Oncogene (2000) 19, 4451 ± 4460 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Activation of the NF-kB pathway by Caspase 8 and its homologs Preet M Chaudhary*,1, Michael T Eby1, Alan Jasmin1, Arvind Kumar1, Li Liu1 and Leroy Hood2 1Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, Dallas, Texas, TX 75390-8593, USA; 2Department of Molecular Biotechnology, University of Washington, Seattle, Washington, WA 98195 USA Caspase 8 is the most proximal caspase in the caspase al., 1996). The prodomain of Caspase 8 consists of two cascade and has been known for its role in the mediation homologous DEDs and serves to keep Caspase 8 in an of cell death by various death receptors belonging to the inactive form (Chinnaiyan and Dixit, 1997). DEDs- TNFR family. We have discovered that Caspase 8 can containing prodomains are also found in two addi- activate the NF-kB pathway independent of its activity tional cellular proteins; Caspase 10 (Mch4, FLICE2), a as a pro-apoptotic protease. This property is localized to proteolytically active Caspase 8 homolog (Fernandes- its N-terminal prodomain, which contains two homo- Alnemri et al., 1996; Vincenz and Dixit, 1997), and logous death eector domains (DEDs). Caspase 10 and MRIT (Casper, c-FLIP, I-FLICE, FLAME, CASH, MRIT, two DEDs-containing homologs of Caspase 8, CLARP), a Caspase 8 homolog which is devoid of can similarly activate the NF-kB pathway. Dominant- protease activity (Goltsev et al., 1997; Han et al., 1997; negative mutants of the Caspase 8 prodomain can block Hu et al., 1997; Inohara et al., 1997; Irmler et al., 1997; NF-kB induced by Caspase 8, FADD and several death Shu et al., 1997; Srinivasula et al., 1997). receptors belonging to the TNFR family. Caspase 8 can In the present study present evidence that the DEDs interact with multiple proteins known to be involved in of Caspase 8 and its homologs are also involved in the the activation of the NF-kB pathway, including the activation of the NF-kB pathway. Our study suggests serine-threonine kinases RIP, NIK, IKK1 and IKK2. the existence of a functional and biochemical link Thus, DEDs-containing caspases and caspase homolog(s) between the caspase and the kinase cascades down- may have functions beyond their known role in the stream of the adaptor molecule TRADD and indicates mediation of cell death. Oncogene (2000) 19, 4451 ± that Caspase 8 and its homologs have roles beyond 4460. their activity as cell death proteases. Keywords: NF-kB; caspase; FADD; death eector domain; MRIT; cFLIP Results Caspase 8 activates NF-kB Introduction We began by testing the ability of Caspase 8 to activate Tumor Necrosis Factor Receptor 1 (TNFR1) is the an NF-kB-driven luciferase reporter construct in 293T best characterized death receptor of the TNFR family cells (Berberich et al., 1994). Expression of Caspase 8 in (Ashkenazi and Dixit, 1998; Baker and Reddy, 1996). these cells had no major toxicity and led to signi®cant On ligand induced aggregation, death domain of activation of the NF-kB/luciferase reporter construct in TNFR1 binds the homologous domain of TRADD, a a dose- and time-dependent manner (Figure 1a ± c). death domain-containing cytoplasmic adaptor protein Caspase 8 failed to activate luciferase reporter constructs (Hsu et al., 1995, 1996; Varfolomeev et al., 1996). driven by mutant NF-kB binding-sites, NF-IL6 or TRADD subsequently activates a kinase cascade, NFAT, which demonstrated the speci®city of the assay consisting of the NF-kB and JNK/SAPK pathways, (Figure 1a and data not shown). The NF-kB inducing via the recruitment of death domain-containing protein ability of Caspase 8 was not limited to the 293T cells RIP (Receptor Interacting Protein) and TRAF2, which since it activated NF-kB in the MCF7 and 293 EBNA lacks a death domain (TNF Receptor Associated cells as well (Figure 1d and data not shown). The ability Factor 1). TRADD also activates a caspase cascade of Caspase 8 to activate the NF-kB pathway was further via the recruitment of FADD/MORT1, another con®rmed by an electrophoretic mobility shift assay, adaptor protein, which possesses a C-terminal death which also con®rmed the speci®city of this response domain and an N-terminal `death eector domain' (Figure 1e). However, Caspase 8 was a relatively weaker (Boldin et al., 1995; Chinnaiyan et al., 1995, 1996; Hsu stimulator of the NF-kB pathway as compared to et al., 1996; Varfolomeev et al., 1996). The DED of TNFR1 (Figure 1e). FADD binds to the N-terminal prodomain of Caspase 8 (also called FLICE, MACH or Mch5), a pro- The prodomain of Caspase 8 mediates NF-kB activation apoptotic apical caspase of the caspase cascade (Boldin et al., 1996; Fernandes-Alnemri et al., 1996; Muzio et The observed ability of Caspase 8 to activate the NF- kB pathway could be secondary to its ability to activate the cell death pathway and the resultant activation of stress-response genes. To separate the *Correspondence: PM Chaudhary, Hamon Center for Therapeutic NF-kB inducing ability of Caspase 8 from its ability to Oncology Research, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas, TX 75390-8593, USA act as a pro-apoptotic caspase, we tested the ability of Received 22 May 2000; revised 30 May 2000; accepted 19 July various deletion and point mutants of Caspase 8 to 2000 activate the NF-kB pathway. Activation of NF-kB by caspases PM Chaudhary et al 4452 Figure 1 Caspase 8 induces NF-kB. (a) 293T cells were transfected with a Caspase 8 expression vector (750 ng) or an empty vector (750 ng) along with an NF-kB/luciferase reporter construct (75 ng) as well as a RSV/LacZ (b-gal) reporter construct (75 ng) in duplicate and reporter assay for NF-kB activation performed as described in the Materials and methods section. A control experiment was performed with a mutant NF-kB/luciferase reporter construct in parallel. The values shown are averages (mean+s.e.m.) of one representative experiment out of three in which each transfection was performed in duplicate. (b) Dose- response of Caspase 8-induced NF-kB activation. 293T cells were transfected with empty vector or indicated amounts (in ng/well) of expression vector for Flag-Caspase 8 C360S in duplicate in each well of a 24-well plate. Western blot analysis with a Flag antibody demonstrates the expression of Caspase 8 at each dose level (right panel). (c) Time course of Caspase 8-induced NF-kB activation. 293T cells were transfected with empty vector or Caspase 8 C360S expression plasmid (250 ng/well) and cell extracts prepared for the measurement of luciferase activity at the indicated time points. (d) Induction of NF-kB by Caspase 8 in the MCF7 cells. MCF7 cells (16105) were transfected with a Caspase 8 expression construct or an empty vector (750 ng each) along with an NF-kB/ luciferase reporter construct (75 ng) in duplicate. Luciferase activity was measured 20 h later. (e) Electrophoretic mobility shift assay. 293T cells were (36105) were transfected with 5 mg of an empty vector (lane 1) or an expression vector encoding the Caspase 8 (lane 2 ± 4) or TNFR1 (lane 5). After 16 h, nuclear extracts were prepared essentially as described previously (Yeh et al., 1997). Nuclear extracts (2 mg) were incubated for 30 min at room temperature with a 32P-labeled NF-kB duplex oligonucleotide (Promega, Madison, WI, USA) in a buer containing 10 mM HEPES (pH 7.9); 50 mM KCl, 0.2 mM EDTA, 2.5 mM DTT, 2 mg poly (dC:dI), 10% glycerol and 0.5% NP-40. Competition was carried out with 100-fold excess of cold NF-kB oligo duplex or a non-speci®c oligo duplex. Protein-DNA complexes were resolved on a 5% native polyacrylamide and run in Tris-glycine buer. Gel was dried and autoradiographed. The position of the induced NF-kB complex is marked by an arrow while * marks the position of a constitutive NF-kB complex. Descriptions of various lanes are as follows: lane 1, empty vector, lane 2, Caspase 8; lane 3, Caspase 8 plus cold non-speci®c competitor duplex; lane 4, Caspase 8 plus cold NF-kB duplex; lane 5, TNFR1 A construct encoding the full-length prodomain of the catalytically active site and is, therefore, proteoly- Caspase 8, containing its two Death Eector Domains tically inactive, was as eective as the wild-type (DEDs) (amino acids 1 ± 180), was able to activate NF- Caspase 8 in activating the NF-kB pathway (Figure 2). kB to an even greater extent than the full-length Caspase 8, whereas deletion constructs encoding either Ability of other caspases to activate NF-kB DED1 (aa 1 ± 103) or DED2 (aa 104 ± 180) alone failed to do so (Figure 2). Similarly, constructs encoding the We also tested the ability of several other caspase full-length protease (caspase homology) domain or the family members to activate the NF-kB pathway. individual p20 or p10 sub-domains failed to activate Unlike Caspase 8, over-expression of Caspase 10 NF-kB (Figure 2 and data not shown). Caspase 8 (Mch4 isoform) led to massive apoptosis in 293T cells C360S, which contains a cysteine to serine mutation at but failed to induce NF-kB (Figure 3a and data not Oncogene Activation of NF-kB by caspases PM Chaudhary et al 4453 the protease activity of both proximal and distal caspases. Both CrmA and p35 were ineective in blocking NF-kB activation by Caspases 8 or 10, thus con®rming that NF-kB activation by these caspases is independent of their ability to activate the caspase cascade (Figure 3d).