Transforming Growth Factor-� and TumorNecrosis Factor-� Cooperate To InduceApoptosis in the Oligodendroglial Cell LineOLI-neu

Norbert Schuster,1 Herdis Bender,1,2 Oliver G. Rossler,3 Anja Philippi,1

Nicole Dunker,2 Gerald Thiel,3 and Kerstin Krieglstein2*1Department of Anatomy and Cell Biology, Medical Faculty, University of Saarland, Homburg/Saar, Germany2Department of Anatomy, University of Goettingen, Goettingen, Germany3Department of Medical Biochemistry and Molecular Biology, University of Saarland, Homburg/Saar, Germany

As shown previously, transforming growth factor-�(TGF-�) plays an important role during the period ofdevelopmental cell death in the nervous system. As withneurons, oligodendrocytes are generated in excess andeliminated by apoptosis. The present study was aimed atinvestigating the possible interaction of TGF-� with tu-mor necrosis factor-� (TNF-�) in the regulation of celldeath in oligodendroglial precursor cells and analyzingthe underlying signaling mechanisms. We show that bothfactors induce apoptosis independently, but cooperatewhen applied together. The investigation of the signalingevents revealed an important role of the JNK pathwayduring induction of apoptosis. TGF-� seemed to be moreefficient at inducing a release in cytochrome c from mi-tochondria than TNF-�. This might be the consequenceof decreased Bcl-xL levels observed in cells treated withTGF-� but not with TNF-�. Both factors stimulatedcaspase-3 activity, which could be inhibited bycaspase-8 or caspase-9 inhibitors. Therefore, we con-clude that TNF-� and TGF-� affect partially commonpathways but also regulate different steps in the apopto-tic cascade. © 2003 Wiley-Liss, Inc.

Key words: O-2A progenitors; apoptosis; proliferation;Bcl-xL; Smad

Neuronal cell death is an important process duringthe development of the nervous system of multicellularorganisms (Oppenheim, 1991; Pettmann and Henderson,1998). Not only neurons but also glial cells undergo ap-optosis as a normal step during development (Knapp et al.,1986; Barres et al., 1992; Casaccia-Bonnefil, 2000).

We have recently shown that transforming growthfactor-� (TGF-�) plays an important role in developmen-tal cell death in the nervous system. The cell numbers ofspecific neuron populations were increased after neutral-ization of endogenous TGF-� (Krieglstein et al., 2000).This increase could be correlated with a decrease ofcaspase activation and apoptosis. Similar results were ob-

tained in the chick retina, where in ovo application ofTGF-�-neutralizing antibodies resulted in reduced apo-ptosis within the central retina (Dunker et al., 2001). Weestablished a cell culture system using the oligodendroglialprecursor cell line OLI-neu to investigate the effects ofTGF-� on the survival of oligodendrocytes and to analyzethe signaling mechanisms involved (Schuster et al., 2002).In our previous study, we could show that TGF-� inducesapoptosis in OLI-neu cells involving Bcl-xL down-regulation and caspase-3 activation.

Tumor necrosis factor-� (TNF-�) is also involved inthe control of glial cell survival, proliferation, and differ-entiation. Cammer and Zhang (1999) could show thatdifferentiation of O4-positive cells into myelin basic pro-tein (MBP)-expressing cells was impaired in the presenceof TNF-�, without affecting the total number of cells inculture. The effect of TNF-� was dose dependent, andwith higher doses a cell loss could be observed (Cammerand Zhang, 1999). Agresti and coworkers (1996) reportedthat TNF-� synergizes with interferon-� (IFN-�) inblocking proliferation and differentiation of primary oli-godendroglial precursor cells as well (Agresti et al., 1996).Oligodendroglial precursors seem to be especially sensitiveto apoptosis (Scurlock and Dawson, 1999). In primaryoligodendrocyte precursor cells, both TNF-� and TGF-�induce growth arrest as well as apoptosis (Yu et al., 2000).

The aim of the present study was to investigate 1) thepossible interaction of TGF-� with TNF-� in the regu-lation of cellular survival in OLI-neu cells and 2) theanalysis of the underlying signaling mechanisms that might

The first two authors contributed equal to this work.

*Correspondence to: Kerstin Krieglstein, Department of Neuroanatomy,Center of Anatomy, University of Goettingen, Kreuzbergring 36, 37075Goettingen, Germany. E-mail: kkriegl@gwdg.de

Received 17 January 2003; Revised 25 March 2003; Accepted 18 April2003

Journal of Neuroscience Research 73:324–333 (2003)

© 2003 Wiley-Liss, Inc.

be involved. We could show that both factors induceapoptosis independently, but cooperate when applied to-gether. The investigation of the signaling events revealedan important role of the JNK pathway during apoptosisinduction. TGF-� seemed to be more efficient at inducingcytochrome c release from mitochondria than TNF-�.This might be a consequence of the decrease in Bcl-xLlevels observed in TGF-�-treated cells but not in cellstreated with TNF-�. Both factors stimulated caspase-3activity, which could be inhibited by the application ofeither specific caspase-8 or specific caspase-9 inhibitor.We conclude that TNF-� and TGF-� affect partiallycommon pathways but also regulate different steps in theapoptotic cascade.

MATERIALS AND METHODSAntibodies and Reagents

Recombinant TGF-�1 was purchased from R&D Sys-tems (Minneapolis, MN). TNF-� was from Calbiochem (BadSoden, Germany). The caspase-8 inhibitor (Z-IETD-FMK) andthe caspase-9 inhibitor (Z-LEHD-FMK) were also purchasedfrom Calbiochem. The cytochrome c-specific antibody wasfrom Pharmingen (San Diego, CA). All antibodies used wereobtained from Santa Cruz (Heidelberg, Germany): actin (sc-1616), Bcl-2 (sc-7382), Bcl-xL (sc-8392), Bax (sc-7480), p27(sc-527), phospho-ERK (sc-7383), p42/p44 (sc-93), pJNK (sc-6254), JNK (sc-7345), p38 (sc-7972), pp38 (sc 7973). Pro-pidium iodide and RNase I were obtained from Sigma (Deisen-hofen, Germany). Secondary anti-mouse, anti-goat, and anti-rabbit antibodies were purchased from Zymed (South SanFrancisco, CA; 115-035-003, 81-1620, 111-035-003).

Expression vectors. The GAL4 expression plasmidspFA2ATF-2 and pFA2Elk1 were purchased from Stratagene (LaJolla, CA). The GAL4-c-jun expression plasmid pGAL4-cjunwas a kind gift of Michael Karin (University of California, SanDiego). The GAL4 fusion proteins encoded by plasmidspFA2Elk1, pFA2ATF-2, and pGAL4-c-jun contained the fol-lowing transcriptional activation sequences: amino acids 1–283of CREB (GAL4-CREB), amino acids 307–428 of Elk-1(Gal4-Elk1), amino acids 1–96 of ATF2 (GAL4-ATF2), andamino acids 1–246 of c-jun (GAL4-c-jun).

Reporter constructs. Plasmid pUAS5luc has beendescribed recently (Thiel et al., 2000). The c-jun promoter/luciferase reporter plasmid pjunwtluc-1,600/�170 has been de-scribed elsewhere (Groot et al., 2000). Plasmid pGL3-HIV LTR(–120/�83; a kind gift of Jakob Troppmair, University ofWurzburg) contains HIV LTR sequences from –120 to �83,including two kB motifs. Plasmid pSBE was kindly provided byPeter ten Dijke (Amsterdam; Jonk et al., 1998).

Cell Culture

OLI-neu, an immortalized oligodendrocyte precursor cellline (Jung et al., 1995), was cultured in Dulbecco’s modifiedEagle’s medium (DMEM; Invitrogen, Karlsruhe, Germany)with 10% fetal calf serum (FCS; Invitrogen), N2 supplement(Invitrogen), and insulin (Sigma). For each experiment, cellswere cultured in OptiMEM medium containing 1% FCS, N2supplement, and insulin. When TGF-�1 or TNF-� was added,5 ng/ml medium of each factor were applied.

Sodium Dodecyl Sulfate-Polyacrylamide GelElectrophoresis and Western Blot Analysis

Cells were lysed in lysis buffer (Tris-HCl, pH 8, 100 mMNaCl, 0.5% Tween 20) supplemented with complete proteaseinhibitor cocktail (Roche, Mannheim, Germany). Proteins wereanalyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) according to the procedure of Laemmli.For Western blot analysis, proteins were transferred to a PVDFmembrane by tank blotting with 20 mM Tris-HCl, pH 8.7,150 mM glycine as transfer buffer. Membranes were blocked inphosphate-buffered saline (PBS) with 0.1% Tween 20 and 5%dry milk for 1 hr at room temperature. The membrane wasincubated overnight with the primary antibody (1:100) in PBS-Tween 20 with 1% dry milk. The membrane was then washedwith PBS-Tween 20 three times before incubatation with theperoxidase-coupled secondary antibody in a dilution of 1:10,000in PBS-Tween 20 with 1% dry milk. Signals were developed bythe Renaissance enhanced luminol reagent from NEN-Kodak(Boston, MA) and visualized in a chemiluminescence imager(Raytest, Straubenhardt, Germany).

Caspase-3 Assay

Cells were harvested in lysis buffer (Tris-HCl, pH 8,100 mM NaCl, 0.5% Tween 20). Twenty microliters per samplewere used for a 100-�l reaction. The caspase-3 fluorimetricassay was performed according to the manufacturer’s instruc-tions (Promega, Mannheim, Germany). For each sample, trip-licate reactions were performed. Additional reactions for eachsample were performed with caspase-3-specific caspase inhibi-tor. Samples were preincubated with or without inhibitor for30 min at 30°C. The fluorogenic caspase-3-specific substratewas added, and the reaction was incubated for additional an30 min at 30°C. For quantification of caspase activity, a flu-orometer was used (Deelux, Goedenstorf, Germany). The back-ground activity from reactions with inhibitor was subtractedfrom the values without inhibitor to obtain specific caspase-3activity.

Fluorescence-Activated Cell Sorting Analysis

Cells were harvested by trypsination for 5 min at roomtemperature, washed three times with PBS (137 mM NaCl,2.7 mM KCl, 1.4 mM KH2PO4, 4.3 mM NaHPO4 � H2O, pH7.4), and resuspended in 300 �l PBS. Resuspended cells werefixed by addition of 700 �l 100% ethanol and left for at least30 min at –20°C. Fixed cells were centrifuged and resuspendedin 800 �l PBS. After addition of 100 �l RNAseA (1 mg/ml) and100 �l propidium iodide (1 mg/ml), cells were incubated at37°C for 30 minutes. Ten thousand freshly stained cells werecounted and analyzed for their cell cycle distribution with a flowcytometer (Facs-Scan; Beckton Dickinson, Heidelberg, Ger-many). Cell cycle distribution analysis was performed with CellQuest software (Beckton Dickinson) based on the characteristicpeaks of 2n, intermediate, and 4n DNA content of the cells.Gates separating the different cell populations were set manually,and the software calculated the percentage of cells present in G1,S, and G2 phases of the cell cycle as well as in the sub-G1 peakrepresenting apoptotic cells.

TGF-� and TNF-� Cooperate To Induce Apoptosis 325

Luciferase Reporter Assay

Twenty thousand cells per well were seeded in a 96-welltissue culture plate. After 24 hr, cells were transfected with0.5 �g of the reporter construct and 50 ng of renilla luciferasecontrol vector (Promega) using the Effectene transfection re-agent (Qiagen, Hilden, Germany). After 3 hr, DNA complexeswere removed, and the experimental conditions were applied.After an additional 18 hr, cells were harvested in passive lysisbuffer (Promega) and assayed for luciferase activity in a lumi-nometer (Lumat B5067; Berthold, Bad Wildbad, Germany)using the dual-luciferase reporter system (Promega).

Cytochrome c Release Assay

For the detection of cytoplasmic cytochrome c as a resultof cytotoxic stimulation cells were treated with TNF-�(5 ng/ml) or TGF-� (5 ng/ml) or with TNF-�/TGF-�(5 ng/ml each). After an additional 24 hr, cells were harvested.Cells were lyzed with digitonin for 4 min on ice. After centrif-ugation for 2 min at 13,000 rpm, the supernatant (cytosolicfraction) was removed, and the pellet was resuspended in PBSwith 0.5% Triton X-100). After incubation for 10 min on ice,the supernantant (mitochondrial fraction) was removed. Pro-teins of the cytosolic fraction were analyzed by SDS-PAGE asdescribed above.

RESULTSTGF-� and TGF-� Induce Apoptosis

Because TGF-� is known as a contextually actingmolecule, we tested the hypothesis whether TGF-� andTNF-�, both known to induce apoptosis of oligodendro-glial cells (Yu et al., 2000; Schuster et al., 2002), mayactually cooperate in their capacity to induce apoptosis inOLI-neu cells, which are cells of oligodendroglial origin.Counts of cells with fragmented nuclei, collected assub-G1 peak by flow cytometry, revealed that TGF-� aswell as of TNF-� induce apoptosis in OLI-neu cells (Fig.1). However, when cells were treated with a combinationof TGF-� and TNF-�, the percentage of cells undergoingapoptosis was highly significantly increased.

Cell Death PathwayTo characterize the cell death pathway mediating the

cooperative actions of TNF-� and TGF-� we first at-tempted to identify the cascade that represents the execu-tion of the apoptotic cell death pathway. The first step thatultimately leads to the activation of the cell death programis the release of cytochrome c from mitochondria. Cyto-chrome c release can be identified and quantified byWestern blot analysis from the cytosolic fraction of treatedcells using cytochrome c-specific antibodies. As shown inFigure 2A, both TGF-� and TNF-� treatment of OLI-neu cells resulted in increased cytosolic concentrations ofcytochrome c. Quantification of cytochrome c release,documented in Figure 2B, revealed that this release ismore efficiently induced by TGF-� than by TNF-�. Thecombination of both factors resulted in an additional in-crease in cytochrome c release from mitochondria.

As has been established, the balance between pro-and antiapoptotic proteins of the Bcl2-family plays a crit-

Fig. 1. TGF-� and TNF-� induce apoptosis in OLI-neu cells.A: FACS analysis of OLI-neu cells following treatment with TNF-�,TGF-�, TNF-� plus TGF-�. Cells were treated incubated for 24 hr,harvested, and fixed in 70% ethanol. Cells were treated with RNAse Aand stained with propidium iodide for 30 min. Stained cells wereanalyzed and counted in a flow cytometer. The diagram shows thequantification of cells in the sub-G1 peak, representing apoptotic cells.B: Diagram showing quantification of cells in G1 phase. Data representthe mean of three independent experiments � SEM. P values derivedfrom Student’s t-test: *P � .01, ***P � .001 compared with controls.

326 Schuster et al.

ical role in the regulation of cytochrome c release frommitochondria (Chao and Korsmeyer, 1998). Therefore,expression levels of Bax (proapoptotic) as well as Bad,Bcl-xL, and Bcl2 (antiapoptotic) were determined. OLI-neu cells were treated for 24 hr with TGF-�, TNF-�, orcombinations of both. Cells were harvested, and proteinextracts were subjected to Western blot analysis using theappropriate antibodies (Fig. 3). The level of the antiapo-ptotic Bad protein remains unchanged under all condi-tions, whereas the Bcl-xL protein level is reduced in cellstreated with TGF-� or both factors. TNF-� does notreduce Bcl-xL levels when applied alone. The Bcl-2 levelremained unchanged when cells were treated with eitherfactor alone, but was decreased when both factors were

applied concomitantly. With all combinations, the level ofthe proapoptotic protein Bax did not change. Therefore,we conclude that TGF-� induces the release of cyto-chrome c more efficiently than TNF-� because of adown-regulation of the antiapoptotic protein Bcl-xL.When cells were treated with both factors together, anadditional down-regulation of Bcl-2 was observed, whichcould be responsible for the cooperative effect of bothfactors. p27 was used as a control for growth arrest inducedby TGF-� but not by TNF-�. As shown in Figure 3, p27is induced by TGF-� and not by TNF-�, which corre-sponds to their different biological effects on the G1 arrestin OLI-neu cells (Fig. 1B).

Because cytochrome c release leads to the activationof a cascade of caspases, we analyzed which caspases areinvolved and in which order they are activated. As Figure4 shows, both factors stimulated caspase-3 activity aloneand more efficiently when OLI-neu cells were treatedwith a combination of both factors. To address the ques-

Fig. 2. Cytochrome c release. A: Cells were treated with TNF-�,TGF-�, or TNF�/TGF� for 24 hr and harvested for cytochrome crelease assay as described in Materials and Methods. One hundredmicrograms of cytosolic fraction extracts were separated in a 12.5% SDSpolyacrylamide gel and blotted onto PVDF membrane. The membranewas incubated overnight with antibodies diluted 1:500 in PBS-T with1% dry milk. After washing, the membrane was incubated for 1 hr withsecondary antibody (diluted 1:10,000 in PBS-T with 1% dry milk). Thesignals were detected with a chemiluminescence imager as described inMaterials and Methods. B: Quantification of cytochrome c release (twoindependent experiments).

Fig. 3. Analysis of p27, Bad, Bcl-2, Bcl-xL, and Bax protein levels.Cells were treated with TNF-�, TGF-�1, or both factors together for24 hr and harvested for Western blot analysis. One hundred micro-grams of extract were separated in a 12.5% SDS-polyacrylamide gel andblotted onto PVDF membrane. The membrane was incubated over-night with antibodies diluted 1:100 in PBS-T with 1% dry milk. Afterwashing, the membrane was incubated for 1 hr with secondary anti-body (diluted 1:10,000 in PBS-T with 1% dry milk). The signals weredetected with a chemiluminescence imager as described in Materialsand Methods. Representative blots from three independent experi-ments.

TGF-� and TNF-� Cooperate To Induce Apoptosis 327

tion of which upstream caspases may be regulated byTGF-� or TNF-�, we used a caspase-3 assay to monitorthe activation of this important effector caspase in thepresence or absence of specific inhibitors of the upstreamkinases caspase-8 and caspase-9. When the cells werepreincubated for 30 min with either specific caspase-8 orspecific caspase-9 inhibitors, the activation of caspase-3was blocked. This indicates that both initiator caspasesseem to be required for efficient activation of caspase-3 inOLI-neu cells.

Regulation of the ERK Signaling Pathway Is anImportant Step in the Regulation of Apoptosis

The transduction of the TGF-�- and TNF-�-generated signals from the plasma membrane to the nu-cleus is accomplished by activation of signal-specific pro-tein kinases that subsequently phosphorylate transcriptionfactors in the nucleus. To measure an increase of proteinkinase activities in the nucleus, we used a transcription-based assay that relies on the fact that the activation do-mains of the transcription factors Elk-1, ATF2, and c-junrequire phosphorylation. The assay is based on the expres-sion of those phosphorylation-dependent transcriptionalactivation domains as fusion proteins together with theDNA-binding domain of the yeast transcription factorGAL4. Transcriptional activation can be monitored bycotransfection of the reporter plasmid pUAS5luc, whichcontains five copies of the GAL4 binding site termedupstream activating sequence (UAS) upstream of a luciferasereporter gene. The reporter plasmid pUAS5luc and one ofthe expression plasmids pFA2Elk1, pFA-ATF2, orpGAL4-c-jun that encoded the GAL4 fusion proteinsGAL4-Elk1, GAL4-CREB, GAL4-ATF2, or GAL4-c-jun were transfected into OLI-neu cells together with theplasmid pRL-TK that encoded renilla luciferase undercontrol of the herpes simplex virus thymidine kinase pro-moter to correct for variations in transfection efficiencies.Cells were treated with either TGF-� or TNF-� for18 hr, and cell extracts were prepared and luciferase ac-tivities determined. The activity of the GAL4-Elk-1 fusionprotein monitoring activity in the p42/p44 MAP kinasepathway was reduced to about 50% compared with con-trols by both factors alone or in combination (Fig. 5A).The investigation of the transcriptional activity of theATF2 activation domain (GAL4-ATF2), monitoring thep38 MAPK pathway, showed, as with the GAL4-c-Elk-1reporter, a slight decrease in reporter activity but no clearsuperimposed effect when both factors were applied to-gether (Fig. 5B).

Next, we analyzed the activity of transcription fac-tors known to be important in regulating life-and-deathdecisions in several cell systems. NF-B has been de-scribed as promoting cell survival in neurons, as a result ofthe up-regulation of antiapoptotic and antioxidant genes.Conversely, an elevated NF-B activity has been pro-posed to trigger cell death. This paradoxical situationmakes it very interesting to identify the signals leading toan enhanced transcription by NF-B and to elucidate thebiological outcome of an activated NF-B transcription

Fig. 4. Analysis of TGF-�- and TNF-�-induced caspase-3 activationin the presence of caspase-8 and -9 inhibitors. A: Cells were treated for24 hr with TNF-� or TGF-� or TNF/TGF-� or vehicle controls.B: Cells were treated as described above but were pretreated for 30 minwith caspase-8 or caspase-9 inhibitor (50 �M). Cell extracts wereanalyzed with regard to their caspase-3 activity. Data represent themean of five independent experiments � SEM.

328 Schuster et al.

factor. NF-B activity was analyzed using an NF-B-responsive HIV promoter/luciferase reporter gene. Figure5C shows that the transcriptional activity of NF-B wasincreased following stimulation of TNF-� but was notinfluenced by TGF-�.

Smad-mediated regulation of transcription has beeninvolved in the regulation of apoptosis. In Figure 5D, wedemonstrate that the Smad cascade was activated byTGF-� but not by TNF-�, insofar as the luciferase activityof the pSBE reporter was increased after TGF-� treat-ment. The cotreatment with TNF-� and TGF-� did notlead to a superstimulation of Smad-dependent reporteractivity, ruling out that the cooperative effect was inducedthrough a superstimulation of the Smad pathway.

Taken together, these data show that, upon TGF-�treatment, Smad proteins are activated by TGF-� but arenot influenced by TNF-�. To the contrary, TNF-� in-duces NF-B activity involved in cellular survival that isnot influenced by TGF-�. The p42/p44 MAP kinasepathway responsible for signaling survival is down-regulated in response to both factors and surprisingly alsop38 activity, which has been shown to be involved inproapoptotic events in many cell systems. However, verystriking is the cooperative effect of both growth factors onthe activity of the JNK cascade (Fig. 5E), which has beendescribed to be responsible for the active induction of celldeath in other cell systems.

Protein kinases of the diverse MAPK pathways re-quire phosphorylation for activation. Thus, detection ofphosphorylated ERK, JNK, or p38 by phosphospecificantibodies can be used as a measure to follow kinaseactivation induced by extracellular signaling molecules. Asshown in Figure 6A, JNK is activated both in TNF-�- andin double-treated cells, whereas TGF-� had no stimula-tory effect. The phosphorylation of p42/p44 MAPK isdecreased as well in TNF-�- and TGF-�-treated cells inthe same way as in double-stimulated cells (Fig. 6B).Phosphorylation of p38 decreases after application ofTNF-� and TGF-� and is more pronounced after co-stimulation with both factors (Fig. 6C), suggesting thatp38 plays no active role in the apoptotic process in OLI-neu cells as an induction of p38 would indicate. The roleof p38, however, is not restricted to the apoptotic pathwaybut has also been described to be of importance for pro-liferation, differentiation, and cytoskeletal organization ofdifferent cell types (Ono and Han, 2000), so an activationof p38 in response to TGF-�-mediated apoptosis is notmandatory.

With these findimgs taken together, we could showthat both TNF-� and TGF-� independently induce ap-optosis, but cooperate when applied in combination. Theinvestigation of the signaling events revealed an importantrole of the JNK pathway during apoptosis induction.TGF-� seemed to be more efficient in inducing a releasein cytochrome c from the mitochondria compared withTNF-�. This may be due to a decrease in Bcl-xL levelsthat could be observed only in TGF-�-treated cells andnot in cells treated with TNF-�. Both factors stimulated

Fig. 5. Analysis of signaling cascades induced by TNF-� and TGF-�.Cells were transfected with reporter constructs as described in Materialand Methods and treated with TNF-�, TGF-�, and TNF-�/TGF-�for 24 hr. Cells were harvested in passive lysis buffer, and 10 �l wereused for luciferase assay. A: Transcriptional activity of Gal4-ELK1fusion protein monitoring p42/p44 MAPK activity. B: Transcriptionalactivity of the Gal4-ATF2 fusion protein monitoring p38 activity. C:Transcriptional activity of HIV promoter luciferase reporter gene mon-itoring NF-B activity. D: pSBE reporter monitoring Smad activation.E: Transcriptional activity of a c-jun promoter luciferase reporter genemonitoring JNK activity. Data are given as relative light units (RLU).Each panel represents mean from three independent experiments �SEM. P values derived from Student’s t-test: *P � .01 compared withvalues from single treatments.

TGF-� and TNF-� Cooperate To Induce Apoptosis 329

caspase-3 activity, which could be inhibited by the appli-cation of either specific caspase-8 or specific caspase-9inhibitor, suggesting that TNF-� and TGF-� affect par-tially common pathways but also regulate different steps inthe apoptosis cascade.

DISCUSSIONIn the present study, we demonstrate that TGF-�

and TNF-� induce apoptosis in OLI-neu cells, a cell lineestablished from oligodendroglial precursor cells (Jung etal., 1995). Both factors cooperate to induce apoptosis inthis cell line. It has already been described that both factorsalone are capable of inducing apoptosis in primary oligo-dendroglial progenitors (Yu et al., 2000). However, themolecular mechanisms and possible interactions of both

growth factors remained unclear. We have shown beforethat OLI-neu cells undergo apoptosis in response toTGF-� and that this cell line is a valid model system withwhich to investigate cell death of oligodendroglial precur-sors in culture compared with primary precursor cells(Schuster et al., 2002). Furthermore, it has been shownthat OLI-neu cells represent oligodendrocytes in manyaspects, among them glia–neuron interaction and migra-tion (Jung et al., 1995; Fok-Seang et al., 1995). ForSchwann cells, a clear synergy between TGF-� andTNF-� has been described (Skoff et al., 1998). Our resultsimply that glial cells of the central nervous system (CNS)and the peripheral nervous system (PNS) seem to react ina similar but not an equal way. In Schwann cells, bothfactors are unable to induce apoptosis alone, but this isdifferent in OLI-neu cells; TGF-� as well as TNF-� caninduce apoptosis alone but are more efficient when ap-plied together.

With regard to the data presented here, we alsotested the hypothesis that the capactity of the individualfactors to induce cell death may rely on the cooperativeeffect with a possibly endogenous other factor. However,the corresponding experiments, applying TGF-� in com-bination with anti-TNF-� antibodies and TNF-� in com-bination with anti-TGF-� antibodies, did not inhibit theinduction of apoptosis (data not shown), suggesting thateach of the factors does have an individual capacity toinduce cell death of OLI-neu cells.

It seems that TGF-� is more efficient at inducing therelease of cytochrome c into the cytoplasm than is TNF-�.This may reflect the ability of both factors to regulatedifferent events in the apoptotic cascade. Especially theability of TGF-� to reduce Bcl-xL protein levels may beresponsible for these differences, insofar as TNF-� doesnot reduce Bcl-xL levels in OLI-neu cells.

In unstimulated cells, Bcl-xL sequesters APAF-1 andinhibits caspase activation (Hu et al., 1998; Pan et al.,1998). When Bcl-xL level is reduced, free APAF-1 canrecruit and activate procaspase-9 in a cytochromec-dependent manner (Li et al., 1997). Because the releaseof cytochrome c precedes the initiation of the caspasecascade at the mitochondrial level, we also investigated therole of the initiator caspases-8 and -9 in the apoptoticprocess. TNF-� is able to initiate apoptosis by directlyactivating caspase-8 at the level of TNF-RI via the deathdomain of the receptor (Baker and Reddy, 1998). Ourdata show that that the specific caspase-8 inhibitor caninhibit the apoptotic process induced by TNF-�. This isalso true for the caspase-9-specific inhibitor, which isexpected, because, in the classical model of caspase action,the induction of caspase-8 requires the following activa-tion of caspase-9 to enhance the apoptotic signal (Kumar,1999; Slee et al., 1999). However, to our surprise, boththe caspase-8 and the caspase-9 inhibitors also blocked theTGF-�-initiated apoptotic program, whereas we expectedto find a specific activation of caspase-9, which is normallyactivated by cytochrome c release at the mitochondriallevel (Li et al., 1997). However, a study investigating

Fig. 6. Analysis of JNK (A), p42/p44 MAPK (B), and p38 (C) activitywith phosphospecific antibodies. Cells were treated with TNF-�,TGF-�, and TNF-�/TGF-� for 30 min as described in Material andMethods. One hundred micrograms of cell extract were separated in a12.5% SDS-polyacrylamide gel and blotted onto PVDF membrane.The membrane was incubated overnight with phospho-ERK or p42/p44 specific antibody diluted 1:100 in PBS-T with 1% dry milk. Afterwashing, the membrane was incubated for 1 hr with secondary anti-body (diluted 1:10,000 in PBS-T with 1% dry milk). The signals weredetected with a chemiluminescence imager as described in Materialsand Methods. Representative blots and their quantification from threeindependent experiments.

330 Schuster et al.

apoptosis in Burkitt’s lymphoma cells revealed that TGF-�can induce apoptosis via a caspase-8-dependent but deathreceptor-independent mechanism (Inman and Allday,2000). In this paradigm, application of a caspase-9 inhib-itor could only partially block TGF-�-mediated apoptosis.The finding that both caspase-8 and caspase-9 inhibitorscan completely block apoptosis in OLI-neu cells could beexplained by a sequential activation in which caspase-8would be upstream of caspase-9. It has already been de-scribed that, in addition to the direct activation ofcaspase-3 by caspase-8, caspase-3 activation can be medi-ated by caspase-8 with caspase-9 as a mediator that finallyprocesses procaspase-3 to the active form (Qin et al.,1999).

The MAP kinase cascades represent another impor-tant regulatory step integrating signals from different pro-and antiapoptotic pathways in the apoptotic process (Xiaet al., 1995; Cross et al., 2000). The analysis with theluciferase reporter constructs led us to the speculation thatthere could be a synergy in the activation of the JNK MAPkinase pathway when both TGF-� and TNF-� wereapplied, although both factors alone seemed to have onlya weak stimulating effect. The investigation with the phos-phospecific antibodies, unlike the reporter studies, re-vealed that TNF-� alone could potently stimulate JNKphosphorylation, whereas TGF-� seemed to have noclear-cut effect. The increased activity in the reporterassays may therefore result from a cooperation furtherdownstream, at the level of transcription factors. This ideais supported by findings that the Smad and JNK pathwayinteract in TGF-�-mediated signal transduction at thelevel of transcription factors by a direct interaction of AP1with Smad proteins at the promoter (Wong et al., 1999;Liberati et al., 1999). The reason why we could not findstrong evidence for a direct activation of JNK by TGF-�as described by others (Engel et al., 1999) may be thedifferent cell systems and other intracellular cofactors re-quired; in a recent study, the adaptor protein Daxx wasfound to be essential for TGF-�-mediated JNK activation(Perlman et al., 2001). JNK activation is not in every cellsystem proapoptotic (for review see Mielke and Herdegen,2000), and JNK1/JNK2 double-knockout mice under-score this fact by showing that inhibition of cell deathhinders the closure of the neural tube, whereas apoptoticdeath in the dorsal plate is activated (Kuan et al., 1999).This underscores that the effect of JNK is cell type specific.

The cooperativity of TGF-� with other growthfactors is one possible explanation for its multifunctionalproperties. Within the last few years, data have accumu-lated suggesting that TGF-� modulates the action of othergrowth factors, i.e., the neurotrophic function of glia-derived neurotrophic factor (GDNF). TGF-� synergizeswith GDNF to promote survival of peripheral and CNSdopaminergic neurons (Krieglstein et al., 1998).

Along these lines, TGF-� has been shown to en-hance the growth-inhibitory effects of dexamethasone(Dex) and 1�,25-dihydroxyvitamin D3 (VD3) in mono-cytoid leukemia U937 cells (Kanatani et al., 1999). To-

gether with VD3, the expression of differentiation specificmarkers such as CD11b and CD14 antigens is enhanced.In cooperation with Dex, these genes are not induced, butthe number of apoptotic cells (Apo2.7-positive cells) isincreased. Both factors induce together with TGF-� anincreased expression of p21waf1, leading to a strong hypo-phosphorylation of the retinoblastoma-susceptibility geneproduct pRb. When cells were treated with TGF-� andDex in combination, the antiapoptotic Bcl-xL protein wasdown-regulated, whereas cotreatment with VD3 blockeddown-regulation of Bcl-xL. These data suggest a model ofTGF-� action as an indispensable cofactor either for dif-ferentiation processes or for elimination of cells, depend-ing on the cooperating growth factor.

In thecal/interstitial cells, TGF-� and TGF-� to-gether induce apoptosis. Each growth factor alone has noeffect on cell viability in these cells (Foghi et al., 1998). InT/I cells treated with both growth factors, bcl-2 mRNAlevel decreased significantly, and the expresion of ICE/caspase-1 was found to be enhanced threefold. Cellstreated only with TGF-� or TGF-� showed unalteredbcl-2 and caspase-1 mRNA levels. Induction and repres-sion processes are involved in the cooperative action ofTGF-� and TGF-�. This perhaps could explain why onlyboth growth factors together can induce cell death in thesecells, one component responsible for induction, the otherfor repression.

One very interesting study in MCF-7 cells under-scores the putative role of the TGF-� pathway in TNF-�-induced apoptosis (Tobin et al., 2001). The authorsoverexpressed a dominant-negative TGF-� receptor todisturb TGF-� signaling in MCF-7 cells. These MCF-7cells were relatively resistant to TNF-�-induced celldeath, possibly mediated by a increased Bcl-2 expression inthese cells. Levels of bax and Bcl-xL remained unchanged.These data suggest that TGF-� sensitizes cells for otherproapoptotic stimuli by down-regulation of the proteinlevels of antiapoptotic Bcl-2 family members and is in linewith our own findings.

Our study provides strong evidence that TGF-� actsas a contextual growth factor and cooperates with TNF-�to induce cell death in OLI-neu cells. Understanding howgrowth factors work together and the intracellular signal-ing events playing a role in these processes is both impor-tant in understanding the cooperation of growth factorsduring development, on the one hand, and during patho-genic situations, on the other. For example, nerve growthfactor (NGF) and TGF-� cooperate to induce cell death inthe developing chick retina (Dunker et al., 2001), butwhich intracellular processes are involved is completelyunclear. It is known that both TNF-� and TGF-� areup-regulated in experimental autoimmune neuritis (EAN)and experimental nerve transection (Rufer et al., 1994;Kiefer et al., 1995; Wagner and Myers, 1996), which mayimply a role during neurodegenerative diseases for bothfactors. At least for multiple sclerosis (MS), there is alsoincreasing evidence that the proinflammatory cytokines,such as TNF-�, directly damage oligodendrocytes in the

TGF-� and TNF-� Cooperate To Induce Apoptosis 331

brains of MS patients (Andrews et al., 1998; Hisahara et al.,2000). Our study might help in understanding howTNF-� and TGF-� induce the cell death program, whichis a prerequisite for developing new therapy strategies totreat neurodegenerative diseases.

ACKNOWLEDGMENTSWe thank Heike Palm for excellent technical assis-

tance. We thank Dr. Jacqueline Trotter for providing theOLI-neu cells and Drs. Peter ten Dijke and Carl-HenrikHeldin for providing several plasmids. This study wassupported by grants from the Deutsche Forschungsge-meinschaft to K.K. (Kr1477/8-1), G.T. (SFB 530/C5),and H.B. (Graduiertenkolleg “Zellulare Regulation undWachstum”).

REFERENCESAgresti C, D’Urso D, Levi G. 1996. Reversible inhibitory effects of

interferon-gamma and tumour necrosis factor � on oligodendroglial lin-eage cell proliferation and differentiation in vitro. Eur J Neurosci 8:1106–1116.

Andrews T, Zhang P, Bhat NR. 1998. TNFalpha potentiates IFNgamma-induced cell death in oligodendrocyte progenitors. J Neurosci Res 54:574–583.

Baker SJ, Reddy EP. 1998 Modulation of life and death by the TNFreceptor superfamily. Oncogene 17:3261–3270.

Barres BA, Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD,Raff MC. 1992. Cell death in the oligodendrocyte lineage. J Neurobiol23:1221–1230.

Cammer W, Zhang H. 1999. Maturation of oligodendrocytes is moresensitive to TNF-� than is survival of precursors and immature oligoden-drocytes. J Neuroimmunol 97:37–42.

Casaccia-Bonnefil P. 2000. Cell death in the oligodendrocyte lineage: amolecular perspective of life/death decicisions in development and dis-ease. Glia 29:124–135.

Chao DT, Korsmeyer SJ. 1998. BCL-2 family: regulators of cell death.Annu Rev Immunol 16:395–419.

Cross TG, Scheel-Toellner D, Henriquez NV, Deacon E, Salmon M, LordJM. 2000. Serine/threonine protein kinases and apoptosis. Exp Cell Res256:34–41.

Dunker N, Schuster N, Krieglstein K. 2001. Transforming growth factorbeta modulates programmed cell death in the retina of developing chick.Development 128:1933–1942.

Engel ME, McDonnell MA, Law BK, Moses HL. 1999. InterdependentSMAD and JNK signaling in transforming growth factor-beta-mediatedtranscription. J Biol Chem 274:37413–37420.

Foghi A ,Teerds KJ, van der Donk H, Moore NC, Dorrington J. 1998.Induction of apoptosis in thecal/intersttial cells: action of transforminggrowth factor (TGF) � plus TGF beta on Bcl-2 and interleukin-1 betaconverting enzyme. J Endocrinol 157:489–494.

Fok-Seang J, Mathews GA, French-Constant C, Trotter J, Fawcett JW.1995. Migration of oligodendrocyte precursors on astrocytes and menin-geal cells. Dev Biol 171:1–15.

Groot M, Boxer LM, Thiel G. 2000. Nerve growth factor and epidermalgrowth factor regulated gene transcription in PC12 pheochromocytomaand INS-1 insulinoma cells. Eur J Cell Biol 79:924–935.

Hisahara S, Takano R, Shoji S, Okano H, Miura M. 2000. Role ofcaspase-1 subfamily in cytotoxic cytokine-induced oligodendrocyte celldeath. J Neural Transm Suppl 58:135–142.

Hu Y, Benedict MA, Wu D, Inohara N, Nunez G. 1998. Bcl-XL interactswith Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation. ProcNatl Acad Sci USA 95:4386–4391.

Inman GJ, Allday MJ. 2000. Apoptosis induced by TGF-beta 1 in Burkitt’s

lymphoma cells is caspase 8 dependent but is death receptor independent.J Immunol 65:2500–2510.

Jonk LJ, Itoh S, Heldin CH, ten Dijke P, Kruijer W. 1998. Identificationand functional characteritation of Smad binding elemen (SBE) in the JunBpromoter that acts as a transforming growth factor-beta, activin, and bonemorphogenetic protein-inducible enhancer. J Biol Chem 173:21145–21152.

Jung M, Kramer E, Grzenkowski M, Tang K, Blakemore W, Aguzzi A,Khazaie K, Chlichlia K, von Blankenfeld G, Kettenmann H, Trotter J.1995. Lines of murine oligodendroglial precursor cells immortalized by anactivated neu tyrosine kinase show distinct degrees of interaction withaxons in vitro and in vivo. Eur J Neurosci 7:1245–1265.

Kanatani Y, Kasukabe T, Okabe-Kado J, Yamamoto-Yamaguchi Y, NagataN, Motoyoshi K, Honma Y. 1999. Role of CD 14 expression in thedifferentiation-apoptosis switch in human monocytic leukemia cellstreated with 1alpha, 25 dihydroxyvitamin D3 or dexamethasone in thepresence of transforming growth factor beta 1. Cell Growth Differ 10:705–712.

Kiefer R, Streit WJ, Toyka KV, Kreutzberg GW, Hartung HP. 1995.Transforming growth factor-beta 1: a lesion-associated cytokine of thenervous system. Int J Dev Neurosci 13:331–339.

Knapp PE, Skoff RP, Redstone DW. 1986. Oligodendroglial cell death injimpy mice: an explanation for the myelin deficit. J Neurosci 6:2813–2822.

Krieglstein K, Henheik P, Farkas L, Jaszai J, Galter D, Krohn K, UnsickerK. 1998. Glial cell line-derived neurotrophic factor requires transforminggrowth factor-beta for exerting its full neurotrophic potential on periph-eral and CNS neurons. J Neurosci 18:9822–9834.

Krieglstein K, Richter S, Farkas L, Schuster N, Dunker N, OppenheimRW, Unsicker K. 2000. Reduction of endogenous transforming growthfactors beta prevents ontogenetic neuron death. Nat Neurosci 3:1085–1090.

Kuan CY, Yang DD, Samanta Roy DR, Davis RJ, Rakic P, Flavell RA.1999. The Jnk1 and Jnk2 protein kinases are required for regional specificapoptosis during early brain development. Neuron 22:667–676.

Kumar S. 1999. Mechanisms mediating caspase activation in cell death. CellDeath Differ 6:1060–1066.

Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES,Wang X. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489.

Liberati NT, Datto MB, Frederick JP, Shen X, Wong C, Rougier-Chapman EM, Wang XF. 1999. Smads bind directly to the Jun family ofAP-1 transcription factors. Proc Natl Acad Sci USA 96:4844–4849.

Mielke K, Herdegen T. 2000. JNK and p38 stress kinases—degenerativeeffectors of signal-transduction-cascades in the nervous system. ProgNeurobiol 61:45–60.

Ono K, Han J. 2000. The p38 signal transduction pathway: activation andfunction. Cell Signal 12:1–13.

Oppenheim RW. 1991. Cell death during development of the nervoussystem. Annu Rev Neurosci 14:453–501.

Pan G, Humke EW, Dixit VM. 1998. Activation of caspases triggered bycytochrome c in vitro. FEBS Lett 426:151–154.

Perlman R, Schiemann WP, Brooks MW, Lodish HF, Weinberg RA.2001. TGF-beta-induced apoptosis is mediated by the adapter proteinDaxx that facilitates JNK activation. Nat Cell Biol 3:708–714.

Pettmann B, Henderson CE. 1998. Neuronal cell death. Neuron 20:633–647.Qin H, Srinivasula SM, Wu G, Fernandes-Alnemri T, Alnemri ES, Shi Y.

1999. Structural basis of procaspase-9 recruitment by the apoptoticprotease-activating factor 1. Nature 399:549–557.

Rufer M, Flanders K, Unsicker K. 1994. Presence and regulation oftransforming growth factor beta mRNA and protein in the normal andlesioned rat sciatic nerve. J Neurosci 39:412–423.

332 Schuster et al.

Schuster N, Bender H, Philippi A, Subramaniam S, Strelau J, Wang Z,Krieglstein K. 2002. TGF-b induces cell death in the oligodendroglial cellline OLI-neu. Glia 40:95–108.

Scurlock B, Dawson G. 1999. Differential responses of oligodendrocytes totumor necrosis factor and other pro-apoptotic agents: role of ceramide inapoptosis. J Neurosci Res 55:514–522.

Skoff AM, Lisak RP, Bealmear B, Benjamins JA. 1998. TNF-alpha andTGF-beta act synergistically to kill Schwann cells. J Neurosci Res 53:747–756.

Slee EA, Adrain C, Martin, SJ. 1999. Serial killers: ordering caspase acti-vation events in apoptosis. Cell Death Differ 6:1667–1674.

Thiel G, Kaufmann K, Magin A, Lietz M, Bach K, Cramer M. 2000. Thehuman transcriptional repressor protein NAB1: expression and biologicalactivity. Biochim Biophys Acta 493:289–301.

Tobin SW, Brown MK, Douville K, Payne DC, Eastman A, Arrick BA.2001. Inhibition of transforming growth factor beta signaling in MCF-7

cells results in MCF-7 cells results in resistance to tumor necrosis factoralpha: a role for Bcl-2. Cell Growth Differ 12:109–117.

Wagner R, Myers RR. 1996. Schwann cells produce tumor necrosis factoralpha: expression in injured and noninjured nerves. Neuroscience 73:625–629.

Wong C, Rougier-Chapman EM, Frederick JP, Datto MB, Liberati NT, LiJM, Wang XF. 1999. Smad3-Smad4 and AP-1 complexes synergize intranscriptional activation of the c-Jun promoter by transforming growthfactor beta. Mol Cell Biol 19:1821–1830.

Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. 1995. Opposingeffects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326–1331.

Yu C, Takeda M, Soliven B. 2000. Regulation of cell cycle proteins byTNF-alpha and TGF-beta in cells of oligodendroglial lineage. J Neuro-immunol 108:2–10.

TGF-� and TNF-� Cooperate To Induce Apoptosis 333