TWEAK Induces Apoptosis through a Death-signaling Complex Comprising Receptor-interacting Protein 1 (RIP1), Fas-associated Death Domain (FADD), and Caspase-8

Avi Ashkenazi,Aminah Ikner

doi:10.1074/jbc.m110.203745

Abstract

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.

Highlights

Two key apoptosis signaling pathways have been identified to date (24 –26)
6C), attenuated apoptosis stimulation (D), and decreased tumor necrosis factor (TNF)␣ production. These results suggest that TWEAK induces apoptosis by driving TNF␣-TNFR1-mediated engagement of receptor-interacting protein 1 (RIP1) to recruit FADD and caspase-8 in a manner that is negatively controlled by cellular inhibitor of apoptosis protein 1 (cIAP1) and positively modulated by CYLD
To further elucidate the mechanisms underlying the cytotoxic action of TWEAK, we chose as our main model a cell line that is sensitive to TWEAK as a single agent, without requirement for exogenous sensitizing factors such as IFN␥ or cycloheximide, namely human squamous cell carcinoma HSC3 cells [42, 43]

Summary

To whom correspondence should be addressed

The cell-extrinsic pathway is activated upon binding of proapoptotic ligands such as Fas/CD95L (TNFSF6) or Apo2L/TRAIL (TNFSF10) to their cognate death domaincontaining receptors on the surface of target cells This leads to receptor clustering and recruitment FADD and caspase-8 to form the death-inducing signaling complex. TRADD subsequently assembles a signaling complex (complex I) at the plasma membrane, which includes RIP1, TRAF2 or 5, and cellular inhibitor of apoptosis proteins cIAP1 and 2 This complex can activate downstream pathways, including the NF-␬B, JNK, and p38 MAPK cascades. We demonstrate that TWEAK stimulates apoptosis via a TNF␣-TNFR1-dependent complex containing RIP1, the adaptor FADD, and the apoptosis-initiating protease caspase-8. We show that TWEAK relies on both the extrinsic and intrinsic apoptosis pathways to trigger cell death

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION