The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling system regulates glioma cell survival via NFkappaB pathway activation and BCL-XL/BCL-W expression.

The Fn14 gene encodes a type Ia transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. We recently showed that fibroblast growth factor-inducible 14 (Fn14) is overexpressed in migrating glioma cells in vitro and in glioblastoma multiforme clinical specimens in vivo. To determine the biological role of Fn14 in brain cancer progression, we examined the activity of Fn14 as a potential mediator of cell survival. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-stimulated glioma cells had increased cellular resistance to cytotoxic therapy-induced apoptosis. Either TWEAK treatment or Fn14 overexpression in glioma cells resulted in the activation of NFkappaB and subsequently the translocation of NFkappaB from the cytoplasm to the nucleus. In addition, Fn14 activation induced BCL-XL and BCL-W mRNA and protein levels, and this effect was dependent upon NFkappaB transcriptional activity. Substitution of a putative NFkappaB binding site identified in the BCL-X promoter significantly decreased Fn14-induced transactivation. Furthermore Fn14-induced transactivation of the BCL-X promoter was also diminished by the super-repressor IkappaBalpha mutant, which specifically inhibits NFkappaB activity, and by mutations in the NFkappaB binding motif of the BCL-X promoter. Additionally small interfering RNA-mediated depletion of either BCL-XL or BCL-W antagonized the TWEAK protective effect on glioma cells. Our results suggest that NFkappaB-mediated up-regulation of BCL-XL and BCL-W expression in glioma cells increases cellular resistance to cytotoxic therapy-induced apoptosis. We propose that the Fn14 protein functions, in part, through the NFkappaB signaling pathway to up-regulate BCL-XL and BCL-W expression to foster malignant glioblastoma cell survival. Targeted therapy against Fn14 as an adjuvant to surgery may improve management of invasive glioma cells and advance the outcome of this devastating cancer.

Members of the tumor necrosis factor (TNF) 1 family are type II transmembrane proteins that are involved in the regulation of various cellular responses, including proliferation, differentiation, and apoptosis (1,2). These proteins can exist as both membrane-associated and soluble forms and generally function as homotrimers (1). TNF-like weak inducer of apoptosis (TWEAK) is a type II membrane protein of the TNF ligand superfamily (3) that induces various cellular responses such as proliferation, survival, apoptosis, and migration (4). In both endothelial cells and astrocytes, TWEAK promotes cell proliferation and not death (5)(6)(7). TWEAK can also stimulate angiogenesis in vivo (8).
The receptor for TWEAK, fibroblast growth factor-inducible 14 (Fn14), is a member of the TNF superfamily of receptors and is characterized as a type Ia transmembrane receptor lacking a cytoplasmic death domain (9 -11). Fn14 is an immediate-early response gene whose expression is directly activated following exposure to growth factors, fetal calf serum, and phorbol ester in fibroblasts (9,10). Human Fn14 contains 129 amino acids, making it the smallest member of the TNF receptor superfamily identified to date (9 -11). The expression of Fn14 is high in a variety of tissues including heart, placenta, kidney, lung, and pancreas and is relatively low in brain and liver (10). In cancerous tissues, Fn14 expression is elevated in hepatocellular carcinomas (10), glioblastoma multiforme (12), and pancreatic cancer (13). In addition, TWEAK binding to Fn14 or overexpression of Fn14 protein promotes nuclear factor-B (NFB) pathway activation that may drive the expression of several NFB-regulated genes (14). In fact, the cytoplasmic domain of the Fn14 receptor contains a single TNF receptor-associated factor binding site flanked by two conserved threonine residues (11,14). TNF receptor-associated factors link transmembrane receptors to the NFB pathway and several serine/threonine protein kinase cascades, including c-Jun NH 2 -terminal kinase, p38, and extracellular signal-regulated kinase, that generally function to promote cellular survival and proliferation (15).
Dysregulated NFB proteins play a role in malignant transformation by either providing continued positive growth stimuli such as that mediated by cytokines or by inhibiting apoptotic pathways (16). NFB functions as a dimer composed of the RelA (p65) and NFB1 (p50) or NFB2 (p52) subunits. In normal resting cells, NFB is sequestered in the cytoplasm by virtue of binding to IB (17,18). Cytokines such as TNF, interleukin-1, and epidermal growth factor trigger a cascade of signaling events after binding to their transmembrane receptors ultimately leading to the activation of IB kinase, which phosphorylates IB at two serine residues, Ser-32/36 (19,20). Phosphorylated IB is rapidly ubiquitinated and degraded through the 26 S proteosome pathway, releasing NFB. Free NFB translocates to the nucleus and binds to the promoter regions of target genes and activates their transcription (17,21).
Both BCL-2 and BCL-X L are NFB-inducible genes. Members of the BCL-2 gene family include the proapoptotic proteins BAD, BIK, and BID and the antiapoptotic proteins BCL-2, BCL-X L , and BCL-W (22). High expression levels of antiapoptotic BCL-2-related proteins have been found in many tumors, and up-regulation of BCL-2 and BCL-X L has been shown to be a key element in malignancy (23) and drug resistance (24,25). BCL-2 overexpression has been observed in several glioma cell lines and in glioma biopsies of various histological grade (26,27) and may confer resistance to radiotherapy and chemotherapy (28 -30). Malignant glioblastoma multiforme displays highly infiltrative behavior and resistance to chemo-and radiotherapy constituting major obstacles for successful therapy and patient outcome (31,32).
To elaborate the role of Fn14 in glioma pathobiology, we examined Fn14 activation as a potential mechanism by which cell survival is fostered. We showed that TWEAK-stimulated glioma cells had increased cellular resistance to cytotoxic therapy-induced apoptosis. In addition, we demonstrated that activation of NFB by the TWEAK-Fn14 ligand-receptor system underlies the molecular basis for resistance to apoptosis induction in glioma cells. Moreover our data indicated that NFB protected glioma cells from cytotoxic therapy-induced apoptosis, in part, by up-regulating expression of the BCL-X L and BCL-W proteins.

MATERIALS AND METHODS
Cell Culture Conditions-Human astrocytoma cell lines T98G (American Type Culture Collection (ATCC), Manassas, VA) and SF767 were maintained in minimum essential medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT) in a 37°C, 5% CO 2 atmosphere at constant humidity. For adenoviral infection, 1.5 ϫ 10 5 cells were plated into a 6-well plate and cultured for 24 h prior to infection.
Antibodies, Reagents, Western Blot Analysis, and Immunofluorescence-Polyclonal antibodies to IB␣ and BCL-2 and monoclonal antibody to BAX were obtained from Cell Signaling Technology Inc. (Beverly, MA). Antibody to the p65 subunit of NFB was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Poly(ADP-ribose) polymerase (PARP) antibodies and the ␣-tubulin monoclonal antibody were obtained from Upstate Biotechnology (Lake Placid, NY). BCL-W polyclonal antibodies were obtained from Stressgen Biotechnologies (San Diego, CA), and monoclonal antibodies specific to BCL-X L were purchased from Zymed Laboratories Inc.. Monoclonal antibodies recognizing both BCL-X L and BCL-X S were obtained from Chemicon International (Temecula, CA). Monoclonal antibody to proliferating cell nuclear antigen was obtained from BD Transduction Laboratories. Human recombinant TWEAK was purchased from PeproTech (Rock Hill, NJ), and human recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was purchased from BIOSOURCE. Laminin from human placenta was obtained from Sigma.
Immunoblotting and protein determination experiments were performed as described previously (33). Briefly monolayers of cells were washed in phosphate-buffered saline containing 1 mmol/liter phenylmethylsulfonyl fluoride and then lysed in 2ϫ SDS sample buffer (0.25 M Tris-HCl, pH 6.8, 2% SDS, 25% glycerol) containing 10 g/ml aprotinin, 10 g/ml leupeptin, 0.7 g/ml pepstatin, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using the BCA assay procedure (Pierce) with bovine serum albumin as a standard. Thirty micrograms of total cellular protein were loaded per lane, separated by 12% SDS-PAGE, and then transferred to nitrocellulose (Schleicher & Schuell) by electroblotting at 4°C. The nitrocellulose membrane was blocked with 5% nonfat dry milk in Tris-buffered saline, pH 8.0, with 0.1% Tween 20 prior to addition of primary antibodies and then horseradish peroxidase-conjugated anti-rabbit or -mouse IgG (Promega, Madison, WI). Protein bands were identified by chemiluminescence and exposed on X-Omat AR film (Eastman Kodak Co.).
Immunofluorescence microscopy was performed as described previously (33). Briefly cells were plated onto 10-well glass slides precoated with 1 g/ml laminin and then cultured for 24 h. In certain experiments, cells were pretreated for 30 min with either 50 M SN50 or SN50M (Calbiochem) prior to TWEAK addition. Cells were fixed for 5 min in 4% paraformaldehyde in phosphate-buffered saline followed by permeabilization with 0.5% Triton X-100 in 10 mM PIPES, pH 6.8, 50 mM NaCl, 3 mM MgCl 2 , 0.3 M sucrose for 5 min at 4°C. Cells were then blocked with 2% bovine serum albumin and 1% goat serum and incubated with primary antibodies followed by secondary Cy3-conjugated anti-mouse or anti-rabbit IgG. Cell nuclei were also stained with 4Ј,6Јdiamidino-2-phenylindole hydrochloride (DAPI) for 15 min at 37°C. Immunofluorescent samples were examined under an LSM 5 Pascal laser scanning confocal microscope (Zeiss, Thornwood, NY) or a microscope equipped with a rhodamine filter for Cy3 fluorescence and a 450 -490 nm band pass excitation filter and 515 nm long pass emission filter for DAPI fluorescence.
Preparation of Recombinant Adenoviruses and Infection-The human Fn14 wild-type (Fn14wt) cDNA in pBluescript, cDNA encoding the murine epitope-tagged truncated Fn14 protein missing amino acid residues 112-129 (pSecTag2/Fn14tCT-myc) (14), and cDNA to the superrepressor IB mutant (S32A/S36A) in pCMV-IB␣M (Clontech) were excised and subcloned into the adenoviral shuttle vector pShuttle-CMV to prepare recombinant E1-deleted adenoviruses using the Ad-Easy system as described previously (34). Expression of untagged Fn14wt and IB␣M proteins were confirmed in transiently transfected COS-7 cells by Western blot analysis using anti-Fn14 (9) and anti-IB␣ (Cell Signaling Technology Inc.) antibodies, respectively. Expression of murine epitope-tagged truncated Fn14 (Fn14tCT-ad) was confirmed by Western blot analysis using an anti-Myc antibody (Cell Signaling Technology Inc.). Viruses were propagated in 293 cells (ATCC CRL 1573), clonally isolated, and titered. Cells were infected at matched multiplicity of infection ranging from 5 to 20.
RNA Isolation and Quantitative Reverse Transcription-PCR-Total RNA was extracted, and quantitative reverse transcription-PCR was performed using a LightCycler (Roche Diagnostics) with SYBR green fluorescence signal detection after each cycle of amplification as described previously (12). Briefly total RNA was isolated from cultured glioma cells using the RNeasy kit (Stratagene). cDNA was synthesized from 1 g of DNase I-treated total RNA in a 20-l reaction volume using the Retroscript kit (Ambion Inc., Austin, TX) for 60 min at 42°C. PCR was performed on 2 l of the cDNA in a final volume of 20 l using the following primers: BCL-W: sense, 5Ј-GAG CCA TAT AGT TCC TTG GGA-3Ј; antisense, 5Ј-TAG AAT AAG TGG GGA GTG GGA-3Ј; BCL-X L : sense, 5Ј-GAA CGG CGG CTG GGA TAC TTT T-3Ј; antisense, 5Ј-GAG AAG GGG GTG GGA GGG TAG A-3Ј (specific to BCL-X L ); BCL-2: sense, 5Ј-TAT CCA ATC CTG TGC TGC TAT C-3Ј; antisense, 5Ј-ACT CTG TGA ATC CCG TTT GAA-3Ј; Bax: sense, 5Ј CCG GAA TTC CGG ATG GAC GGG TCC GGG GAG CAG-3Ј; antisense, 5Ј TGC TCT AGA GCA TCA GCC CAT CTT CTT CCA G-3Ј; and histone H3.3: sense, 5Ј-CCA CTG AAC TTC TGA TTC GC-3Ј; antisense, 5Ј-GCG TGC TAG CTG GAT GTC TT-3Ј. To distinguish the BCL-X splice variants, the following primers were used to amplify BCL-X L and BCL-X S simultaneously: sense, 5Ј-TTG GAC AAT GGA CTG GTT GA-3Ј; antisense, 5Ј-GTA GAG TGG ATG GTC AGT G-3Ј as previously published by Bargou et al. (35). The PCR data were analyzed with the LightCycler analysis software, and quantification based on the number of cycles necessary to produce a detectable amount of product above background was performed as described previously (12,36). Specificity of the PCR product amplification was verified by analyzing the melting curves for standard and sample products (37) along with electrophoresis of the PCR products in a 2% agarose gel followed by ethidium bromide staining for determination of amplicon size.
Transient transfection of siRNA was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were plated in a 6-well plate at 2.0 ϫ 10 5 cells/well in 1.5 ml of Dulbecco's modified Eagle's medium supplemented with 10% serum and without antibiotics. Transfections were carried out according to the manufacturer's protocol after cells were fully spread (6 h postplating). BCL-X L and BCL-W small interfering RNAs were transfected at 20 nmol/liter. No cell toxicity was observed at 20 nmol/liter siRNA. Using quantitative PCR, we verified that the siRNA oligos to BCL-X L and BCL-W specifically inhibited the expression of BCL-X L and BCL-W, respectively, but not other members of the BCL-2 family (i.e. BCL-2 and BAX). Maximum inhibition was achieved by day 2-3 after transfection, and cells were assayed at day 3 or 4 post-transfection.
Apoptosis Assays-Apoptotic cells were evaluated by nuclear morphology of DAPI-stained cells as described previously (40). Briefly cells with condensed, fragmented chromatin were manually scored as apoptotic cells. At least five fields (total of 1000 cells) were evaluated, and data are reported as apoptotic cells/total cells ϫ 100. Verification of apoptotic cells was conducted by co-immunofluorescence staining using a monoclonal antibody against activated cleaved caspase 3 (Promega). At least 1000 cells per treatment were evaluated for condensed chromatin and activated caspase 3. In addition, nuclear PARP proteolytic cleavage was assessed by immunoblotting analysis of cellular lysates as described above using antibodies recognizing both the intact (116-kDa) and proteolytic (85-kDa) forms of PARP. In certain experiments, TWEAK (100 ng/ml) was preincubated with Fn14-Fc decoy receptor (2.5 g/ml) or control mouse IgG as described previously (7,12).
Transfection and Dual Luciferase Reporter Assays-Dual luciferase reporter assays were performed according to the manufacturer's protocols (Promega). Cells were plated in a 24-well tissue culture dish and then incubated in normal growth medium for 24 -48 h until 70% confluency was reached. Cells were transiently transfected with 0.5 g of 3ϫ tandem NFB luciferase reporter gene (Clontech), BCL-X promoter/ luciferase reporter gene pGL2(848) or mutant pGL2BM (41), or pGL basic promoter/enhancerless luciferase reporter gene (Promega) using Effectene reagent (Qiagen). In some cases, cells were co-transfected with pCMV-IB␣M or control pcDNA3.1 (1 g). As an internal standard, all plasmids were co-transfected with 50 ng of pRL-TK (Promega), which contains the Renilla luciferase gene. At 6 h post-transfection, the medium was replaced with Dulbecco's modified Eagle's medium supplemented with 0.1% fetal bovine serum. In certain experiments cells were either treated with 100 ng/ml TWEAK or infected with adenoviruses expressing Fn14wt, Fn14tCT, or LacZ. All cells were harvested 48 h after transfection, washed two times with phosphate-buffered saline, and lysed in passive lysis buffer. All treatments were done in triplicate for each experiment. Luciferase activity was measured using a TD-20/20 luminometer (Turner Designs, Sunnydale, CA) and normalized against the activity of the Renilla luciferase gene for differences in transfection efficiency. The results were expressed as relative light units of luciferase activity to Renilla activity.

TWEAK Protects Glioma Cells from Cytotoxic Therapy-induced Apoptosis through Activation of the Fn14
Receptor-TRAIL, a member of the TNF family, exhibits selective cytotoxicity for glioma cells versus non-neoplastic astrocytes in vitro (42). Unlike TRAIL, TWEAK induces cell proliferation and not apoptosis when added to astrocytes (5,6,8,43). Here we investigated the effect of TWEAK on glioma cell survival. TRAIL treatment of T98G glioma cells induced apoptosis (Fig.  1A) indicated by cells showing condensed, fragmented chromatin revealed using a DAPI nuclear morphology staining technique (40). Apoptotic cells were further validated by co-immunofluorescent detection of activated caspase 3 (data not shown). Pretreatment of cells with TWEAK for 2 h prior to TRAIL addition reduced TRAIL-induced apoptosis by 50% (Fig. 1A). Progressive reduction of the percentage of cells undergoing TRAIL-induced apoptosis was observed using longer preincubations with TWEAK. No changes in base-line apoptosis was observed in cells treated with TWEAK alone (Fig. 1A). Immuno-blot analysis examining nuclear PARP proteolytic cleavage revealed the presence of the 85-kDa proteolytic fragment of PARP in TRAIL-treated T98G cells (Fig. 1B). PARP cleavage was not observed in cells treated with TWEAK. To investigate whether TWEAK protection of glioma cells from TRAIL-induced apoptosis was a result of Fn14 receptor interaction, a soluble Fn14-Fc decoy receptor was applied (7). When TWEAK was preincubated with the Fn14-Fc decoy receptor before application to the cells TWEAK did not prevent TRAIL-mediated proteolytic cleavage of PARP. In contrast, control mouse IgG did not prevent the TWEAK protective effect (Fig. 1B). These results suggest that TWEAK induces glioma cell survival via activation of the Fn14 receptor. We also observed that TWEAK treatment of the TRAIL-resistant glioma cell line SF767 inhibited camptothecin-induced apoptosis (Fig. 1C).
To confirm that the biological effect of TWEAK on glioma cell survival was a result of signaling initiated by interaction with the Fn14 receptor, we infected both T98G and SF767 glioma cells with recombinant replication-deficient adenoviruses expressing either Fn14wt or a truncated Fn14 protein missing amino acids 112-129 (Fn14tCT), a region containing the TNF receptor-associated factor binding sequence motif as described by Brown et al. (14). It has been reported previously that Fn14tCT cannot activate downstream pathways such as the NFB pathway (14). We found that overexpression of Fn14tCT inhibited TWEAK suppression of cytotoxic therapy-induced apoptosis as compared with overexpression of Fn14wt (Fig. 2, A, lanes e and f, and B, lanes o and p). Whereas expression of Fn14wt had no cytotoxic effect on glioma cells (Fig. 2, A, lane g, and B, lane q), apoptosis was detected in cells expressing Fn14tCT (Fig. 2, A, lane j, and B, lane t). To determine whether Fn14 overexpression could promote cell survival, glioma cells were infected with Fn14wt adenoviruses prior to addition of cytotoxic agents. Overexpression of Fn14 in the absence of TWEAK resulted in the suppression of cytotoxic therapy-induced apoptosis (Fig. 2, A, lane h, and B, lane r). Co-infection of glioma cells with both Fn14wt and Fn14tCT sensitized glioma cells to cytotoxic therapy-induced apoptosis (Fig. 2, A, lane i,  and B, lane s). These findings indicate that Fn14 signaling can enhance glioma cell survival.
TWEAK Promotes NFB Activation and IB␣ Phosphorylation in Glioma Cells-Previous studies have shown that TWEAK treatment of various cell types stimulates NFB activation (7, 14, 44 -47). To determine whether TWEAK promotes NFB activation in glioma cells, we examined the localization of NFB by immunofluorescence in glioma cells following TWEAK stimulation. T98G cells were cultured under low serum conditions (0.1% fetal bovine serum) for 16 h prior to TWEAK addition. Cells were fixed and immunostained for the p65 subunit of NFB. Localization of NFB was observed in the cytoplasm of untreated cells (Fig. 3A, a, asterisks). Cell nuclei were identified by co-staining with DAPI (data not shown). Cells treated with TWEAK showed translocation of the p65 subunit of NFB from the cytoplasm to the nucleus after 2-4 h (Fig. 3A, b and c, arrows) similar to treatment with the positive control NFB activator phorbol 12-myristate 13-acetate (Fig.  3A, d). Blocking TWEAK binding to endogenous Fn14 by preincubation with Fn14-Fc decoy receptor prevented TWEAKinduced NFB translocation to the nucleus (Fig. 3A, e). However, preincubation of TWEAK with control mouse IgG did not prevent NFB translocation to the nucleus (Fig. 3A, f). To further validate TWEAK induction of NFB nuclear translocation, we isolated the nuclear fraction of cells stimulated with TWEAK and immunoblotted for the NFB p65 protein subunit. Densitometric analysis revealed an increased level of p65 protein in the nuclear lysates of cells exposed to TWEAK with a 2-fold increase after 30 min compared with untreated cells (Fig.  3B), consistent with the immunofluorescence findings.
NFB translocation and transcriptional activity are inhibited by its association with IB␣ proteins (48). For NFB nuclear translocation and transcriptional activation to occur, phosphorylation of IB has to occur at serine residues 32 and 36, which results in proteosome-mediated IB␣ degradation and liberation of NFB (17). Immunoblot analysis of whole cellular lysates of T98G cells after TWEAK treatment using an anti-IB␣ antibody showed an induction of IB␣ phosphorylation over the indicated time (Fig. 3C). Phosphorylation of IB␣ was detected after 30 min and persisted to the 4-h time point. Concomitantly the level of endogenous IB␣ protein decreased, as expected, upon TWEAK treatment, corresponding to IB␣ phosphorylation. In addition, translocation of NFB to the nucleus in glioma cells promoted high transcriptional activity as shown in Fig. 3D. T98G cells were transfected with the NFB enhancer/luciferase reporter plasmid and the Renilla luciferase gene to correct for transfection efficiency. Luciferase assays revealed that upon TWEAK treatment there was a 45-fold induction of NFB transcriptional activity over the activation level detected in the vector-transfected cells. However, TWEAK-stimulated NFB transcriptional activity was sup- pressed when cells were co-transfected with the super-repressor form of IB␣ mutant (IB␣M), which specifically suppresses NFB activity (Fig. 3D).
Regulation of BCL-X L and BCL-W Expression by the TWEAK-Fn14 Ligand-Receptor System Is Dependent upon NFB Activity-NFkB has been shown to regulate the expression of genes that actively participate in controlling cell survival (BCL-2, BCL-X L , survivin, and the inhibitor of apoptosis protein family) (17,21). To understand the mechanisms of TWEAK-induced inhibition of apoptosis in glioma cells, we examined the mRNA levels of several BCL-2-related genes, including both antiapoptotic genes (BCL-2, BCL-W, and BCL-X L ) and proapoptotic genes (BCL-X S and BAX) using real time quantitative PCR analysis. We reasoned that increased resistance to apoptosis might be attributed to increased expression of the antiapoptotic genes. Cells treated with TWEAK for various lengths of time showed no changes in BCL-2 and BAX mRNA levels, and BCL-X S mRNA expression could not be detected in unstimulated or TWEAK-stimulated cells (data not shown). However, BCL-X L and BCL-W transcripts were induced in a time-dependent manner upon TWEAK treatment. Increased BCL-X L and BCL-W mRNA expression was detected at 4 h, and maximal induction was noted at the last time point examined, 24 h (Fig. 4A). The effect of TWEAK on BCL-X L and BCL-W mRNA expression was inhibited when cells were infected with an adenovirus expressing the Fn14tCT protein (Fig. 5A, lane d).
To determine whether the regulation of BCL-X L and BCL-W

FIG. 3. Effect of TWEAK treatment on NFB cellular localization, IB␣ phosphorylation, and NFB activation in glioma cells.
A, T98G cells were cultured under reduced serum and then treated with solvent control phosphate-buffered saline (a) TWEAK (100 ng/ml) for 2 (b) or 4 h (c) or with TPA (100 nM) for 2 h (d). In certain experiments, TWEAK was preincubated with soluble Fn14-Fc decoy receptor (e) or control mouse IgG (f) prior to addition. Cells were fixed and stained for the p65 subunit of NFB. Arrows indicate NFB p65 immunostaining in the cell nucleus (N), and asterisks indicate NFB p65 in the cytoplasm. Bar, 20 M. B, T98G cells were treated with 100 ng/ml TWEAK for various time periods as indicated above. Following treatment the cells were lysed, and nuclear extracts were prepared and immunoblotted for either the p65 subunit of NFB or proliferating cell nuclear antigen (PCNA). C, whole cell lysates of TWEAK-treated cells were immunoblotted for phospho-IB␣, total IB␣, or ␣-tubulin. D, cells were co-transfected with the NFB enhancer/luciferase reporter plasmid and the pRL-TK plasmid in combination with either the pCDNA3.1 vector (V) or the pCMV-IB␣M (IB␣M) plasmid. Cells were then cultured for 16 h under reduced serum conditions prior to TWEAK addition. After 6 h, cell lysates were prepared and assayed for luciferase and Renilla activity using the dual luciferase reporter assay system. The promoter activity was normalized to the activity of the Renilla and further normalized to untreated cells. The results are expressed as relative light units of luciferase activity to Renilla activity. Values represent mean and S.D. of observations from three independent experiments, each carried out in duplicate. WB, Western blot.

TWEAK-Fn14 Regulates Glioma Cell Survival
mRNA expression by TWEAK was dependent upon NFB activity, we inhibited NFB activation using a cell-permeable pharmacological peptide inhibitor, SN50. SN50 contains a nuclear localization sequence of the p50 subunit of NFB and functions to inhibit the translocation of the NFB active complex into the nucleus (49). TWEAK-stimulated BCL-X L and BCL-W mRNA expression was repressed in the presence of SN50 (Fig. 5A, lane e), while the cell-permeable inactive control peptide, SN50M, had no effect on TWEAK-stimulated BCL-X L and BCL-W mRNA expression (Fig. 5A, lane f). Similarly cells infected with IB␣M also had reduced TWEAK-stimulated BCL-X L and BCL-W mRNA expression (Fig. 5A, lane g).
Changes in BCL-X L and BCL-W protein levels following TWEAK stimulation were analyzed by Western blot analysis. As shown in Fig. 4B, BCL-X L protein levels increased in a time-dependent manner upon TWEAK addition. Densitometric analysis of the BCL-X L signal intensity revealed a 4-fold induction after 24 h of TWEAK treatment. Interestingly an increase in BCL-X L protein levels was observed as early as 30 min. In comparison, little change in BCL-W protein expression was detected over 8 h of TWEAK exposure, but a maximal 2-fold increase was detected at the 16-and 24-h time points (Fig. 4B). In contrast, TWEAK did not change BAX or BCL-2 protein levels (Fig. 4B); in addition, BCL-X S protein expression was not detected in unstimulated or TWEAK-stimulated cells (data not shown). Moreover both inhibition of Fn14 signaling by Fn14tCT and NFB inactivation by IB␣M suppressed TWEAK-elevated BCL-X L and BCL-W protein expression after 24 h (Fig. 5B), corroborating the changes in mRNA expression.
TWEAK Addition or Fn14 Overexpression Transactivates the BCL-X Promoter via the NFB Pathway-We assessed whether TWEAK could enhance BCL-X promoter activity via NFB activation. Studies by Tsukahara et al. (41) identified the NFB binding motif at positions Ϫ848 to Ϫ840 of the BCL-X promoter. This putative NFB element is reported to be a binding site for the p65 and p50 subunits of NFB (41). T98G glioma cells were co-transfected with a luciferase reporter proceeded by a 5Ј deleted portion of the BCL-X promoter containing the NFB binding site (pGL(848)) along with the Renilla luciferase plasmid. As shown in Fig. 6, the luciferase activity in cells stimulated with TWEAK was ϳ8-fold higher than that in nontreated cells (compare lanes a and b). However, the luciferase activity in TWEAK-stimulated cells infected with Fn14tCT (Fig. 6, lane d) was almost equivalent to that in control cells. Similarly forced overexpression of Fn14 receptor independent of TWEAK resulted in a 6-fold induction of luciferase activity as compared with LacZ control (Fig. 6, compare lanes a and e). Furthermore a mutated NFB-luc plasmid, pGL2BM, which has the same length as pGL2(848) but possesses CC-to-GG mutations at positions Ϫ841 and Ϫ840 within the NFB motif,  6. Transactivation of the BCL-X promoter by TWEAK addition or Fn14 overexpression. T98G cells were transfected with the luciferase reporter plasmid pGL2(848) driven by a portion of the BCL-X promoter containing the NFB binding motif or with the pGL2BM construct, which contains mutations within the NFB binding motif of the BCL-X promoter. Cells were also co-transfected with the pTK Renilla to correct for transfection efficiency. Cells were then cultured under reduced serum for 16 h and then treated with or without TWEAK for an additional 6 h. In certain experiments, cells were infected with Fn14wt, Fn14tCT, or control LacZ adenoviruses. Cells were harvested in passive lysis buffer and analyzed for luciferase activity using the dual luciferase reporter system. The promoter activity was normalized to the activity of the Renilla and further normalized to untreated cells. The results are expressed as relative light units of luciferase activity to Renilla activity. Values represent mean and S.D. of observations from three independent experiments, each carried out in duplicate. displayed a reduced level of transactivation (Fig. 6, lanes c and  f). These results indicate that the TWEAK-Fn14 ligand-receptor system transactivates BCL-X promoter activity via the NFB pathway.
Inhibition of NFB Activity Antagonizes TWEAK-induced Cell Survival-To investigate the role of NFB in TWEAKinduced glioma cell survival, we antagonized NFB function using the super-repressor IB␣M mutant. T98G cells were infected with IB␣M or control LacZ adenoviruses. Cells were then pretreated with TWEAK for 6 h prior to addition of TRAIL. As described earlier, TWEAK conferred resistance to TRAIL-induced apoptosis (Fig. 7, lanes c and d). However, inhibition of NFB function by IB␣M suppressed TWEAKinduced cell survival (Fig. 7, lane e). Cell survival induced by forced overexpression of Fn14 was also inhibited by IB␣M (Fig. 7, lanes f and g). Similar data were observed for SF767 cells (data not shown). Taken together, our results show that TWEAK-Fn14 ligand-receptor signaling to the NFB transcription factor fosters glioma cell resistance toward cytotoxic therapy-induced apoptosis.
Small Interfering RNA-mediated Depletion of BCL-X L or BCL-W Inhibits TWEAK-induced Cell Survival-To further examine whether BCL-X L and BCL-W are critical for TWEAKmediated glioma cell survival, we inhibited the expression of BCL-X L and BCL-W by transient transfection of small interfering RNA oligonucleotide duplexes. Three independent siRNA oligos were designed against BCL-X L and BCL-W. The level of mRNA inhibition for each siRNA oligo to BCL-X L was ϳ75%, whereas the level of inhibition of BCL-W was ϳ80%, indicating that the inhibitory effect of the oligos is specific (Fig.  8A). The siRNA oligos to BCL-X L did not affect the expression levels of BCL-2 and BCL-W; similarly BCL-W siRNA oligos did not affect the expression levels of BCL-2 and BCL-X L (Fig. 8A). Inhibition of protein expression was also verified by Western blot analysis using antibodies specific to either BCL-X L (Fig.  8B) or BCL-W (Fig. 8C). Small interfering RNA-mediated depletion of BCL-X L resulted in a 2-fold induction of apoptosis in T98G (Fig. 8D, lanes k and o) and SF767 (Fig. 8E, lanes k and  o) cells in the presence of cytotoxic drugs as compared with cytotoxic drug-treated untransfected (Fig. 8, D and E, lane c) or control siRNA luciferase-transfected cells (Fig. 8, D and E, lane  g). Likewise depletion of BCL-W antagonized TWEAK-induced cellular survival in both T98G and SF767 cells (Fig. 8, D and E,  lane p). Interestingly, in T98G BCL-X L -depleted cells, TWEAK conferred a semiprotective effect on TRAIL-induced apoptosis, suggesting that BCL-W may compensate for the TWEAK survival effect (Fig. 8D, lane l). However, in SF767 cells, shutdown of BCL-X L suppressed camptothecin induced-apoptosis (Fig.  8E, lane l).
We next determined whether suppression of both BCL-X L and BCL-W would result in the enhancement of cytotoxic therapyinduced apoptosis. Shutdown of both BCL-X L and BCL-W expression in untreated or control luciferase siRNA-treated cells resulted in ϳ20 -25% cellular apoptosis (Fig. 8, D and E, lane q), highlighting the constitutive survival function of these mediators. In the presence of cytotoxic drugs, ϳ60 -80% apoptotic cells were observed, which was 20% higher compared with shutdown of either BCL-X L or BCL-W alone (Fig. 8, D and E, lane s). In addition, these cells were refractory to TWEAK-induced cell survival. Thus, these results suggest that both BCL-X L and BCL-W are critical for the TWEAK survival response.

DISCUSSION
In the present study we described a role for the TWEAK-Fn14 ligand-receptor system in promoting survival of glioma cells through the transcriptional regulation of two antiapoptotic BCL-2 family members, BCL-X L and BCL-W. We demonstrated that the NFB pathway is an important downstream target of TWEAK-Fn14 signaling using both a pharmacological peptide inhibitor (SN50) and a super-repressor IB␣ mutant. Activation of the Fn14 receptor resulted in elevated expression of BCL-X L and BCL-W. Inhibition of NFB function diminished Fn14-induced expression of BCL-X L and BCL-W. Likewise inhibition of BCL-X L and BCL-W by small interfering RNA oligos antagonized TWEAK regulation of cell survival, subsequently making glioma cells susceptible to cytotoxic therapy-induced apoptosis. Moreover we showed that the activation of the BCL-X promoter by Fn14 was regulated through NFB, supporting the notion that the TWEAK-Fn14 ligand-receptor system plays a role in apoptosis prevention in glioma cells.
Elevated NFB activity has been observed in various carcinoma cells and glioblastoma multiforme (50 -52). NFB activation in malignant cells can result in resistance to certain chemotherapeutic agents, irradiation, and cytokines, an important characteristic of malignant glioma (53)(54)(55)(56). This activation may contribute to cellular resistance to cytotoxic interventions by preventing apoptosis. We speculate that inhibition of NFB may confer sensitivity of glioma cells to these agents. TWEAK has been shown to induce NFB activation via the Fn14 receptor, which results in a rapid (14) and long lasting NFB activation via IB␣ and p100 regulation (45). Our results are consistent with this latter report; indeed both IB␣ phosphorylation and NFB nuclear translocation were observed for as long as 4 h post-TWEAK stimulation and then diminished over time. Previously we demonstrated that Fn14 mRNA is highly expressed in glioma cells in vivo and enhanced in migrating cells in vitro (12). It is possible that the pathophysiological roles of TWEAK-Fn14 signaling may contribute to constitutive NFB activity in invasive glioma cells and hence lead to resistance to chemo-and irradiation therapies.
Both BCL-X L and BCL-W belong to the subfamily of antiapoptotic BCL-2 family members that share several antiapoptotic features with BCL-2. These proteins are able to differentially block chemo-and irradiation therapy-induced cell death (24). The balance between antiapoptotic and proantiapoptotic BCL-2 family members has been described as a pri- mary event in determining the susceptibility to apoptosis through maintaining the integrity of the mitochondria and inhibiting activation of the caspase cascade (22). High expression levels of antiapoptotic BCL-2-related proteins have been found in many tumors, and up-regulation of these proteins has been shown to be a key element in tumor malignancy and drug resistance (22,24). BCL-2 and BCL-X L overexpression has been observed in several glioma cell lines and in glioma biopsies regardless of histological grade (26,27,57). Down-regulation of BCL-2 and/or BCL-X L expression using antisense oligonucleotides abolishes tumorigenicity and enhances chemosensitivity in human malignant glioma cells (57)(58)(59). In addition, overexpression of BCL-2 inhibits TRAIL-induced apoptosis in various carcinoma cell lines and also in gliomas (59). However, in this study, we observed changes in BCL-X L and BCL-W expression consequent to TWEAK-Fn14 signaling but no alterations in BCL-2 or BCL-X S expression. In addition, depletion of BCL-X L and BCL-W levels antagonized the TWEAK survival response, suggesting that up-regulation of BCL-X L and BCL-W may be critical for TWEAK protection against cytotoxic therapy-induced apoptosis.
The BCL-X promoter is distinct from the BCL-2 promoter and is regulated by different transcriptional activators (60,61). The BCL-X gene encodes a full-length pre-mRNA tran-script that is capable via alternative RNA splicing to produce several protein products with either antiapoptotic (BCL-X long) or proapoptotic (BCL-X short) activity (60). Different promoter regions have been described in the regulation of the expression of these splice variants (60). Differences in the use of the promoter region resulting in the increased expression of BCL-X L or BCL-X S are attributed to cell type and differentiation status of the cell (62). In certain cell types, transcription of the BCL-X gene is controlled by NFB (41,63). Binding sites for the active NFB subunits p56/RelA and c-Rel have been demonstrated using functional analysis of the BCL-X promoter (41,63,64). Our results demonstrate that TWEAK-Fn14 signaling is able to increase the promoter activity of the BCL-X gene and that this response is dependent upon NFB activation. TWEAK-mediated BCL-X promoter activation was profoundly inhibited by a super-repressor mutant of IB␣ (Fig. 6) or by introduction of mutations in the NFB-like element in the mouse BCL-X promoter constructs. Thus, these findings further support the role of NFB in TWEAK-induced BCL-X L expression.
The highest level of BCL-X L mRNA expression was observed at 24 h post-TWEAK stimulation. Although the protein level of BCL-X L corresponded to the mRNA expression at 24 h, rapid increase of this protein level was observed 30 min after TWEAK addition (Fig. 5A). These data argue for the presence of additional signal(s) from the TWEAK-Fn14 ligand-receptor system that potentially influence the protein stability of BCL-X L . One mechanism by which the cellular level of BCL-X L can be regulated is through the activity of the AKT kinase. Activated AKT increases BCL-X L protein stability through the phosphorylation of the proapoptotic protein BAD on Ser-136 (22). In the absence of activated AKT, BAD forms heterodimers with BCL-X L and prevents the release of cytochrome c from the mitochondria (65,66). This complex formation abrogates the antiapoptotic function of BCL-X L (67, 68), thus facilitating apoptosis via a cytochrome c-dependent pathway. Conversely when AKT is activated, BAD becomes phosphorylated and is sequestered in the cytoplasm by interacting with 14-3-3 scaffolding proteins; this in turn suppresses apoptosis (69). Preliminarily we observed phosphorylation of AKT on Ser-473 upon TWEAK stimulation and subsequently BAD phosphorylation on Ser-136. 2 Current investigations are exploring the signaling pathway(s) from the Fn14 receptor that may impact the stability and function of BCL-X L at the protein level.
There is presently little information available on the regulation of BCL-W and the mechanism by which it suppresses cell death. Although the promoter of BCL-W is not characterized, our data suggests that BCL-W expression is regulated through the NFB pathway since inhibition of NFB activity suppresses TWEAK induction of BCL-W expression. It has been proposed that BCL-W is localized to the mitochondria and nuclear envelopes, the same sites where BCL-X L and BCL-2 reside (70,71). Like BCL-2, increased levels of BCL-W can suppress cell death by blocking stress-activated protein kinase/ c-Jun NH 2 -terminal kinase activation (72). In addition, BCL-W is expressed in various tissues including the brain, testis, heart, and intestines (70,71) and plays an important antiapoptotic role in regulating the survival of neurons (73). Furthermore BCL-W expression is elevated in certain tumor cell lines of epithelial origin such as colonic, cervical, and breast cancer cells (71). In fact, our gene expression profiling of glioma cells from patient biopsy specimens identified BCL-W as a candidate gene up-regulated in invasive glioma cells (74). Immunohistochemical analysis of BCL-W in glioma biopsy specimens confirmed that BCL-W was expressed in the invading cancer cells but not in the neighboring normal brain cells, implying that BCL-W expression may be important for invasive glioma cell survival. This result is similar to those reported in infiltrative morphotypes of gastric cancer by Lee and colleagues (72).
Our study further indicates that Fn14 overexpression independent of TWEAK may drive the promoter activity of BCL-X, and this activity is not observed if there are mutations in the NFB binding motif. In fact, we found that glioma cells overexpressing Fn14 were able to suppress cytotoxic therapy-induced apoptosis, and cell survival was diminished when NFB activity was suppressed. These data are consistent with a previous study demonstrating that Fn14 overexpression in NIH 3T3 cells resulted in increased NFB transcriptional activity (14). In our earlier report, we demonstrated that Fn14 expression is induced in migration-activated glioma cells in vitro and significantly increases according to tumor grade with the highest levels in glioblastoma tissue specimens (12). In comparison, TWEAK mRNA levels are low in glioblastoma samples relative to normal brain tissue (12). It is possible that overexpression of Fn14 in glioblastoma multiforme may result in the aberrant activation of NFB resulting in increased transcriptional activity of survival factors such as BCL-X L and BCL-W. This may possibly explain how invasive glioma cells affect resistance toward chemotherapeutic and cytotoxic agents.
In the treatment of glioma, sensitivity or resistance of tumor cells to cytotoxic therapy has substantial clinical consequences. However, the molecular mechanisms and/or intrinsic factors controlling cellular resistance are not well understood. In the present study, regulation of two key NFB genes, BCL-X L and BCL-W, by the TWEAK-Fn14 ligand-receptor system enhanced glioma cell resistance to both TRAIL-and camptothecin-induced apoptosis. Our results offer a potential mechanism by which the TWEAK-Fn14 signaling system can contribute to the regulation of glioma cell survival potentially by up-regulation of BCL-X L and BCL-W expression. Thus, understanding the function of Fn14 may lead to the development of effective therapies against invasive gliomas.