or an agonistic Fas antibody, or against apoptosis induced by the chemotherapeutic agent doxorubicin. Incubation with TAPF does not affect the quantity of TNF that binds to the cell. TAPF prevents TNF-induced cleavage of caspases 8, 9, 3 and 7 and the apoptotic substrate PARP (poly-ADP ribose polymerase), but has no effect when these molecules are induced by an agonistic Fas antibody. TAPF induces rapid phosphorylation of the NF-κB/p65 (nuclear factor-κB) transcription factor at serine 536 which is indicative of its activation. TAPF increases the expression of cFLIP (cellular FLICE-inhibitory protein) which is a potent inhibitor of apoptosis that acts by preventing the cleavage of caspase 8. This increase in cFLIP is coincident with protection from TNF-induced apoptosis. Decreasing cFLIP levels using shRNA (short hairpin RNA) decreases protection by TAPF. TAPF also induced the anti-apoptotic A20 protein. These data indicate that TAPF protects human dermal fibroblasts from TNF-induced apoptosis by induction of cFLIP and subsequent inhibition of caspase 8 cleavage.