Two Different Mechanisms in Jurkat T Cells (Fas/APO-1)

Protein Kinase C Inhibits CD95 (Fas/APO-1)-Mediated Apoptosis by at Least Two Different Mechanisms in Jurkat T Cells

This information is current as Carmen Ruiz-Ruiz, Gema Robledo, Jovita Font, Manuel of September 26, 2021. Izquierdo and Abelardo López-Rivas J Immunol 1999; 163:4737-4746; ; http://www.jimmunol.org/content/163/9/4737 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Protein Kinase C Inhibits CD95 (Fas/APO-1)-Mediated Apoptosis by at Least Two Different Mechanisms in Jurkat T Cells1

Carmen Ruiz-Ruiz,* Gema Robledo,* Jovita Font,† Manuel Izquierdo,‡ and Abelardo Lo´pez-Rivas2*

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we dem-

onstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has Downloaded from been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane. The Journal of Immunology, 1999, 163: 4737–4746. http://www.jimmunol.org/ poptosis is an active form of cell death that plays a fun- Upon CD95 stimulation and trimerization, a set of effector proteins damental role in normal development, tissue homeosta- is recruited to this receptor, forming the death-inducing signaling A sis, and pathological situations (1, 2). Apoptosis is also complex (DISC) (18). FADD/MORT-1 is the first protein that implicated in negative selection of immature thymocytes, T cell- binds to CD95 by interacting with a cytoplasmic region known mediated cytotoxicity, and establishment of tolerance to self-Ag as death domain, which is essential for the induction of apo- (3–5). One of the most potent effectors of apoptosis in T lympho- ptosis (6, 19, 20). In turn, FADD/MORT-1 recruits FLICE/ 3 cytes is the CD95/CD95L system. The CD95 receptor, a member MACH/caspase-8 through its death effector domain, initiating a of the TNF/NGF receptor superfamily (6, 7), is constitutively ex- cascade of events involving the family of Cys proteases known pressed in a majority of T cell lines and in previously activated as caspases (21, 22). by guest on September 26, 2021 ϩ CD45RO T cells from peripheral blood (8, 9). CD95L is a type Activation of the serine/threonine protein kinase PKC by phor- II membrane protein homologous to TNF (10) whose expression is bol ester can repress apoptosis in several cell systems (23–25). In very low in peripheral blood T cells but is markedly up-regulated this respect, it is interesting that protein phosphorylation could also after activation of TCRs (11–13). CD95L can also be present in play a role in the regulation of CD95-induced apoptosis (26–28). soluble form (14). These proteins are involved in the maintenance Furthermore, activation of PKC induces a CD95-resistant pheno- of peripheral self tolerance, regulation of immune responses, and T type in T cells (29–31) and antagonized the sensitization induced cell-mediated cytotoxicity (15, 16). Multivalent cross-linking of by different agents to CD95-mediated apoptosis in other cells (32). the CD95 receptor is required for CD95-induced apoptosis (17). We have previously shown that PKC plays a negative role in CD95-mediated apoptosis in Jurkat T cells by counteracting at a very early stage the signals generated after cross-linking of this *Instituto de Parasitologıá y Biomedicina, Consejo Superior de Investigaciones Ci- entificas, Granada, Spain; †Departamento de Gene´tica, Facultad de Biologia, Univer- receptor (33). Activation of PKC can induce the sequential acti- sidad de Barcelona, Barcelona, Spain; and ‡Centro Nacional de Biotecnologıá, Con- vation of p21ras, the protein kinases c-Raf and MAPKK/ERKK sejo Superior de Investigaciones Cientificas, Madrid, Spain (MEK)1/2, and the MAPK ERK1/2 (34). Moreover, activation of Received for publication April 23, 1999. Accepted for publication August 13, 1999. the ERK pathway prevents apoptosis and promotes cell survival The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance (35). A role for the MAPK pathway in preventing the early and late with 18 U.S.C. Section 1734 solely to indicate this fact. features of CD95-mediated apoptosis has recently been proposed 1 This work was supported by a grant from the Comisioń Interministerial de Ciencia in T cells (36, 37). y Tecnologıá (SAF97-0064-C03-01; to A.L.-R.) and by fellowships from the Minis- UV irradiation of cells induces a rather complex cellular re- terio de Educacioń y Ciencia and the Fundacioń Cientı´fica de la Asociacioń Espanõla Contra el Cańcer (to C.R.-R.). sponse, which includes the stimulation of protein kinases cascades 2 Address correspondence and reprint requests to Dr. Abelardo Lo´pez-Rivas, Instituto (38), the activation of transcription factors (39), and the induction de Parasitologıá y Biomedicina, Consejo Superior de Investigaciones Cientificas, of proinflammatory cytokines (40). Moreover, it is well known that calle Ventanilla 11, 18001 Granada, Spain. E-mail address: [email protected] UV irradiation induces an apoptotic cell death program (41), al- 3 Abbreviations used in this paper: CD95L, CD95 ligand; NGF, nerve growth factor; though the mechanism of the apoptotic action of UV is not com- MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; DISC, death-inducing signal- pletely understood. Recent works have indicated that ceramide is ing complex; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; BIM, bisin- generated in cells after different stress stimuli (42, 43). Further- dolylmaleimide; FLICE, Fas-associated death domain protein-like IL-1␤-converting enzyme-like protease; FADD, Fas-associated death domain protein; MORT, Mediator more, it was demonstrated that ceramide up-regulated the expres- of receptor-induced toxicity; PARP, poly(ADP-ribose) polymerase. sion of CD95L and induced cell death by apoptosis, which was

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 4738 PKC ACTIVATION INHIBITS CD95 RECEPTOR AGGREGATION mediated by CD95/CD95L interactions (43). Although ceramide- DNA transfections mediated CD95L up-regulation may be responsible for the apo- Cells were transfected by electroporation (BTX Electroporation System, ptosis induced under certain stress conditions, there are recent pub- San Diego, CA). Cells (2 ϫ 107) were resuspended in 0.65 ml of medium lished results that suggest the existence of CD95L-independent with the indicated concentration of plasmid DNA and then pulsed at 960 mechanisms of stress-induced apoptosis (44). Furthermore, it has ␮F, 186 Oh, and 300 V. Transfected cells were diluted with culture me- 6 been reported that UV light induces clustering of CD95 receptors dium to 10 cells/ml and cultured for 20 h. After incubation, cell suspen- sions were subjected to Ficoll gradient separation to remove dead cells. in the cell membrane by an unknown mechanism in the absence of CD95L or anti-CD95 Abs (45, 46). This results in the recruitment Analysis of ERK kinase activity of the death adapter molecule FADD/MORT1 and the induction of Cells were transfected by electroporation with pEF-Bos ERK2-Myc tag caspase 8-mediated apoptosis. and incubated with the appropriate stimuli. Immunoprecipitates of ERK2- In this report we have tried to determine more precisely the Myc tag and in vitro kinase assays to analyze ERK2 activity were con- mechanism by which activation of PKC inhibits apoptotic cell ducted as described previously (48). Briefly, cell lysates were precleared with insoluble protein A-Sepharose suspension and incubated for 1 h with death induced upon CD95 ligation in the membrane of human 2 ␮g of 9E10 mAb and then for another hour with 20 ␮l of a 50% sus- Jurkat T cells. We show that the inhibition of PKC-mediated pension of protein G-Sepharose beads (Sigma). After several washes, in MAPK activation either by a specific inhibitor or by a dominant vitro kinase assays were conducted for 30 min at room temperature in 20 inhibitory mutant of MEK-1 only partially blocks PKC attenuation ␮l of a kinase assay buffer supplemented with 10 ␮M ATP, 5 ␮Ci of ␥ 32 ␮ of CD95-mediated apoptosis. Furthermore, our results indicate for [ - P]ATP, and 10 g of MBP as a substrate. The reaction was stopped with 2ϫ sample buffer, and samples were run in 15% SDS-PAGE minigels. the first time that activation of PKC inhibits anti-CD95- and UV Quantitation of 32P incorporated into the MBP protein band was performed light-induced CD95 receptor aggregation through a MAPK-inde- by electronic autoradiography (InstantImager, Packard Instrument, Downloaded from pendent mechanism. We also present data indicating that activa- Meriden, CT). tion of PKC reduces apoptosis triggered by UVB irradiation of Cytofluorometric analysis of CD2 expression and apoptosis Jurkat T cells. Jurkat cells (2 ϫ 105) transfected with the rat CD2 plasmid were washed and resuspended in 100 ␮l of PBS, and 6 ␮g/ml of anti-CD2 mAb was Materials and Methods added. Conditioned medium from myeloma X63 was used as a negative Materials control. After 30 min on ice, cells were washed once with cold PBS and http://www.jimmunol.org/ incubated with FITC-conjugated rabbit anti-mouse Igs (Dako, Carpinteria, RPMI 1640 medium and FCS were obtained from Life Technologies Eu- CA) for 30 min on ice. After this incubation, cells were treated as described rope (Paisley, U.K.). PDBu, carbachol, and myelin basic protein (MBP) above for the analysis of apoptotic cells. CD2-positive cells and hypodip- were purchased from Sigma (St. Louis, MO). BIM, Ro-318220, and H-89 loid apoptotic cells were analyzed in a FACScan cytofluorometer (Becton were obtained from Calbiochem (La Jolla, CA). CH-11 mAb (IgM) react- Dickinson, San Jose, CA). ing with CD95 was purchased from Medical & Biological Laboratories (Nagoya, Japan). Anti-Apo-1 (IgG3) was obtained from Kamiya Biomed- Immunoblot detection of PARP ical (Thousand Oaks, CA). Antagonistic anti-CD95 DX2 mAb (IgG1) and Cells (5 ϫ 105) incubated under the indicated conditions, were pelleted, anti-CD95L NOK-1 mAb (IgG1) were obtained from PharMingen (San resuspended in 20 ␮l of sample buffer (50 mM Tris-HCl (pH 6.8), 6 M Diego, CA). Anti-CD95 rabbit polyclonal IgG Ab (C-20), anti-CD95L rab- urea, 6% 2-ME, 3% SDS, and 0.003% bromophenol blue) and sonicated. by guest on September 26, 2021 bit polyclonal IgG Ab (Q-20), and anti-Myc mAb (9E10; IgG1) were pur- Proteins were resolved on SDS-10% polyacrylamide minigels and trans- chased from Santa Cruz Biotechnology (Santa Cruz, CA). MEK1 inhibitor, ferred onto Immobilon membranes (Millipore, Bedford, MA). The blot was PD098059, was purchased from New England Biolabs (Beverly, MA). blocked with 5% milk powder in PBS/0.1% Tween 20 (PBS/Tween) for Rabbit polyclonal antiserum against poly(ADP-ribose) polymerase 1 h, washed with PBS/Tween, and incubated with antiserum anti-FII (1/ (PARP), anti-FII, was provided by Dr. G. de Murcia (Ecole Supe´rieure de 2000) for 1 h. The blot was again washed with PBS/Tween and developed Biotechnologie de Strasbourg, Centre National de la Recherche Scienti- with HRP-coupled goat anti-rabbit (1/2000; Dako) followed by enhanced fique, Strasbourg, France). Wild-type MEK (pEXV3 MAPKKwt), domi- chemiluminescence (Amersham, Aylesbury, U.K.). nant inhibitory mutant MEK (pEXV3 MAPKK 221A), and constitutively active MEK (pEXV3 MAPPK 217E/221E) plasmids were gifts from Dr. Immunoblot detection of CD95L C. J. Marshall (Institute of Cancer Research, London, U.K.). We are grateful to Dr. D. Cantrell (Imperial Cancer Research Fund, London, U.K.) for Acetone-precipitated proteins from cell lysates corresponding to 5 ϫ 105 the pEF BOS ERK-2-Myc tag and CMV-rat CD2 plasmids and the anti- cells were resolved in 10% SDS-PAGE minigels. Blots were probed with ratCD2 Ab (OX-34). We are also grateful to Dr. Michael Hahne (Univer- rabbit polyclonal anti-CD95L Ab (Q-20; Santa Cruz Biotechnology). To sity of Lausanne, Lausanne, Switzerland) for the gift of recombinant hu- detect immunocomplexes, the blots were probed with HRP-coupled goat man CD95L and CD95L cross-linker. anti-rabbit IgG followed by enhanced chemiluminescence. Analysis of monomeric and aggregated CD95 receptors Cell culture To detect CD95 aggregation upon anti-CD95 mAb stimulation, Jurkat cells Cells of the human leukemic T cell lines Jurkat and J-HM1-2.2 Jurkat (5 ϫ 106) were incubated in complete growth medium at 37°C with 1 expressing the human muscarinic acetylcholine type 1 (HM1) receptor and ␮g/ml CH11 IgM anti-CD95 for 10 min, washed with PBS buffer, and the human Raji B lymphoblastoid cell line were maintained in culture in lysed in 20 ␮l of ice-cold lysis buffer (50 mM HEPES (pH 7.4), 150 mM RPMI 1640 medium containing 10% FCS and 1 mM L-glutamine at 37°C ␮ NaCl, 10 mM NaF, 10 mM iodoacetamide, 200 MNa3VO4, and 1% in a humidified 5% CO2/95% air incubator. Nonidet P-40) for 10 min. Postnuclear supernatants were resolved on SDS- 7.5% PAGE minigels. UVB irradiation of Jurkat cells For the detection of CD95 oligomerization after UVB light exposure, cells (5 ϫ 106) were irradiated for 5 min, incubated at 37°C for 5 min, Jurkat cells were irradiated at room temperature in complete growth me- washed with PBS, and treated as described previously (45). In brief, cells dium with a UVB transilluminator source (310 nm, Fotodyne, New Berlin, were resuspended in 500 ␮l of PBS and incubated with 2 mM cleavable WI). Briefly, Jurkat cells (106/ml) were seeded in plates and exposed from cross-linker 3,3Ј-dithiobis-[sulfosuccinimidyl propionate] (Sigma) for 15 below to a UVB light source at a distance of 2.5 cm for different times. The min on ice. The reaction was quenched with 10 mM ammonium acetate for energy applied in these experiments ranged from 2–10 J. After irradiation, 10 min, and cells were washed with PBS and lysed in 150 ␮l of ice-cold the cells were incubated for the indicated time. lysis buffer for 10 min. Postnuclear supernatants were incubated at 4°C for 1 h with either 0.1 ␮g/ml (Ab limiting) or 2 ␮g/ml (Ab excess) Apo-1 IgG3 Determination of apoptotic cells anti-CD95 mAb, and immune complexes were precipitated using protein A-Sepharose (Sigma). After washing three times in lysis buffer, immuno- Hypodiploid apoptotic cells were determined by cytofluorometric analysis of precipitates were resuspended in Laemmli buffer, boiled for 5 min, and DNA content after extraction of the degraded DNA from apoptotic cells (47). resolved on SDS-10% polyacrylamide minigel. The Journal of Immunology 4739

Resolved samples were transferred onto Immobilon membranes, and the blots were treated as described previously (immunoblot detection of PARP) using a rabbit polyclonal anti-CD95 Ab (0.2 ␮g/ml) followed by HRP-conjugated goat anti-rabbit (1/2000; Dako) for the detection of CD95.

Results Activation of MEK1 is partially involved in the prevention of CD95-induced apoptosis by a PKC activator in Jurkat T cells Several reports have described that T cell activation can inhibit apoptosis induced by ligation of CD95 in the cell surface with CD95 Abs (36, 37). It has also been suggested that the mechanism underlying this inhibitory effect involves the activation of the MAPK pathway (36, 37). T cell activation induces the expression of FLIP (FLICE inhibitory protein), a potent inhibitor of CD95- induced apoptosis (37, 49). Induction of FLIP in Jurkat T cells was dependent on the stimulation of the MAPK pathway and could be observed after several hours of activation (37). Our previous results indicated that inhibition of CD95-induced signaling is observed immediately after addition of PKC activators (33), thus Downloaded from making unlikely the involvement of a newly synthesized inhibitor. In this report we have tried to determine the mechanism of PKC- mediated prevention of CD95-induced apoptosis and to deﬁne the step(s) in CD95 signaling at which PKC exerts its inhibitory role. Inhibition of CD95-mediated apoptosis by PKC activation oc-

curs in a dose-dependent manner (Fig. 1A). Thus, when Jurkat cells http://www.jimmunol.org/ were incubated in the presence of an anti-CD95 Ab, apoptosis was clearly prevented at PDBu concentrations ranging from 2–20 ng/ ml. Because this inhibitory action of PKC has been mainly studied in apoptosis induced by CD95 Abs (29–31, 33) we further investigated this effect, and here we show that PDBu also inhibited CD95L-induced apoptosis in Jurkat cells (Fig. 1B). To analyze the role of the MAPK pathway in mediating the inhibition of CD95-induced apoptosis by PDBu, PD 098059, a rather specific inhibitor of the activation of MEK1/2 (50), was by guest on September 26, 2021 used. As shown in Fig. 2A, treatment of Jurkat cells with either 2 or 20 ng/ml PDBu prevented CD95 Ab-induced cell death. In these experiments, pretreatment of cells with PD 098059 significantly reduced the anti-apoptotic effect of 2 ng/ml PDBu (Fig. 2A). In- terestingly, this inhibitor of MEK1/2 activation did not affect the prevention of CD95-induced apoptosis by 20 ng/ml PDBu (Fig. 2A). These results suggested that aside from MEK1/2 activation, other signals generated upon PKC activation might be involved in the inhibition by phorbol esters of Fas-mediated cell death. How- ever, it could also be possible that PD 098059 was not able to inhibit MEK1/2-mediated MAPK activation at high PDBu concentrations, because it has been reported that the extent of inhibi- FIGURE 1. PKC activation by a phorbol ester inhibited CD95 Ab- and tion by PD 098059 might depend on how potently MEK1/2 was CD95L-induced apoptosis in Jurkat cells. A, Jurkat cells were incubated for activated by agonists (50). To determine whether this was the case, 8 h without or with 100 ng/ml CD95 Ab CH11 in the absence or the we analyzed the activation of the MAPK pathway in cells tran- presence of different concentrations of PDBu. Error bars represent the SD siently transfected with ERK2 tag (48) and stimulated with two from four independent experiments. B, Jurkat cells were incubated for 8 h different concentrations of PDBu. As shown in Fig. 2B, ERK-2 in the presence or the absence of recombinant human CD95L (20 ng/ml) and CD95L cross-linker (XL; 40 ng/ml). PDBu (20 ng/ml) was also added was activated by the phorbol ester in a dose-dependent manner. to some cultures at the beginning of the experiment. Results are represen- Moreover, the presence of PD 098059 completely inhibited ERK2 tative of two different experiments. In all experiments, hypodiploid apo- activation induced by PDBu even when a high concentration of ptotic cells were determined by cytofluorometric analysis of DNA content. phorbol ester was used (Fig. 2B). These results indicated the existence in Jurkat cells of at least two different PKC-dependent mechanisms that inhibit Fas-mediated cell death: a MAPK-depen- siently cotransfected Jurkat cells with a plasmid encoding the rat dent mechanism, as we have shown in this study and has been CD2 receptor and a plasmid encoding either a dominant inhibitory recently proposed (36), that can be of significance under conditions mutant of MEK1 or the wild-type protein. After incubation with of suboptimal stimulation of PKC by agonists, and a MAPK-in- the appropriate stimuli we determined the percentage of apoptotic dependent pathway that is activated when PKC is maximally cells within the population of transfected cells (CD2ϩ cells). As stimulated. shown in Fig. 2C, when cells transfected with wild-type MEK1 To confirm the importance of MEK1 activation in the apoptosis were treated with CD95 mAb, they underwent apoptosis (32% inhibitory signals induced at a submaximal dose of PDBu, we tran- sub-G1 cells). In the presence of 2 ng/ml PDBu there was a marked 4740 PKC ACTIVATION INHIBITS CD95 RECEPTOR AGGREGATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Inhibition of MEK1 reduced the abrogation of CD95-induced apoptosis by PDBu. A, Jurkat cells were incubated for 2 h with or without 50 ␮M of the MEK1 inhibitor PD 098059. Following this treatment, cells were incubated for 8 h either without or with 100 ng/ml CH11 in the absence or the presence of two different concentrations of PDBu (2 and 20 ng/ml). The percentage of apoptotic cells in the population was determined by flow cytometry. B, PD 098059 blocked the activation of ERK2 caused by PDBu. Jurkat cells transfected with 10 ␮g of pEF-BOS ERK2 tag were either unstimulated or stimulated for 30 min with 2 or 20 ng/ml PDBu after1hofpreincubation in the presence or the absence of 50 ␮M PD 098059. MBP kinase assays were performed on the 9E10 immunoprecipitates from cell lysates. ERK activation is expressed as fold induction of MBP phosphorylation as determined by electronic autoradiography. C, Jurkat cells were cotransfected with 10 ␮g of rat CD2 DNA and either 30 ␮g of dominant inhibitory mutant MEK (pEXV3 MAPKK 221A; DN) or 30 ␮g of wild-type MEK (pEXV3 MAPKKwt; WT) as described in Materials and Methods. Twenty hours after transfections cells were incubated with or without 100 ng/ml CH11 in the presence or the absence of 2 ng/ml PDBu for 8 h. Hypodiploid apoptotic cells in the CD2ϩ population were determined by flow cytometry. Results are presented as the percentage of apoptotic cells in the population after subtracting the values of apoptosis in untreated cultures. The levels of apoptosis in untreated WT and DN cells were 17 Ϯ 2% and 14 Ϯ 3%, respectively. D, Jurkat cells were cotransfected with 10 ␮g of rat CD2 DNA and either 30 ␮g of constitutively active MEK (pEXV3 MAPPK 217E/221E; CA) or 30 ␮gof wild-type MEK (pEXV3 MAPKKwt; WT) and incubated for 20 h. Apoptotic cells in the CD2ϩ population were determined by flow cytometry after stimulation without or with 100 ng/ml CH11 for 8 h. In all experiments error bars represent the SD from at least three independent experiments. E, Cell extracts from Jurkat cells treated as described in A were subjected to Western blotting, and the formation of the 29-kDa fragment of PARP was determined as described in Materials and Methods. Results are representative of at least two different experiments.

inhibition of the apoptotic program (12% sub-G1 cells). However, tially protected these cells from CD95-mediated apoptosis (33% cells transfected with a dominant inhibitory mutant MEK1 were sub-G1 cells). These results conﬁrmed those obtained using the more susceptible to CD95-mediated apoptosis (45% sub-G1 cells) inhibitor of MEK1 activation PD098059 and suggested that acti- than those transfected with wild-type MEK1 and PDBu only par- vation of MEK1 could play a certain role in the abrogation of The Journal of Immunology 4741

apoptosis by suboptimal doses of PKC activators. To determine whether the activation of MEK1 is sufﬁcient to block CD95 mAb- induced apoptosis, we cotransfected Jurkat cells with the CD2 DNA and with DNA encoding either a constitutively activated MEK1 or the wild-type protein. After analysis of the CD2ϩ population we observed that the percentage of apoptotic cells after stimulation with CD95 mAb was reduced in those expressing constitutively activated MEK1 (Fig. 2D). These data support the idea that the inhibitory effect of low doses of PDBu on CD95-induced cell death is mediated in part by activation of MEK1. A key event in CD95-mediated apoptosis is the sequential activation of several members of the family of Cys proteases, caspases, which, in turn, induce the cleavage of different substrates (51, 52). One of these substrates is the 113- to 116-kDa nuclear enzyme PARP, which is proteolitically cleaved by caspases within the bipartite nuclear location signal to produce two fragments of ϳ85 and 29 kDa (53). In a previous study we have demonstrated that PKC activation inhibited caspase-3 activation and PARP

cleavage induced by anti-CD95 mAb in Jurkat cells (33). In this Downloaded from report, we have analyzed the possible involvement of MEK1 activation in mediating the inhibitory action of PKC on CD95-induced activation of caspase-3-like proteases. To this end we have determined the generation of the 29-kDa fragment of PARP as previously described (33). The experiment shown in Fig. 2E indi-

cates that the phorbol ester, PDBu, inhibited CD95-mediated http://www.jimmunol.org/ cleavage of PARP in Jurkat cells in a dose-dependent manner. Similar to what was observed in cell death experiments (Fig. 2A), the inhibitor of MEK1 activation, PD 098059, was able to prevent the inhibitory effect on PARP cleavage of different concentrations of PDBu (Fig. 2E). Furthermore, in agreement with the experiments analyzing apoptosis (Fig. 2A), PD 098059 did not affect PDBu-induced halt of CD95-mediated PARP cleavage at PDBu 20 ng/ml (Fig. 2E). by guest on September 26, 2021 PKC activation inhibits the aggregation of CD95 receptors FIGURE 3. CD95 receptor aggregation induced upon CD95 ligation by induced by CD95 mAbs or UVB radiation IgM mAb was inhibited by PDBu. A, After preincubation for 30 min without or with 20 ng/ml PDBu, Jurkat cells were treated for 10 min in the In a previous report we reported that PKC activation blocked absence or the presence of 1 ␮g/ml CH11. Cell lysates were analyzed by CD95-elicited signals that occurred immediately after CD95 re- Western blotting to detect CD95 aggregates as described in Materials and ceptor ligation by specific mAb (33). It has been demonstrated that Methods. The arrows indicate CD95 aggregates and monomeric CD95. B, the first event upon CD95 stimulation is the trimerization of the After incubation for1hintheabsence or the presence of either 6 ␮M BIM ␮ intracellular death domain, which, in turn, recruits FADD and or 50 M PD 098059, Jurkat cells were treated as described in A, and FLICE to form the DISC (18, 21, 22). On the other hand, it was CD95 aggregation was analyzed. All results are representative of at least three different experiments. reported that high molecular mass (Ͼ200 kDa) CD95 aggregates are formed immediately after receptor cross-linking with CD95 Abs in different human B and T cell lines (54). To further char- caused by the PKC activator (Fig. 3B). Similar results were ob- acterize the inhibition of CD95 signaling pathway by PKC acti- tained with the PKC inhibitor Ro-318220 (not shown). However, vation we have analyzed CD95 aggregation in Jurkat cells. As we did not observe a prevention of the suppressor effect of PDBu shown in Fig. 3A, stimulation of Jurkat cells with an IgM CD95 on CD95 aggregation when using the PKA inhibitor H-89 (results mAb induced the formation of SDS-stable high m.w. aggregates of not shown). The role of the MAPK pathway in mediating PDBu- CD95 as previously reported (18, 54). These aggregates were not induced attenuation of CD95 aggregation was assessed with the seen if the cells were incubated with the Ab at 4°C, which is below inhibitor of MEK1 activation, PD 098059. The results obtained the transition temperature of the membrane (results not shown). indicated that the PDBu effect was independent of MEK1 activa- Moreover, CD95 aggregation by Ab required an intact cell, as it tion (Fig. 3B). All these experiments were performed at the con- was not observed when the Ab was added to lysed cells (not centration of PDBu (20 ng/ml) that caused maximal reduction in shown). aggregate formation. At lower concentration of PDBu (2 ng/ml), Interestingly, in the presence of PDBu, CD95 mAb-induced re- which partially inhibits CD95-induced apoptosis in a MAPK-de- ceptor aggregation was significantly reduced (Fig. 3A). We ob- pendent way (Fig. 2A), PDBu was not able to inhibit CD95 ag- served the same results when Jurkat cells were stimulated with gregation induced by anti-CD95 mAb (not shown). These results IgG3 CD95 mAb (unpublished results). Importantly, this acute suggested that PKC activation, following treatment with maximal treatment with PDBu did not affect the expression of CD95 Ag in concentrations of phorbol ester, may prevent CD95-induced apo- the membrane of Jurkat cells (33). To determine whether the effect ptosis by inhibiting the first step in CD95 signaling pathway, that of PDBu was due to PKC activation, we used the PKC inhibitor is, receptor multimerization. BIM. The presence of BIM during the incubation with phorbol It has been reported that UVB radiation induces the oligomer- ester abrogated the reduction in CD95 aggregates formation ization of CD95 receptors, which triggers the downstream caspase 4742 PKC ACTIVATION INHIBITS CD95 RECEPTOR AGGREGATION Downloaded from http://www.jimmunol.org/

FIGURE 4. Induction of CD95 receptor aggregation upon UVB irradiation was prevented by PDBu. Jurkat cells (A) or Raji cells (B) were incubated in the presence or the absence of 20 ng/ml PDBu for 30 min. After this incubation, cells were UVB irradiated for 5 min and incubated for 5 min. Cells were washed, treated with cleavable cross-linker reagent, and ␮ lysed. CD95 was immunoprecipitated with limiting (0.1 g/ml) concen- FIGURE 5. PKC activation by a phorbol ester inhibited UVB radiation- by guest on September 26, 2021 trations of IgG3 CD95 Ab. Immunoprecipitated CD95 was determined as induced apoptosis in Jurkat cells. A, Jurkat cells were UVB irradiated for described in Materials and Methods. For comparison, an excess of CD95 2, 5, or 10 min and then incubated for4hinthepresence or the absence mAb (2 ␮g/ml) was used to immunoprecipitate CD95 molecules in un- of 20 ng/ml PDBu before analyzing the percentage of apoptotic cells. B, treated cells (A). Results are representative of at least three independent After 1-h treatment in the absence or the presence of either 50 ␮MPD experiments. 098059 or 6 ␮M BIM, Jurkat cells were UVB irradiated for 5 min and incubated with or without 20 ng/ml PDBu for 4 h. In all experiments hypodiploid apoptotic cells were determined by cytoﬂuorometric analysis effector pathway and apoptosis (45, 46). We investigated in two of DNA content. Error bars represent the SD from two independent different cell lines, Jurkat and Raji, whether UVB light induced experiments. CD95 aggregation and if it could be inhibited by PKC activation. To this end, we followed an experimental design previously described (45). The basic principle is that at limiting concentrations determined by the generation of hypodiploid cells. The percentage (0.1 ␮g/ml) of IgG3 anti-APO-1 mAb more molecules of the of apoptotic cells after radiation was a function of the irradiation CD95 receptor will be immunoprecipitated in the presence of a time length (Fig. 5A). Similar to what was observed in Jurkat cells reversible cross-linker if aggregation has taken place upon UVB treated with CD95 mAb, we found that in the presence of PDBu irradiation of cells. The results presented in Fig. 4, A and B, indi- there was a marked reduction in the percentage of apoptotic cells cate that in UVB-treated Jurkat and Raji cells there was a marked induced upon UVB irradiation (Fig. 5A). Moreover, the effect of increase in the amount of CD95 protein immunoprecipitated by PDBu on UVB light-induced apoptosis was due to the activation of limiting Ab compared with that in untreated cells. These results PKC. As shown in Fig. 5B, the suppression of apoptosis caused by indicated that UVB irradiation of Jurkat and Raji cells caused ag- the phorbol ester was not observed in the presence of the PKC gregation of CD95 receptors as previously demonstrated in other inhibitor BIM. The PKC inhibitor Ro-318220 produced similar cell lines (45, 46). Interestingly, UVB radiation-induced CD95 re- results as BIM (not shown). In contrast, the PKA inhibitor H-89 ceptor oligomerization was signiﬁcantly inhibited in the presence did not abolish the PDBu effect on apoptosis (results not shown). of 20 ng/ml PDBu (Fig. 4, A and B). We also examined the role of the MAPK pathway in the prevention of UVB light-induced apoptosis by PKC activation. As shown Activation of PKC reduces UV irradiation-induced apoptosis in in Fig. 5B, PD 098059 did not affect the inhibition of UVB light- Jurkat T cells induced apoptosis caused by 20 ng/ml PDBu. These results sug- Based on the above data, we tried to determine whether the acti- gested that, similar to what has been observed in CD95 mAb- vation of PKC by phorbol ester was also able to inhibit UVB induced apoptosis, activation of PKC with maximal doses of PDBu light-induced apoptosis in Jurkat cells. When UVB irradiated for prevented CD95 oligomerization and cell death induced upon different times, Jurkat T cells underwent apoptotic cell death, as UVB radiation by a mechanism independent of MEK1 activation. The Journal of Immunology 4743

However, we cannot exclude the possibility that other CD95-independent mechanisms might also contribute to UV radiation-induced apoptosis (42).

Induction of CD95L expression upon UVB irradiation of Jurkat T cells does not mediate UVB-induced apoptosis It has been recently demonstrated that certain stress treatments such as anticancer drugs and gamma irradiation up-regulate CD95L expression and induce apoptosis through a CD95-dependent pathway involving the formation of a CD95/CD95L complex (43). In addition, it was shown that ceramide, which accumulates in response to stress, mediates the up-regulation of CD95L expression and apoptosis observed following drug addition or gamma irradiation. However, more recent data have indicated that not all stress stimuli use the same cell death pathways (44). It was shown that UV, gamma irradiation, and anisomycin stimulated c-Jun N- terminal kinase activity and induced CD95L expression in Jurkat cells. Nevertheless, only anisomycin-induced apoptosis was dependent on CD95/CD95L interactions (44). To further investigate Downloaded from the role of CD95L in UVB-induced apoptosis we used J-HM1-2.2 Jurkat cells expressing the human muscarinic acetylcholine type 1 (HM1) receptor. We have recently shown that these cells undergo apoptosis upon activation of the muscarinic receptor with the ag- onist carbachol (55). This cell death process involved the up-regulation of CD95L (Fig. 6A) and required the formation of a CD95/ http://www.jimmunol.org/ CD95L complex, as it could be blocked by either an antagonistic CD95 Ab (DX2) or an anti-CD95L Ab (NOK-1; Fig. 6B). There- FIGURE 6. Induction of CD95L expression upon UVB irradiation of fore, these cells provide a positive control for the Abs used to J-HM1-2.2 Jurkat cells does not mediate UVB-induced apoptosis. A, prevent CD95-CD95L interactions. We observed that UVB irra- J-HM1-2.2 Jurkat cells were UVB irradiated for 5 min and then incubated diation induced the expression of CD95L in J-HM1-2.2 Jurkat for the times indicated in the figure. Other cultures were incubated in the cells (Fig. 6A) as previously reported in Jurkat cells (44). However, absence (C) or the presence of 500 ␮M carbachol (Car) for 6 h. After the cell death resulting from UVB irradiation was not prevented by incubations, CD95L expression in the cells was determined as described in blocking CD95/CD95L interactions with antagonistic CD95 Ab or Materials and Methods. The data shown are representative of two inde- by guest on September 26, 2021 CD95L Ab (Fig. 6B), which suggests that UV-induced CD95L is pendent experiments. B, Cultures of J-HM1-2.2 Jurkat cells were UVB irradiated for 5 min and then incubated for4hintheabsence (UVB) or the not involved in UV-mediated apoptosis of J-HM1-2.2 cells. Sim- presence of antagonistic CD95 mAb (UVBϩDX2) or CD95L mAb ilar results were obtained in Jurkat cells (not shown). (UVBϩNOK-1). Other cultures were incubated for 8 h with 500 ␮M carbachol in the absence (carbachol) or the presence of antagonistic CD95 Discussion mAb (carbacholϩDX2) or CD95L mAb (carbacholϩNOK-1). Finally, Mechanisms of cellular resistance to CD95-mediated apoptosis some cultures were treated for 15 h with agonistic CD95 mAb CH-11 (20 in T cells: role of protein kinase C and receptor oligomerization ng/ml) in the absence (anti-CD95) or the presence of antagonistic CD95 mAb (anti-CD95ϩDX2) or CD95L mAb (anti-CD95ϩNOK-1). Untreated Cellular expression of CD95 receptor and ligand is not always cultures (control) were also incubated for 15 h. Hypodiploid apoptotic cells sufficient to elicit an apoptotic response. There are examples of were determined by cytofluorometric analysis of DNA content. Error bars CD95-resistant cells that express high levels of CD95 receptor in represent the SD from two independent experiments. their plasma membrane and yet they are not killed by CD95L or CD95 mAbs (27, 28). This is the case of peripheral T lymphocytes, which in the initial phases of antigenic stimulation up-regulate the (24, 33, 51, 52). We and others have recently shown that activation expression of CD95 and CD95L, but proliferate instead of dying. of PKC antagonizes CD95-mediated signaling and apoptosis in These cells undergo CD95-mediated apoptosis only after they are Jurkat cells and in other cell lines (29, 30, 33, 59). Furthermore, repetitively stimulated through the TCR (56). Mechanisms involv- inhibitors of this kinase enhance the susceptibility of cells to anti- ing the up-regulation of FLIP (49) or Bcl-xL (57) during the early CD95 mAbs (59, 60). PKC activates the MAPK pathway by stim- stage of T cell activation have been proposed to explain the resis- ulating p21ras and Raf kinase in different cell types (61, 62). It has tance of mature T cells to CD95-mediated apoptosis. Lack of re- been proposed that activation of the ERK pathway prevents apo- cruitment of FLICE to the CD95 death-inducing signaling com- ptosis and promotes cellular survival (35). On the other hand, plex has also been observed in these cells (57). Resistance can be CD95 ligation can induce the activation of JNK/SAPK (63, 64), overcome by treatment with inhibitors of macromolecular synthe- which may contribute to the induction of apoptosis. These obser- sis (58), which suggests the presence of short-lived inhibitory pro- vations suggest that different MAP kinase family members could teins. In this respect, it is interesting to mention the existence of pro- contribute in different ways to the decision for cellular life or teins associated with the cytosolic part of CD95 Ag that may regulate death. In this report we have demonstrated that under certain cir- the capacity of this receptor to transmit apoptotic signals (28). cumstances, the PKC-induced ERK pathway may be important in The human T cell line Jurkat has been widely used as a model negatively controlling CD95-mediated cell death. Our results con- to study activation-induced cell death and Fas-mediated apoptosis. firmed recently published data indicating that the MAPK kinase Jurkat T lymphocytes are normally sensitive to CD95-mediated (MEK1) is a negative regulator of CD95-mediated apoptosis in T apoptosis when CD95 receptor is cross-linked by anti-CD95 Abs cells (37). This protective mechanism may have a role in the early 4744 PKC ACTIVATION INHIBITS CD95 RECEPTOR AGGREGATION stages of T cell activation, preventing the onset of CD95-depen- membrane level upon irradiation of cells may lead to the aggregation dent, activation-induced cell death (36). How activation of the of receptors required for complete activation in the absence of ligands MAPK pathway leads to resistance to CD95-mediated apoptosis is (73). More recently, it was reported that the apoptotic response of not known, although members of this cascade can phosphorylate different cell types to UV irradiation is mediated at least in part by Bad and inhibit apoptosis (65). Moreover, as MAPK signals seems activation of CD95/Fas/APO-1 (45, 46). This activation occurs via to inhibit the initial phases of CD95-mediated apoptosis (36), it CD95 receptor oligomerization, subsequent recruitment of the may potentially interfere in the steps proximal to formation of the death adapter molecule FADD/MORT1, and induction of death complex (18, 21, 22). In this respect, induction of FLICE caspase-8 activity. In this report we have demonstrated that upon inhibitory protein by stimulation of the MAPK pathway was re- irradiation of Jurkat cells with UVB light there is aggregation of cently reported in T cells activated with Con A (37). However, our CD95 receptors. Furthermore, our results have shown for the first data demonstrated that the dependence on MEK1 activation for time that activation of PKC prevents UV light-induced aggregation survival was only observed when submaximal concentrations of of CD95 receptors and apoptosis. Similar results were obtained in PKC activator were used. In this report we show that in the pres- the human lymphoma B cell line Raji. Consistent with these results ence of maximal concentrations of phorbol ester, a PKC-regulated, was the finding that CD95 Ab-induced aggregation of CD95 re- MAPK-independent pathway is activated that inhibits CD95 ag- ceptors was also reduced in Jurkat cells incubated in the presence gregation in the membrane and CD95-mediated apoptosis. It is of a PKC activator. To confirm the early abrogation by PKC of interesting that apoptosis induced by a human CD40/CD95 chi- CD95 signaling we have tried to determine the association of the meric receptor is not inhibited by PKC activation (66). This re- adapter molecule FADD/MORT1 with CD95. However, we have ceptor contains the CD95 transmembrane/intracellular domain failed to detect the recruitment of this adapter by CD95 aggregates. Downloaded from fused to the CD40 extracellular domain. As the extracellular do- This is not unexpected in view of recent data (74) indicating the main could play an important role in regulating multimerization of existence of two different CD95/Fas/APO-1 signaling pathways receptors at the cell membrane (67), its replacement by a heterol- (type I and II cells). According to these authors Jurkat cells are ogous receptor domain might suppress specific constraints derived type II cells in which DISC formation is strongly reduced, al- from intracellular signaling, such as PKC activation. On the other though they express high levels of FADD and caspase-8. However,

hand, it remains to be demonstrated whether PKC directly modu- although lower amounts of FADD and caspase-8 are recruited to http://www.jimmunol.org/ lates CD95 receptor oligomerization or requires additional effector the aggregated receptor in Jurkat cells compared with type I cells, activities. A recent report (68) has indicated that phosphatidylino- it appears that this is sufficient to mediate apoptosis upon CD95 sitol 3Ј-kinase can have a suppressor effect on CD95-mediated ligation (74). apoptosis in T cells. Moreover, it is known that phosphatidylino- Our results indicate that a likely target for PKC-mediated inhi- sitol 3Ј-kinase is a downstream effector of p21ras (69), which, in bition of CD95 mAb and UVB radiation-induced apoptosis is the turn, can be activated by PKC (61). Further investigation is needed mechanism of CD95 Ag oligomerization in the membrane (18). to demonstrate the role of phosphatidylinositol 3Ј-kinase in PKC- However, at present we should not exclude the possibility that dependent, MAPK-independent inhibition of CD95-induced other targets downstream of CD95 oligomerization might also co- apoptosis. operate in the regulation by PKC of CD95 signaling. In this re- by guest on September 26, 2021 spect, it has been proposed that expression of c-Myc sensitizes Regulation of UV light-induced CD95 aggregation and fibroblasts to CD95-induced apoptosis (75), and PKC-␤ could ab- apoptosis by PKC rogate Myc-induced apoptosis in some cell types (76), although The apoptotic response of cells to UV radiation may include the this blocking action seemed to be related to changes in the cell accumulation of the tumor suppressor protein p53 and the tran- cycle. On the other hand, the inhibitory protein of CD95 signaling scriptional activation of p53-regulated genes (70). However, other FLIP is induced upon T cell activation (37, 49). The role of FLIP mechanisms are also involved (39). Among these p53-independent in PKC-induced inhibition of CD95-mediated apoptosis remains to mechanisms, the activation of the sphingomyelin pathway by UV be elucidated. light has been reported (42). This pathway, initiated by hydrolysis of sphingomyelin in cell membranes, generates the second mes- Protein phosphorylation and the control of CD95 receptor senger ceramide, which, in turn, stimulates a stress-activated pro- clustering in the plasma membrane tein kinase (SAPK/JNK) pathway. It has been shown that SAPK/ Although CD95/APO-1 phosphorylation has not been observed JNK activation by various stress treatments is implicated in stress- upon activation of this receptor (18), phosphorylation sites have induced apoptosis (42). Our unpublished observations indicate that been found in the membrane-proximal cytoplasmic domain of the rapid activation by UVB light of the SAPK/JNK pathway, CD95/APO-1 (77). Therefore we speculate that phosphorylation of which is normally a result of ceramide generation, is not inhibited these sites by either PKC or associated kinases (77) may generate by phorbol ester pretreatment of Jurkat cells, suggesting that the negatively charged residues that could function to impede oli- attenuation of UVB-induced apoptosis by PKC should occur at a gomerization of receptors and consequently prevent CD95-medi- different point. More recent evidence has suggested a role of the ated signaling and apoptosis. Regulation of CD95 function and CD95/CD95L system in stress-activated apoptotic cell death (43). sensitivity by negatively charged molecules has been proposed in However, there are contradictory findings about the role of CD95L the case of the surface sialylation of CD95 (18). in UV radiation-induced apoptosis (44, 71). Our data demonstrated On the other hand, agents that inhibit actin polymerization may that blocking CD95/CD95L interactions in Jurkat cells did not di- have profound effects on receptors patching and capping (78). In minish UVB-induced apoptosis, although CD95L was induced af- this respect, it has been reported that nitric oxide, which may de- ter UVB irradiation. These results also implied that PKC activation crease filamentous actin formation (78), is an inhibitor of CD95- must be interfering at a different step in the pathway(s), leading to mediated apoptosis (79). PKC may induce drastic alterations in apoptosis upon UVB irradiation of Jurkat T cells. cell morphology and membrane dynamics, which correlates with The activation of growth factor and cytokine receptors by UV the reorganization of submembranous actin (80). This action is light has been recently demonstrated (72, 73). These findings have mediated by phosphorylation and inhibition of the filamentous ac- prompted the hypothesis that the physical stress elicited at the plasma tin cross-linking protein MARCKS (81), a widely expressed PKC The Journal of Immunology 4745 substrate (82). Therefore, an explanation for the observed effect of 22. Boldin, M. P., T. M. Goncharov, Y. V. Goltsev, and D. Wallach. 1996. Involve- PKC activation on CD95-induced apoptosis would be that phos- ment of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85:803. phorylation of cellular substrates involved in regulating actin cy- 23. 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