Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Death-inducing Signaling Complex and Its Modulation by c-FLIP and PED/PEA-15 in Glioma Cells*

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can trigger apoptosis in some tumor cells but not other tumor cells. To explore the signal transduction events in TRAIL-triggered apoptosis and its modulation in nontransfected tumor cells, we analyzed TRAIL-induced death-inducing signaling complex (DISC) in TRAIL-sensitive and -resistant glioma cells. Caspase-8 and caspase-10 were recruited to the DISC, where they were proteolytically activated to initiate apoptosis in TRAIL-sensitive glioma cells. Caspase-8 and caspase-10 were also recruited to the DISC in TRAIL-resistant cells, but their further activation was inhibited by two antiapoptotic proteins termed cellular Fas-associated death domain-like interleukin-1 -converting enzyme-inhibitory protein (c-FLIP) and phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15kDa (PED/PEA-15). Both long and short forms of c-FLIP were recruited to the DISC, where the long form c-FLIP was cleaved to produce intermediate fragments. Of the three isoforms of PED/ PEA-15 proteins, only the doubly phosphorylated form was expressed and recruited to the DISC in TRAILresistant cells, indicating that the phosphorylation status of PED/PEA-15 determines its recruitment in the cells. Treatment with calcium/calmodulin-dependent protein kinase inhibitor rescued TRAIL sensitivity in TRAIL-resistant cells, providing a potential new approach to sensitize the cells to TRAIL-induced apoptosis.

TRAIL 1 is a member of tumor necrosis factor family that triggers apoptosis in human tumor cells but not normal cells (1), providing therapeutic potential for tumors. TRAIL signals apoptosis through death receptors DR4 (TRAIL receptor 1) (2) and DR5 (TRAIL receptor 2) (3,4), as demonstrated in transfectants. DR4 and DR5 are type I transmembrane proteins with a cytoplasmic death domain, which binds to the death domain of the intracellular adaptor Fas-associated death domain (FADD) (5)(6)(7)(8). The death effector domain (DED) of FADD, in turn, interacts with the DED of caspase-8 to recruit this apoptosis-initiating protease to the death receptors (9,10), leading to the assembly of the DISC (11). Caspase-8 is activated through trans-and autocatalytical cleavage in the DISC due to a close proximity of the molecules (12) and initiates apoptosis by subsequent cleavage of downstream effector caspases (caspase-3 and caspase-7) (9, 10).
Sensitivity of tumor cells to TRAIL is modulated by various DED-containing antiapoptotic proteins (18,19). One such protein is c-FLIP (20), and also termed CASH (21), CASPER (22), CLARP (23), FLAME-1 (24), I-FLICE (25), MRIT (26), and Usurpin (27). c-FLIP proteins are expressed in two isoforms: (a) c-FLIP S , a shorter form (M r ϳ28 kDa), contains two DEDs; and (b) the longer form, c-FLIP L (M r ϳ55 kDa), has two DEDs and a caspase-like domain in which the active center cysteine residue is substituted by a tyrosine residue (20,22,28). Both forms of c-FLIP interact with FADD and caspase-8 through DED-DED interaction and potently block caspase-8 binding to the DISC (20). Recent analysis of Fas-mediated DISC in transfectants further revealed that c-FLIP L and c-FLIP S are recruited to the DISC to inhibit caspase-8 cleavage in the DISC (28,29).
TRAIL-induced signal transduction has been well characterized in transfectants, but it remains largely elusive in nontransfected tumor cells. Caspase-8 and caspase-10 have been shown to initiate TRAIL-induced apoptosis, which appears to be modulated by c-FLIP and PED/PEA-15. However, signal events of these proteins in TRAIL-induced apoptosis have not yet been established. Therefore, we examined the TRAIL-induced DISC to explore the molecular mechanisms in malignant glioma cells. Our studies showed for the first time that caspase-8 and caspase-10 are recruited to the DISC in both TRAIL-sensitive and -resistant glioma cells but that caspase activation is inhibited by c-FLIP and PED/PEA-15 in the DISC in TRAIL-resistant cells. In these cells, phosphorylation of PED/PEA-15 influences its recruitment and implementation of its antiapoptotic function.
Cell Death Assay and Detection of Cleavage of Caspases and c-FLIP-For cell death, cells were seeded in 96-well plates at 2 ϫ 10 4 cells/well and treated at 37°C for 16 h with TRAIL-FLAG (starting at 1 g/ml) in 3-fold serial dilutions in the presence of 3 g/ml anti-FLAG M2. Some cells were also treated with KN-93 (starting at 100 M), z-IETD-fmk (20 M), or Z-AEVD-fmk (20 M). For glioma cells, cell death was determined by crystal violet assay (18). For Jurkat cells, dead cells were stained with trypan blue and counted by cytometry. For cleavage of caspases and c-FLIP, subconfluent cells were treated with 1 g/ml TRAIL-FLAG and 3 g/ml anti-FLAG M2 alone or in the presence of KN-93 (100 M) or caspases inhibitors at 37°C for the times indicated. At each time point, cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail). After centrifugation at 16,000 ϫ g for 15 min at 4°C, supernatants were subjected to Western blotting.
DISC Analysis by Immunoprecipitation-Immunoprecipitation for DISC analysis was performed according to a modified protocol as reported previously (6,7). Cells (5 ϫ 10 7 ) were stimulated with 1 g/ml TRAIL-FLAG and 3 g/ml anti-FLAG M2 for 30 min at 37°C with or without 20 M z-IETD-fmk pretreatment. The cells were lysed for 30 min on ice with lysis buffer. In unstimulated controls, 1 g of TRAIL-FLAG and 1 g of anti-FLAG M2 were added after lysis to immunoprecipitate nonstimulated death receptors. The soluble fraction was immunoprecipitated with 20 l of goat anti-mouse IgG1-agarose overnight at 4°C and analyzed by Western blotting and two-dimensional PAGE immunoblotting.

TRAIL-induced Recruitment of Caspase-10 to the DISC in
Jurkat Cells-Jurkat cells express DR5, but not DR4, and can be killed by TRAIL through apoptosis (16). DISC analysis in parental Jurkat cells and their FADD def and caspase-8 def clones has established that TRAIL induces incorporation of DR5, FADD, and caspase-8 into the DISC (6,7,17). Taking advantage of this system, we examined these cells to determine if caspase-10 is recruited to the DISC to initiate apoptosis independent of caspase-8. We stimulated the cells with TRAIL-FLAG/M2 for 30 min, at which time a maximal recruitment of all the signaling proteins was observed (6,7,17). The DISC was immunoprecipitated and examined by Western blot to identify the DISC proteins.
Western blots revealed DR5, FADD, caspase-8, and caspase-10 in the TRAIL-induced DISC in the stimulated parental Jurkat cells (Fig. 1A). Four caspase-10 bands were detected: caspase-10 precursors (p59 and p54) and their corresponding fragments that contain prodomain still attached to the large subunit (p47 and p43), indicating that caspase-10 is recruited to and completes its first-step cleavage in the DISC. This was quickly followed by the further cleavage of caspase-8 and caspase-10, as shown by the appearance of the active p18 form of caspase-8 (Fig. 1B) and the p27 prodomain form of caspase-10 (Fig. 1D). We further examined downstream caspase-3 and found that caspase-3 was proteolytically processed to produce the active p20 and p18 forms (Fig. 1D). Stimulation of the Jurkat cells with TRAIL-FLAG/M2 resulted in extensive cell death, as demonstrated by crystal violet analysis (Fig. 1C).
Caspase-10 proteins (p59, p54, p47, and p43) were also detected in TRAIL-induced DISC in the caspase-8 def clone, but not in the FADD def clone (Fig. 1A), indicating that FADD is required for caspase-10 recruitment. TRAIL-FLAG/M2 triggered cell death in the caspase-8 def clone, but it was delayed and inefficient as compared with that seen in the parental cells (Fig. 1C). This delayed cell death occurred through activation of caspase-10 and caspase-3, as evidenced by the appearance of p27 caspase-10 prodomain and caspase-3 p20 and p18 forms on Western blot at 48 h (Fig. 1D). Treatment of caspase-8 def cells with z-AVED-fmk, a caspase-10-specific inhibitor, abolished TRAIL-FLAG/M2-induced cell death (Fig. 1C) and activation of caspase-3 (Fig. 1D), further indicating that caspase-10 is an initiator of TRAIL-induced apoptosis independent of caspase-8.
Molecular Dissection of TRAIL-induced DISC in TRAIL-sensitive Glioma Cells-We further investigated TRAIL-induced recruitment of caspase-8 and caspase-10 to initiate apoptosis in tumor cells. To this end, we analyzed the TRAIL-induced DISC in malignant glioma cell lines (LN-18, T98G, and U343MG) that express DR5 and undergo extensive apoptosis upon TRAIL stimulation (18). In TRAIL-FLAG/M2-stimulated cells, Western blots detected caspase-8 precursors (p55 and p53) and their first cleavage products (p43 and p41) and caspase-10 precursors (p59 and p54) and their cleavage fragments (p47 and p43) (Fig. 2A). These findings indicated that both caspase-8 and caspase-10 are recruited and complete their first-step cleavage in the DISC. DR5 proteins (49 and 43 kDa) and FADD (25 kDa) were also detected in the DISC, indicating that caspase-8 and caspase-10 recruitment to DR5 requires FADD. This DISC formation was quickly followed by further cleavage of caspase-8 to produce active p18 subunits and of caspase-10 to produce p27 fragments (Fig. 2B). Caspase-3 was also cleaved into active p20 and p18 products (Fig. 2B), and extensive cell death was observed in all cell lines tested (Fig. 2C).
Our previous studies showed that z-IETD-fmk, a caspase-8specific inhibitor, partially protects glioma cells from TRAILinduced apoptosis (18), suggesting that caspase-10 may be responsible for apoptosis in these cells. To test this hypothesis, we treated U343MG cells with 20 M z-IETD-fmk for 2 h and then stimulated the cells with TRAIL-FLAG/M2. DISC analysis revealed recruitment of caspase-8 to the DISC ( Fig. 2A), but its further cleavage was inhibited by z-IETD-fmk (Fig. 2B). Caspase-10 was also recruited to the DISC, where it was proteolytically processed ( Fig. 2A) and further activated downstream caspase-3 (Fig. 2B), resulting in a maximal 50% cell death (Fig. 2C). The activation of these caspases and cell death were abolished by the presence of z-AEVD-fmk, a caspase-10 inhibitor (Fig. 2, B and C). These findings indicate that caspase-10 is an independent initiator of TRAIL-induced apoptosis in glioma cells as well.
Caspase-8 and Caspase-10 Recruitment to the DISC in TRAIL-resistant Glioma Cells-Many glioma cell lines are resistant to TRAIL (18), but the molecular mechanism of this resistance remains unclear. To investigate this, we analyzed the DISC in glioma cell lines (LN-215, LN-464, and LN-443) that are resistant to TRAIL-FLAG/M2-induced apoptosis, as determined by crystal violet assay for cell death and by Western blotting for caspase-3 activation (data not shown). We first examined caspase-8 to determine if its function as an apoptosis initiator is impaired in TRAIL-resistant cells. Western blots detected capase-8 precursors (p55 and p53) and its first-step cleavage fragments (p43 and p41) in the DISC (Fig. 3A). It appeared, therefore, that caspase-8 is recruited to and proteolytically cleaved in the DISC. Western blots further revealed DR5 and FADD in the DISC, indicating that this first-step cleavage of caspase-8 requires DISC assembly. However, failure to detect active p18 subunits of caspase-8 in these cells upon TRAIL-FLAG/M2 stimulation (data not shown) suggested that further cleavage of caspase-8 in the DISC is inhibited.
In contrast to caspase-8, caspase-10 was not detected by Western blotting in the TRAIL-resistant LN-215, LN-464, and LN-443 lines (Fig. 3A). To investigate the molecular mechanisms of caspase-10 absence, we first compared the protein expression levels of caspase-8 and caspase-10 in TRAIL-sensitive and -resistant cells. Western blot showed that caspase-8 precursors (p55 and p53) were expressed at similar levels in both TRAIL-sensitive and -resistant cell lines but that much lower levels of caspase-10 precursors (p59 and p54) were detected in TRAIL-resistant cells (Fig. 3B), suggesting that the protein expression levels of caspase-8 and caspase-10 influence their recruitment to the DISC by competing for DED binding sites on FADD. To further examine this hypothesis, we examined the LN-405 cell line that is resistant to TRAIL (data not shown) but expresses high levels of caspase-10 (Fig. 3B). Indeed, DISC analysis of LN-405 cells showed that both caspase-8 and caspase-10 were recruited to and cleaved in the DISC (Fig. 3A).
c-FLIP Recruitment to and Cleavage in the DISC-The DEDcontaining protein c-FLIP interacts with FADD to block caspase-8 recruitment to the DISC in transfectants (20 -27). Our finding that caspase-8 is recruited to the DISC suggested an alternative mechanism. We examined TRAIL-FLAG/M2 immunoprecipitated samples to look for c-FLIP in the DISC. Both c-FLIP L and c-FLIP S were expressed in TRAIL-resistant cells, as detected by Western blot analysis of cell extracts (Fig. 3A). Detection of a weaker band of p55 c-FLIP L and a dense new band of p43 products in the DISC indicated that c-FLIP L was recruited to and proteolytically cleaved in the DISC to produce its intermediate p43 form. The short form, c-FLIP S (p25), was also detected in the DISC (Fig. 3A).
Kinetic analysis on Western blots of c-FLIP L cleavage revealed that its intermediate p43 forms were produced in the TRAIL-resistant cells, but not in the TRAIL-sensitive cells, upon TRAIL stimulation (Fig. 4). Both TRAIL-sensitive and -resistant cells showed first-step cleavage of caspase-8 in the DISC ( Figs. 2A and 3A), but it appeared that simultaneous recruitment of caspase-8 and c-FLIP L to the DISC resulted in c-FLIP L cleavage into intermediate products in TRAIL-resistant cells (Fig. 3A). Detection of caspase-8 precursors (p55 and p53) and its first-step cleavage products (p43 and p41) (Fig.  3A), together with c-FLIP S and c-FLIP L intermediate p43 (Fig.  3A), suggested that both forms of c-FLIP might be responsible for inhibiting further cleavage of caspase-8 in the DISC.
PED/PEA-15 Recruitment Dependent on Its Phosphorylation Status-PED/PEA-15 is an additional DED-containing protein that inhibits TRAIL-induced apoptosis (18). Further analysis of the DISC revealed PED/PEA-15 in the TRAIL-resistant glioma cells (Fig. 3A), but not in the TRAIL-sensitive cells (data not shown). We reported previously that PED/PEA-15 antiapoptotic function depends on its phosphorylation (18), and therefore we proposed that the phosphorylation status of PED/ PEA-15 protein may influence its recruitment. To test this hypothesis, we first analyzed TRAIL-sensitive and -resistant glioma cells for their expression of PED/PEA-15 isoforms. Twodimensional PAGE immunoblotting detected unphosphorylated (N), singly phosphorylated (Pa), and doubly phosphorylated Pb forms of PED/PEA-15 in TRAIL-resistant LN-215 cells, but detected only N and Pa forms in TRAIL-sensitive U343MG cells.
Next, we examined the TRAIL-FLAG/M2 immunoprecipitated samples from LN-215 cells on two-dimensional PAGE immunoblot to determine the isoform(s) of PED/PEA-15 in the DISC. As shown in Fig. 5A, only the Pb form was present in the DISC, indicating that double phosphorylation of PED/PEA-15 is required for its recruitment to the DISC. We then treated LN-215 cells with KN-93, a CaMK inhibitor (37), to inhibit PED/PEA-15 phosphorylation and analyzed the isoform(s) of PED/PEA-15 present in the cells. Exposure of LN-215 cells to 100 M KN-93 for 16 h greatly reduced the Pb form (Fig. 5A).  5B) and significantly increased cell death in this TRAIL-resistant cell line (Fig. 5C). DISCUSSION The signal transduction in TRAIL-induced apoptosis and its inhibition in nontransfected tumor cells remain largely un-known. Understanding of these signaling events that control tumor cell death could provide therapeutic potentials for tumors. In the present study, we dissected TRAIL-induced DISC for the first time in TRAIL-sensitive and -resistant malignant glioma cells to reveal the molecular mechanisms in TRAILinduced apoptosis and its modulation. Apoptosis-initiating protease caspase-8 molecules are recruited via FADD to the DR5 to form DISCs, where the caspase-8 is proteolytically cleaved to produce its first-step cleavage products in both TRAIL-sensitive and -resistant cells. Caspase-8 is further cleaved to the products that activate downstream caspase-3, resulting in cell death in TRAIL-sensitive glioma cells. These results are in accord with DISC analysis in transfectants and Jurkat cells (5)(6)(7)(8). However, in TRAIL-resistant glioma cells, further cleavage of caspase-8 is inhibited.
We further showed for the first time that caspase-10 is also recruited to the DISC in both TRAIL-sensitive and -resistant glioma cells. Caspase-10 was reported to have four isoforms, three of which (caspase-10/a, caspase-10/b, and caspase-10/d) contain a protease domain that is functional in transfectants (38). However, recent studies revealed only caspase-10/a and caspase-10/d in nontransfected B and T cells (17). Failure to detect caspase-10 in the DISC in previous studies (6,7) was explained recently by the observation that the antibodies used in the studies recognize only caspase-10/b and cross-react with Hsp60 (17). We selected an antibody that recognizes three isoforms of caspase-10 (17,39) and showed that caspase-10 is recruited to and cleaved in the TRAIL-induced DISC. Caspase-10 recruitment is detected in some but not other cell lines, which correlates well with its protein expression levels in the cells. Recruitment of caspase-10 to the DISC triggers apoptosis in the presence of caspase-8-specific inhibitor, indicating that caspase-10 is an apoptosis initiator independent of caspase-8.
Our observation that c-FLIP and PED/PEA-15 are recruited to the DISC may explain why further cleavage of caspase-8 and caspase-10 is inhibited in TRAIL-resistant cells. A large number of c-FLIP and PED/PEA-15 molecules in the proximity of caspase-8 and caspase-10 in the DISC may prevent further cleavage of the caspases according to the induced proximity model (12). c-FLIP L is recruited to and cleaved into intermediate fragments in the DISC. This occurs simultaneously with caspase-8 recruitment and first-step cleavage, suggesting that both events may contribute to each other. Indeed, a recent study with transfectants showed that c-FLIP L recruitment contributes to caspase-8 first-step cleavage in the DISC, where the c-FLIP L intermediate form inhibits further cleavage of caspase-8 (17).
Very little is known about the molecular basis for the difference in c-FLIP and PED/PEA-15 recruitment in TRAIL-resistant and -sensitive tumor cells. Expression of c-FLIP in melanoma cells was reported to correlate with their sensitivity to TRAIL (19), but additional studies in melanoma (40) and glioma cells (18) failed to confirm this correlation. It appears that c-FLIP antiapoptotic functions are modulated by its phosphorylation through the phosphatidylinositol 3-kinase signaling pathway in tumor cells (41), but the mechanism by which this signaling pathway regulates c-FLIP expression status and/or antiapoptotic function remains to be investigated.
We have reported that inhibition of PED/PEA-15 phosphorylation restored TRAIL sensitivity in TRAIL-resistant cells (18). Here, we further showed that treatment with KN-93 (a CaMK inhibitor) (37) largely rescued the cell sensitivity to TRAIL. A doubly phosphorylated form of PED/PEA-15 is recruited to the DISC in TRAIL-resistant cells, indicating that phosphorylation of PED/PEA-15 is required for its recruitment to the DISC. Doubly phosphorylated PED/PEA-15 proteins are expressed in TRAIL-resistant but not TRAIL-sensitive cells, further indicating a significant difference in the upstream mechanisms that control phosphorylation of PED/PEA-15 in TRAIL-resistant cells compared with TRAIL-sensitive cells. Further investigation of functions of CaMK, protein kinase C, and phosphatidylinositol 3-kinase signaling pathways in glioma cells will shed some light on the complex regulatory mechanisms that control tumor cell death and survival.