Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Fas ligand (FasL) 1 and its receptor Fas are prototypic members of the tumor necrosis factor (TNF) ligand family and the death domain-containing receptor subgroup of the TNF recep-tor superfamily, respectively. Fas and FasL have been predominately investigated in respect to their death-inducing capabilities. For example, Fas and FasL are involved in T-cell mediated cytotoxicity (1,2), apoptosis induction in activated lymphocytes (3)(4)(5)(6), and maintainance of the immunoprivileged state of eyes and testis (7,8). Consequently mouse strains bearing loss of function mutations in FasL (gld) or Fas (lpr) show lymphoproliferation, lymphadenopathy, and production of autoimmune antibodies (9,10). Similarly many forms of the human autoimmune lymphoproliferative syndrome are caused by dominant interfering Fas or FasL gene mutations (11)(12)(13).
The molecular mechanisms of Fas/FasL-induced apoptosis have been studied in detail during the last years. The initial event of FasL-induced apoptosis is the multimerization of Fas by the membrane-bound form of its ligand. This receptor oligomerization leads to the formation of a death-inducing signaling complex comprising the adaptor molecule FADD/MORT, which binds with its C-terminal death domain to the death domain of Fas, and the FADD-interacting initiator caspase-8, which interacts with the N-terminal death effector domain (DED) of FADD via its own DEDs (14). The death-inducing signaling complex-induced proximity of procaspase-8 molecules leads to autoproteolytic activation and initiation of the apoptotic caspase cascade (15). This process, however, can be inhibited by FLICE inhibitory protein (FLIP) (16), also designated as casper (17), CLARP (18), FLAME-1 (19), I-FLICE (20), CASH (21), MRIT (22), or ursupin (23). FLIP exists in two splice forms: FLIP-long (FLIP-L) comprising two N-terminal DEDs and an enzymatically inactive pseudo caspase domain and FLIP-short (FLIP-S) containing only the two DEDs. Both splice forms of FLIP seem to act as dominant negative molecules in respect to Fas-mediated caspase-8 processing at an intermediate state (24). Although the apoptotic features of the FasL/Fas system are well established, there is now also increasing evidence for functions that are not related to induction of apoptosis such as activation of NF-B and c-Jun N-terminal kinase (JNK) (25)(26)(27) or induction of proliferation (28 -30) and differentiation (31). We show here that stimulation of Jurkat T-cells with FasL or the closely related TNF-related apoptosisinducing ligand (TRAIL) leads to up-regulation of the protooncogene c-fos, a target gene of mitogenic stimuli. The FasL/ TRAIL-induced up-regulation of c-Fos is regulated by FLIP and requires FADD and caspase-8, but the protease activity of the latter is dispensable.

EXPERIMENTAL PROCEDURES
Antibodies and Reagents-Chemicals and secondary antibodies were obtained from Sigma, and cell culture reagents were from Life Technologies, Inc. The Jurkat T-cell lines that are deficient for FADD and caspase-8 and the respective parental control cell line as well as Jurkat clones overexpressing FLIP-L and FLIP-S, respectively, were described elsewhere (16,32,33). An additional Jurkat clone (JB-6) deficient in caspase-8 expression was a kind gift from Shigekazu Nagata (Osaka University Medical School, Osaka, Japan). All Jurkat clones were maintained in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum in a humidified 5.0% CO 2 environment. The caspase-8-specific mAb was a gift from Klaus Schulze-Osthoff (Universitä t Mü nster, Mü nster, Germany). The broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) was purchased from Bachem (Heidelberg, Germany). The anti-FLIP rat mAb Dave-2 was from Apotech (Lausanne, Switzerland), and the anti-c-Fos polyclonal rabbit IgG was from Santa Cruz Biotechnology (Heidelberg, Germany).
RNase Protection Assay-Jurkat cells (10 ϫ 10 6 ) were treated with the indicated reagents for the given times, and total RNAs were isolated with the RNA INSTAPURE kit (Eurogentech, Seraing, Belgium) according to the manufacturer's recommendations. The presence of transcripts of the indicated apoptosis-related genes as well as the internal controls L32 and glyceraldehyde-3-phosphate dehydrogenase were analyzed using a custom Multi-Probe template set (PharMingen, Hamburg, Germany). Probe synthesis, hybridization, and RNase treatment were performed with the RiboQuant Multi-Probe RNase Protection Assay System (PharMingen). Protected transcripts were resolved by electrophoresis on denaturing polyacrylamide gels (5%) and visualized on a PhosphorImager with the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Western Blotting of Caspase-8, cFLIP, and c-Fos-For Western blot analysis of caspase-8 and FLIP lysates were prepared in radioimmune precipitation buffer supplemented with a protease inhibitor mixture stock solution (Roche Molecular Biochemicals) as recommended by the supplier. Cell debris was removed by centrifugation (10,000 ϫ g, 10 min), and the protein concentration was determined by the Bradford assay. For detection of c-Fos protein nuclear extracts were prepared as described elsewhere (26) for electrophoretic mobility shift assay analysis. Proteins (50 g) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by electroblotting. Blots were blocked for 1 h at room temperature in Trisbuffered saline containing 0.05% Tween 20 and 3% (w/v) dry milk, washed, and incubated with anti-caspase-8 mouse mAb, anti-FLIP rat mAb Dave-2 (Apotech, Epalinges, Switzerland) or anti-c-Fos rabbit IgG (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h at room temperature. Bound antibodies were visualized with alkaline phosphatase-conjugated goat anti-mouse/rat/rabbit IgG (Sigma) and nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrate.
Immunocomplex JNK Assay-c-Jun N-terminal kinase assays were performed using an anti-JNK1 rabbit antiserum for immunoprecipitation (Santa Cruz Biotechnology) and glutathione S-transferase-c-Jun as substrate. The in vitro kinase assay has been described elsewhere (34).
In Vitro Caspase-3 Activity Assay-Caspase-3 activity was measured

FIG. 1. c-Fos is up-regulated by FasL independently of activation of NF-B and JNK.
A-C, Jurkat cells were treated as indicated with soluble FasL cross-linked with FLAG-specific mAb M2 (1 g/ml) and z-VAD-fmk (Z; 10 M). If not otherwise stated 300 ng/ml FasL were used for 6 h. Total RNAs were isolated for RNase protection analysis, and 10 g of each RNA sample were analyzed with a custom Multi-Probe template set to detect the indicated mRNAs. L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in each template set as internal controls. D, Jurkat cells were treated with cross-linked FasL (100 ng/ml) for the indicated time in the absence or presence of z-VAD-fmk. Cells were lysed in radioimmune precipitation buffer, and proteins were immunoblotted with a caspase-8-specific mAb to detect cleavage of procaspase-8 that is indicative of activation of this enzyme. In parallel cell lysates (30 g) were assayed for caspase-3 activity using the fluorogenic substrate Ac-DEVD-AMC (20 M). E, Jurkat cells were stimulated for 6 h with cross-linked (1 g/ml M2) soluble FasL (300 ng/ml) in the presence of z-VAD-fmk (Z; 10 M) or with 100 nM phorbol 12-myristate 13-acetate (P) or remained untreated.
Nuclear extracts were prepared, and proteins were immunoblotted with anti-c-Fos antibody (Santa Cruz Biotechnology). The relative protein expression levels were determined with the Bio Profil software (Vilber Lourmat, Torcy, France). Alternatively, total RNAs were isolated for RNase protection analysis, and 10 g of each RNA sample were analyzed with a custom Multi-Probe template set to detect the indicated mRNAs. L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in each template set as internal controls. The indicated relative expression levels of c-Fos were already normalized with respect to the endogenous L32 control. F, cell lysates were prepared from Jurkat cells that had been stimulated for the indicated times with M2-cross-linked (1 g/l) soluble FasL (100 ng/ml) in the absence or presence of z-VAD-fmk (10 M). JNK activity was measured by the immunocomplex kinase assay with glutathione S-transferase (GST)-c-Jun-(1-79) as a substrate. G, Jurkat cells were transiently transfected with an AP-1 reporter gene construct by electroporation. The next day cells were split and stimulated with 100 nM phorbol 12-myristate 13acetate (P) or FasL (300 ng/ml) cross-linked with FLAG-specific mAb M2 (1 g/ml) in the presence of z-VAD-fmk (Z; 10 M). Finally reporter gene activity was determined as described under "Experimental Procedures." n.s., nonspecific.

RESULTS AND DISCUSSION
Recent studies of FADD-deficient mice (35)  (1 g/ml) and z-VAD-fmk (Z; 10 M). Total RNAs were isolated for RNase protection analysis, and 10 g of each RNA sample were analyzed with a custom Multi-Probe template set to detect the indicated mRNAs. L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were included in each template set as internal controls. Similar results were also obtained with an independent FADD-deficient clone (clone I.6.2) and the caspase-8-deficient Jurkat clone JB-6 from the laboratory of Shigekazu Nagata.

FIG. 3. FasL-and TRAIL-induced up-regulation of c-Fos is regulated by FLIP. A, lysates of parental Jurkat cells and clones overex-
pressing FLIP-L and FLIP-S, respectively, were analyzed by Western blotting with the FLIP-L/S-specific rat mAb Dave-2 and alkaline phosphatase-conjugated anti-rat antiserum. B and C, parental Jurkat cells and derived clones that stably overexpress the long and short splice forms of FLIP, respectively, were incubated with the indicated combinations of FasL (300 ng/ml) (B) or soluble TRAIL (100 ng/ml) (C), each cross-linked with M2 (1 g/ml) and z-VAD-fmk (Z; 10 M). Total RNAs were analyzed as described in the legend for Fig. 2. of Fas in mitogenic signaling pathways was also suggested from reports demonstrating Fas-dependent proliferation of fibroblasts (28 -30). We have analyzed the expression of the early mitogenic response marker c-Fos in Jurkat T-cells upon TRAIL and FasL stimulation using RNase protection analysis assays with a Multi-Probe template set containing apoptosisand proliferation-related templates including c-Fos. FasL induced c-Fos mRNA in a dose-dependent manner in Jurkat cells (Fig. 1A). Induction of c-Fos was independent from the activation of caspase-8 and the subsequent triggering of the apoptotic machinery because the pan caspase-inhibitor z-VAD-fmk had no inhibitory effect on FasL-induced up-regulation of c-Fos (Fig. 1B) at concentrations at which processing of procaspase-8 and activation of caspase-3 were completely blocked (Fig. 1D). Up-regulation of c-Fos mRNA by the well known potent c-Fos inducer phorbol 12-myristate 13-acetate was readily detectable already after 30 min, peaked after 1 h, and reached almost background levels after 3 h (data not shown). In contrast, FasL-induced up-regulation of c-Fos occurred with delayed kinetics, starting to become detectable after 1 h and reaching a plateau after 6 h (Fig. 1C). Maximal phorbol 12-myristate 13-acetate-induced c-Fos mRNA levels were 5 times higher than maximal mRNA levels induced by FasL (Fig. 1E). Similarly, maximal c-Fos protein induced by phorbol 12-myristate 13-acetate was 5.5 times higher than maximal c-Fos protein induced by FasL (Fig. 1E). However, one should take into account that prolonged FasL-induced up-regulation of c-Fos might have another quality compared with the rapid transient up-regulation by phorbol 12-myristate 13-acetate.
Some recent reports have demonstrated the capability of Fas to induce NF-B activation (25,26). We here therefore tested whether this pathway is involved in c-Fos up-regulation. Overexpression of a nondegradable mutant of I-B did not affect Fas-induced up-regulation of c-Fos in Jurkat T-cells (data not shown) suggesting that the NF-B pathway is not involved in this process. The c-fos gene is a target of AP-1 that is transcriptionally up-regulated when AP-1 is activated by JNKs (40). However, JNK is not involved in FasL-induced up-regulation of c-Fos because the pan caspase inhibitor z-VAD-fmk completely prevented FasL-mediated JNK activation (Fig. 1F) but not induction of c-Fos mRNA (Fig. 1, A and C). AP-1 is a dimeric transcription factor composed of Jun and Fos (or activating transcription factor) subunits. We therefore looked at whether FasL-induced up-regulation of c-Fos is accompanied by activation of an AP-1 reporter gene plasmid. Although phorbol 12myristate 13-acetate was able to up-regulate the AP-1 reporter gene more than 35-fold, we found no evidence for any activation after FasL stimulation (Fig. 1G). However, this was not unexpected because AP-1 activity depends on phosphorylation of its transactivation domain by JNK, but FasL does not induce this pathway in the presence of z-VAD-fmk (Fig. 1F).
FADD and caspase-8 are essential components of the ligandinduced Fas receptor-signaling complex (14). We have further analyzed FasL-induced up-regulation of c-Fos in Jurkat clones deficient in these molecules (32,33,41). In two independent FADD-deficient Jurkat clones we found no significant c-Fos up-regulation upon treatment with FasL ( Fig. 2A and data not  shown). Moreover, caspase-8-deficient Jurkat clones were also found to be deficient in FasL-induced up-regulation of c-Fos ( Fig. 2B and data not shown). In two clones, we observed that induction of c-Fos by phorbol ester was unaffected (data not shown). Because the pan caspase-inhibitor z-VAD-fmk had no effect on FasL-induced up-regulation of c-Fos but in parallel the lack of caspase-8 completely prevented this event, our data point to a signaling function of caspase-8 that is independent from its proteolytic activity. A function of caspase-8 to act as a signal-transducing molecule independent of its role as an effector caspase of the apoptotic program was also observed in two recent studies that demonstrated activation of the NF-B and JNK pathways in response to transient overexpression of caspase-8 (42,43). However, the caspase-8-dependent activation of the JNK pathway shown in the latter study was not activated by FasL in Jurkat cells. Indeed as already mentioned, FasL-induced JNK activation was completely blocked by z-VAD-fmk (Fig. 1E) in Jurkat cells. Thus, because JNK and NF-B activation are not critically involved in FasL-induced up-regulation of c-Fos (see above), these data suggest that caspase-8 links a novel non-caspase-activating signaling pathway to Fas.
Data from the literature indicate that the death domaincontaining TRAIL receptors TRAIL-R1 and TRAIL-R2 engage similar or at least closely related intracellular signaling pathways as Fas leading to the induction of apoptosis (14, 44 -47), NF-B, and JNK activation (26,48). We have found that the same is true for up-regulation of c-Fos. Like FasL, TRAIL induced c-Fos in Jurkat cells via a FADD/caspase-8-dependent pathway independent of the protease activity of caspase-8 (Fig. 2B).
FLIP is a major regulator of apoptotic and non-apoptotic signaling pathways engaged by FasL and TRAIL. We have therefore analyzed the FasL/TRAIL-induced up-regulation of c-Fos in Jurkat clones (Fig. 3A) stably expressing the long slice form FLIP-L or the short splice form FLIP-S. Up-regulation of c-Fos was found to be differentially regulated by FLIP-S and FLIP-L (Fig. 3, B and C). In Jurkat FLIP-L cells, FasL-and TRAIL-induced c-Fos up-regulation was totally blocked (Fig. 3, B and C). However, in Jurkat FLIP-S cells FasL-and TRAILinduced c-Fos expression was not reduced but was even enhanced (Fig. 3, B and C). We regularly found a weak but significant induction of TNF mRNA by TRAIL and FasL (Fig.  1), opening the possibility that TRAIL-and FasL-induced upregulation of c-Fos is mediated by the induction of endogenous TNF. However, in contrast to TRAIL and FasL, the closely related TNF did not affect c-Fos expression in Jurkat cells (data not shown). Thus, although TRAIL, FasL, and TNF act via stimulation of members of the death domain-containing receptor subgroup of the TNF receptor superfamily, only TRAIL and FasL, but not TNF, induce a common pathway leading to up-regulation of c-Fos.
Based on the data described in this study and data from the literature, a model of Fas/TRAIL signaling appears possible in which the expression level of FLIP and in particular the balance of FLIP-S and FLIP-L expression determines the outcome of Fas and TRAIL receptor triggering. It has been shown that in the absence of FLIP expression FasL/TRAIL induces formation of a FADD-containing complex that "catalyzes" repeated cycles of procaspase-8 (p55/53) recruitment, proximity-induced activation of p55/53 to the p18/p10 form of caspase-8 via a p43/41 intermediate, and release of the receptor-bound prodomain of caspase-8 (24). Recruitment of FLIP-L arrested this cycling death-inducing signaling complex in a state containing procaspase-8 or the p43/41 intermediate product of caspase-8 processing and FLIP-L (24) (Fig. 4A). It is now tempting to speculate that in this FLIP-L/FLIP-S-arrested state the receptor-signaling complexes of Fas, TRAIL-R1, and TRAIL-R2 acquire new signaling capabilities that are not related to apoptosis induction. According to our data, a FLIP-S-arrested complex may acquire the capability to interact with a yet unknown protein X that couples this FLIP-S-arrested complex to the activation of the c-fos gene (Fig. 4B). This protein X might be replaced by TNF receptor-associated factor (TRAF) proteins and/or receptor interacting protein when FLIP-S is substituted by FLIP-L because FLIP-L, but not FLIP-S, activates NF-B (42) and interacts with TRAF1, TRAF2 (17,49), and receptor interacting protein (49) (Fig. 4C).
It has been shown that c-Fos is an important regulator of cell proliferation and has a specific function in thymocyte development (50). Thus, it is tempting to speculate that FasL/TRAIL-induced up-regulation of c-Fos is part of the proliferative response of thymocytes and T-cells upon T-cell receptor triggering, which is impaired in FADD-deficient mice (35) or transgenic mice overexpressing a dominant-negative deletion mutant of FADD (36 -38). However, in lpr mice harboring a defective Fas molecule or gld mice expressing dysfunctional FasL, no inhibition of T-cell proliferation was found. This is at the first glance contradicting to a role of Fas in T-cell proliferation. However, it cannot be ruled out that in these mice TRAIL or other death ligands substitute for Fas/FasL interaction with regard to c-Fos-induction but not with regard to apoptosis induction. Furthermore, a recent study showing that lpr mice in a nonselecting background exert reduced proliferation in thymic T-cell development suggests that Fas/FasL contributes to thymocyte proliferation under defined circumstances (51). Moreover, recent studies have also shown that FasL augmented CD3-induced proliferation, whereas Fas-Fc blocked T-cell proliferation (49,(52)(53)(54). In contrast to the c-Fos activation described here, T-cell proliferation was dependent on active caspases in these studies (53,55). Thus, up-regulation of c-Fos may reflect a death receptor-mediated proliferative response that is, in contrast to other components of the proliferative response, caspase-independent. However, it cannot be ruled out that the FasL/TRAIL-induced up-regulation of c-Fos shown in this study is not related to T-cell receptor-dependent proliferation of T-cells. Further analysis with primary T-cells will be necessary to clarify that.