Mol Cell Biol 21(16): 5299-5305

NF-κB Signals Induce the Expression of c-FLIP

Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center,^{and Apotech Biochemicals, Biopôle,}^{CH-1066 Epalinges, Switzerland}

^{Corresponding author. Mailing address: Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. Phone: 41 21 692 5738. Fax: 41 21 692 5705. E-mail:}hc.linu.bi@ppohcst.gruj.

Received 2001 Feb 1; Revisions requested 2001 Mar 16; Accepted 2001 May 24.

Abstract

Activation of the transcription factor NF-κB is a major effector of the inducible resistance to death receptor-mediated apoptosis. Previous evidence indicates that the combined transcriptional activation of TRAF-1, TRAF-2, IAP-1, and IAP-2 is required to suppress cell death by tumor necrosis factor (TNF). Here we show that NF-κB activation upregulates the caspase 8 inhibitor FLIP, resulting in increased resistance to Fas ligand (FasL) or TNF. Restoration of either the full-length 55-kDa long form of FLIP or an alternatively spliced short form of FLIP in NF-κB null cells inhibits TNF- and FasL-induced cell death efficiently, whereas the expression of IAP or TRAF family members only partially rescues cells from death. Resistance to either FasL- or TNF-induced apoptosis is overcome when cells are incubated in the presence of the protein synthesis inhibitor cycloheximide. This treatment leads to the rapid downregulation of FLIP but not to that of TRAF2. Our findings suggest that FLIP is an important mediator of NF-κB-controlled antiapoptotic signals.

Abstract

Members of the tumor necrosis factor (TNF) receptor family and their corresponding ligands are critical regulators of apoptosis and various other cellular processes. Some of the receptors (Fas, TNF-R1, TRAIL-R1, TRAIL-R2, TRAMP/DR3, DR6, and EDA-R) contain a cytoplasmic region, called the death domain (DD), which is essential for cell death signaling (18, 22). Signals emanating from Fas and TNF-R1 have been intensively studied (14). Upon receptor activation, the DD of Fas undergoes direct homotypic interaction with a DD in the adapter protein FADD, while FADD recruitment is indirect (via TRADD) in the case of TNF-R1 (4). The death effector domain (DED) at the amino terminus of FADD then recruits pro-caspase 8 via homotypic interaction with its two DEDs. The high local concentration of caspase 8 zymogens facilitates self-processing and assembly of the mature enzyme. Activated caspase 8 initiates apoptosis by subsequent cleavage of downstream caspases (caspase-3, -6, and -7).

Death induced by death receptors is tightly regulated by genes that are activated by the transcription factor NF-κB (25). Modulation of the response in favor of NF-κB protects cells from apoptosis; failure to do so results in increased cell death. At least six NF-κB-responsive genes are involved in this survival amplification loop (26), i.e., those that encode IAP-1 and IAP-2, which block caspase activity (7); that which encodes the Bcl-2 family member A1 (25); and those that encode TRAF-1, TRAF-2 (1), and A20 (21), which are themselves implicated in the NF-κB signaling pathway. However, overexpression of all of these genes affords, at best, partial protection, in particular from death triggered by TNF (25). The only known potent inhibitor of death receptor signals is c-FLIP. Two c-FLIPs have been characterized (23, 24). The full-length 55-kDa-long form of FLIP (FLIP_L) exhibits overall structural homology to caspase 8, containing two DEDs that interact with FADD and an inactive caspase-like domain. An alternatively spliced short form of FLIP (FLIP_S) contains only the two DEDs and displays reduced antiapoptotic capacity.

We undertook a series of experiments to investigate whether c-FLIP is implicated in the antiapoptotic NF-κB response. Here we provide evidence that FLIP expression is upregulated upon the stimulation of several signaling pathways that are known to trigger activation of the transcription factor NF-κB. Moreover, we found that cells that were rendered highly sensitive to death ligand-induced apoptosis by blocking NF-κB activation could be rescued by expressing FLIP. FLIP may therefore play a key role in the NF-κB-mediated control of death signals.

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