Combined Action of ERK and NFκB Mediates the Protective Effect of Phorbol Ester on Fas-induced Apoptosis in Jurkat Cells*

The mechanisms whereby phorbol esters antagonize Fas-induced apoptosis in Jurkat T cells are poorly defined. In the present study, we report that protection from Fas-induced apoptosis by 12-O-tetradecanoylphorbol 13-acetate (TPA) is dependent on both ERK and NFκB activation. First, we showed that two specific mitogen-activated protein kinase/ERK kinase-inhibitors, PD98059 and U0126, both counteracted TPA-mediated suppression of Fas-induced apoptosis. Moreover, the dose-dependence of U0126-mediated inhibition of ERK phosphorylation correlated with that of reversion of the anti-apoptotic effect of TPA. Second, we observed an excellent correlation between repression of TPA-induced NFκB activation by an irreversible inhibitor of IκBα phosphorylation, BAY11–7082, and its ability to abrogate TPA-induced suppression of Fas-mediated apoptosis. Furthermore, we located the anti-apoptotic effect of both ERK and NFκB to lie upstream of the mitochondrial membrane potential depolarization event. Finally, although each inhibitor at optimal, non-toxic concentration by itself only partly reversed TPA-mediated repression of apoptosis, the combination of U0126 and BAY11–7082 completely abolished the anti-apoptotic effect of TPA. Together these findings suggest that TPA-induced activation of ERK and NFκB are parallel events that are both required for maximal inhibition of Fas-induced apoptosis in Jurkat T cells.

Apoptosis is an evolutionarily conserved and highly controlled form of cell death that plays a crucial role in development, maintenance of homeostasis, and immunological responses in multicellular organisms. Dysregulation of apoptosis is implicated in a range of diseases, including cancer, neurodegenerative disorders, and autoimmune diseases (1). Apoptosisinducing signals can be divided into two main groups: (i) those that lead to the formation of a death-inducing signaling complex at the cytoplasmic side of death receptors, resulting in the direct activation of a cascade of caspases responsible for cell death, and (ii) those that initially lead to the release of apoptogenic molecules from the mitochondria (2). In certain cell types, however, even death receptor-initiated apoptotic signals may be dependent on the mitochondria to induce cell death (3). Such cell types are termed "type II"-cells, in contrast to "type I"-cells where death receptor-induced cell death is independent of the mitochondria (3). One of the best char-acterized type II-cells is the Jurkat T cell (4), where apoptosis induced by ligation of the Fas/CD95 death receptor (5) is inhibited by interference with pro-apoptotic mitochondrial changes (3,6,7).
Fas-mediated apoptosis plays a pivotal role in a number of physiological processes and seems to be particularly important for immune function (8). Inhibition of Fas-mediated cell death is critical for T lymphocytes to survive activation, because activation of T cells leads to a markedly increased expression of both the Fas-receptor and its ligand (9 -12). Accordingly, stimulation through the T cell receptor (TCR) 1 has been shown to confer resistance of mature T lymphocytes toward Fas-induced apoptosis (9,11,13). The mechanism of this anti-apoptotic effect has been studied in Jurkat T cells, where Fas-induced apoptosis can be inhibited by stimulation with antibodies against the TCR (14), the TCR-binding and mitogenic lectin concanavalin A (ConA) (15), or by the PKC-activating phorbol ester TPA (16 -23).
In two early reports (15,21), the inhibiting effect of TPA or ConA on Fas-mediated apoptosis in Jurkat cells was ascribed solely to the MEK-ERK pathway. However, a number of studies have challenged the significance of this pathway in protection against Fas-mediated cell death (13, 16, 20, 22, 24 -27).
The aim of the current study was therefore to (i) assess the importance of the MEK-ERK pathway in protection against Fas-mediated apoptosis and (ii) determine possible contributions from other anti-apoptotic signaling pathways that are activated independently of MEK. We report that the ERK pathway plays a significant, but not exclusive, role in both TPA-and ConA-mediated suppression of Fas-induced apoptosis in Jurkat cells. Furthermore, we identify NFB as an equally important anti-apoptotic player and show that ERK and NFB are activated independently of each other. Finally, we demonstrate that simultaneous inhibition of ERK and NFB fully abrogates the ability of TPA to antagonize Fasinduced cell death. These results suggest that T cells make use of (at least) two independently activated signaling pathways to inhibit Fas-induced apoptosis.

MATERIALS AND METHODS
Reagents, Antibodies, and Plasmids-CH11 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). TPA, ConA, LY294002, wortmannin, and calphostin C were obtained from Sigma. PD98059, U0126, BAY11-7082, SB202190, and bisindolylmaleimide I were purchased from Calbiochem. TPA, calphostin C, and bisindolylmaleimide I were dissolved in ethanol, and ConA was dissolved in phosphate-buff-* This work was supported by the Norwegian Cancer Society. 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 with 18 U.S.C. Section 1734 solely to indicate this fact.
Cell Culture and Treatment-The Jurkat E6 -1 cell line (28) was purchased from American Type Culture Collection (Manassas, VA) and was cultured in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum (Invitrogen), 2 mM glutamine, 125 units/ml penicillin, and 125 g/ml streptomycin at 37°C in a humidified incubator with 5% CO 2 . Except for transfection studies, experiments were performed on cells that had been diluted to 2 ϫ 10 5 cells/ml 1 day earlier. Cell-permeable protein kinase inhibitors were added 1-2 h prior to TPA or ConA, which were added 5 min before CH11.
Determination of Apoptotic Cells by Morphological Criteria-Jurkat cells were pipetted to single cell suspension, and putatively 10,000 cells were analyzed for size (forward scatter) and granularity (side scatter) using a FACSCalibur flow cytometer (BD Biosciences). Based on the scatter profiles two populations, viable cells and apoptotic cells, were identified, where the apoptotic cells exhibit decreased forward scatter (size) and increased side scatter (granularity) compared with the viable cells. In our experiments, the percentage of necrotic cells (exhibiting increased forward scatter compared with viable cells) was always less than 0.5%.
TUNEL Assay-The in situ cell death detection kit Fluorescein (Roche Molecular Biochemicals) was used to detect DNA strand breaks that are associated with apoptosis (36). The assay was performed as recommended by the manufacturer, with the exception that cells were permeabilized with 0.1% saponin (in phosphate-buffered saline) instead of Triton X-100.
Analysis of Mitochondrial Membrane Potential-Jurkat cells were stained for 10 min at 37°C with 10 g/ml of the cationic, mitochondrial membrane potential sensor dye, JC-1, and 10,000 cells were analyzed by fluorescence-activated cell sorter (FACS). In advance, compensation for the overlap between FL1 and FL2 had been set by FACS analyses of Jurkat cells stained with anti-CD3-FITC-Ab (BD Biosciences) for FL1 and anti-CXCR4-PE-Ab (BD PharMingen) for FL2.
Western Blot Analysis-Jurkat cells (2 ml) were washed once with phosphate-buffered saline and lysed by gentle rotation at 4°C for 15 min in 50 l of lysis buffer (50 mM Tris-Cl, pH 7.4, 0.1% Triton X-100, 250 mM NaCl, 10 g/ml leupeptin, 9.5 g/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM NaF, 0.1 mM Na 3 VO 4 , 10 mM ␤-glycerophosphate). Cell lysates were cleared by centrifugation at 4°C for 10 min at 14 000 ϫ g and boiled for 5 min in 1ϫ Laemmli sample buffer (29). Equal amounts of protein (ϳ25 g) were separated by 12% (for ERK) or 9% (for PARP) SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Amersham Biosciences) using a semi-dry transfer cell (Bio-Rad), and the membranes were probed with antibodies from Cell Signaling Technologies as recommended by the manufacturer. Immunoreactive proteins were visualized with the ECL-plus detection system (Amersham Biosciences).
Transfections-Exponentially growing Jurkat cells (1 ϫ 10 7 ) were washed in RPMI 1640 (saving the conditioned medium), resuspended in 400 l of RPMI, mixed with a total of 10 -20 g of DNA, and electroporated at 250 V and 950 F using a Bio-Rad GenePulser. The cells were then transferred to 2 ml of conditioned medium on ice. Subsequently, 8 ml of fresh medium (with 10% fetal bovine serum) was added, and the cells were allowed to rest for 24 h before experimental treatments were initiated.
Green Fluorescent Protein (GFP) Cotransfection Studies-Jurkat cells were cotransfected with pEGFP-N1 and pCMV 4 -IB␣-SS32/36AA or pRL-CMV as a control. After stimulation with TPA and/or CH11, cells were analyzed by flow cytometry. Cells that expressed high levels of pEGFP-N1 (i.e. showing high FL-1 intensity) were also expected to express high levels of the mutant IB-construct, thus resulting in a high degree of NFB inhibition. These cells were gated and analyzed for forward and side scatter properties.
Reporter Assays-A dual luciferase reporter assay system (Promega) was used as described in the manufacturer's protocol. Luciferase activities were measured with a Turner Designs (Sunnyvale, CA) luminometer.

MEK-inhibitors PD98059 and U0126 Decrease the Ability of TPA and ConA to Suppress Fas-mediated Apoptosis-Fas-in-
duced apoptosis of Jurkat cells can be inhibited by the phorbol ester TPA or by the TCR-binding lectin ConA. Investigations of the role of the MEK-ERK pathway as a potential anti-apoptotic mediator in this process have yielded conflicting results (15,16,21,22,27). Therefore, to understand how Fas-induced apoptosis is regulated in Jurkat cells, we first assessed the importance of the MEK-ERK pathway in both TPA-and ConA-mediated protection against apoptosis. ERK is a serine/threonine protein kinase that is activated through phosphorylation by the dual-specificity protein kinase MEK (30). To date, the only MEK substrate recognized is ERK (31), and therefore specific inhibition of MEK will also specifically inhibit ERK phosphorylation and thus ERK activity. We tested the effect of two structurally unrelated cell-permeable MEK-inhibitors, PD98059 (32,33) and U0126 (34), on various features of apoptosis. First we examined the overall morphology of the cells by flow cytometric analysis of cell size (forward scatter) and granularity (side scatter), apoptotic cells being smaller and more granular than viable cells. Dot blots from one such experiment are shown in Fig. 1A, and the result from three independent experiments is graphically presented in Fig. 1B. Stimulation of Jurkat cells with agonistic anti-Fas antibodies (CH11) alone increased the percentage of apoptotic cells from 4 to 65% after 12 h. In the presence of TPA or ConA, however, CH11-induced apoptosis was reduced to 24%. Both PD98059 and U0126 partly reversed the anti-apoptotic effect of TPA and, to a lesser extent, that of ConA. The largest effect was seen when cells were pretreated with U0126 before TPA. Under these circumstances, CH-11-induced apoptosis was increased to 52%, i.e. the TPA effect was reversed by 68%. When examining the levels of the apoptosis-specific 89-kDa C-terminal caspase cleavage product of PARP (35) as a second means of analyzing apoptotic features, qualitatively the same results were obtained. As shown in Fig As a third means of analyzing apoptotic features, we examined the cleavage of genomic DNA, a hallmark of apoptosis (36), by the TUNEL assay. In this assay, DNA strand breaks generated by DNA fragmentation are labeled with fluorescein dUTP, and the amount of DNA fragmentation (reflected by fluorescence intensity) can then be determined at the singlecell level by flow cytometry. Under the same experimental conditions as described in Fig. 1, A and B, cells were subjected to the TUNEL assay after 6 h of CH11 treatment. An example of dot plots from one such experiment is shown in Fig. 1C, and the results from three independent experiments are graphically presented in Fig. 1D. Whereas only 5% of the cells were gated as positive for DNA fragmentation in untreated Jurkat cells, 51% of the cells were TUNEL-positive after treatment with CH11 alone. Pretreatment with TPA reduced the amount of TUNEL-positive cells to 18%. Both PD98059 and U0126 were able to substantially counteract the TPA effect. Again, U0126 was the most efficient inhibitor. In the presence of U0126 the percentage of TUNEL-positive cells rose to 39%, i.e. the TPA-effect was reversed by 64%.
To ensure that the MEK-inhibitors actually inhibited ERK activity in the cells, we examined the levels of phosphorylated ERK protein by Western blot analyses. Indeed, as shown in Fig. 1B, both PD98059 and U0126 substantially inhibited both TPA-and ConA-induced phosphorylation of ERK after 30 min of stimulation. The inhibiting effect was also observed at earlier time points (10 and 20 min) and was sustained for at least 2 h (data not shown). U0126 was a more efficient inhibitor of ERK phosphorylation than PD98059 (Fig. 1B, compare lanes 4 and 7 with lanes 5 and 8, respectively). This could be the reason why U0126 also was observed to be superior to PD98059 in abrogating the anti-apoptotic effects of TPA and ConA. To test whether there was a connection between the extent of ERK inhibition and augmentation of apoptosis, we examined the dose-dependence of U0126mediated reversion of TPA's protective effect against Fasinduced apoptosis with that of ERK inhibition. Indeed, as shown in Fig. 2, there was an excellent correlation between U0126-mediated ERK inhibition and abolishment of the antiapoptotic effect of TPA. We could therefore conclude that the MEK-ERK pathway was involved in both TPA-and ConA-mediated inhibition of Fas-induced apoptosis in Jurkat cells. However, even at concentrations of U0126 that almost completely inhibited ERK phosphorylation, the MEK-inhibitor was never able to fully abolish the protective effect of TPA or ConA against Fas-induced apoptosis, as determined by examination of cell morphology, PARP-cleavage, and DNA fragmentation. This suggested that at least one signaling pathway, other than the MEK-ERK pathway, is involved in the anti-apoptotic effect of TPA and ConA in Jurkat cells.
Inhibition of NFB Reverses TPA-and ConA-mediated Suppression of Fas-induced Apoptosis-In addition to the MEK-ERK pathway, three other signaling pathways have been reported to be involved in regulation of Fas-induced apoptosis in A, after treatment with CH11 for 12 h, cells were harvested and their forward-and side-scatter profiles were analyzed by flow cytometry as described under "Materials and Methods." The percentages of cells gated as apoptotic are indicated. One representative experiment is shown. B, average percentages of apoptotic cells from three independent experiments like the one described above are presented graphically with S.E. of the mean as indicated. Protein levels of (phosphorylated and total) ERK and cleaved PARP were determined by Western blot analyses after 0.5 and 2 h of CH11 treatment, respectively. TPA or ConA treatment alone led to a slight induction of apoptosis (5-10%), which was not significantly altered by MEK inhibition (data not shown). C, after treatment with CH11 for 6 h, cells were subjected to the TUNEL assay as described under "Material and Methods." The percentages of cells gated as TUNEL-positive are indicated. One representative experiment is shown. D, average percentages of TUNEL-positive cells from three independent experiments like the one described in panel C are presented graphically with S.E. of the mean as indicated.
human T cells, i.e. those that lead to the activation of PI3K/Akt, p38/MAPK, and NFB, respectively (13, 24 -26). We therefore tested whether cell-permeable inhibitors of PI3K, p38/MAPK, or NFB would prevent TPA-or ConA-mediated suppression of Fas-induced apoptosis in Jurkat cells. The inhibitors were used at concentrations that are known to specifically inhibit PI3K (10 M LY294002 or 100 nM wortmannin), p38/MAPK (5 M SB202190), or NFB (2.5 M BAY11-7082), respectively, and apoptosis was analyzed by examination of cellular morphology. Whereas inhibitors against PI3K and p38/MAPK had no effect, the NFB inhibitor BAY11-7082 markedly abrogated both TPA-and ConA-mediated protection against Fas-induced apoptosis (Fig. 3). To examine whether this effect could be attributed to specific NFB inhibition, we assessed whether there was a correlation between the ability of BAY11-7082 to inhibit NFB and its ability to reverse TPA-mediated suppression of Fas-induced apoptosis. To determine NFB activity, Jurkat cells were transiently transfected with a luciferase reporter plasmid controlled by three repeated NFB response elements (3ϫ B-TATA), together with a constitutively active renilla luciferase reporter (pRL-CMV) to normalize for variability in transfection efficiencies. Treatment with TPA and CH11 stimulated the NFB reporter about 6-fold above basal levels, and BAY11-7082 inhibited this activation in a dose-dependent manner (Fig. 4A). Thus at 3 M, BAY11-7082 inhibited NFB activity by 86%. At concentrations above 3 M, BAY11-7082 was toxic to Jurkat cells (data not shown). Treatment with CH11 alone did not increase NFB reporter activity (rather a slight inhibition was observed), and TPA-induced activation of the reporter was not significantly influenced by CH11 (Fig. 4A). When we tested the same concentrations of BAY11-7082 for its ability to inhibit the anti-apoptotic effect of TPA (Fig. 4B), we observed that there was excellent correlation between BAY11-7082-induced inhibition of NFB activity and reversion of TPA-mediated suppression of Fas-induced apoptosis (compare Fig. 4A and B).
To further establish the role of NFB in the anti-apoptotic effect of TPA, Jurkat cells were transiently transfected with an expression vector encoding a non-degradable IB␣ mutant (␦IB␣). To be able to detect transfected cells, the cells were cotransfected with pEGFP-N1, an expression vector encoding GFP. As a control, cells were transfected with pEGFP-N1 and a plasmid expressing renilla luciferase. As shown in Fig. 4C, the ability of TPA to suppress Fas-induced apoptosis was reduced in cells transfected with the ␦IB expression vector. Thus, the percentage of apoptotic cells in the presence of TPA and CH11 increased from 18% in control-transfected cells to 31% in cells transfected with the ␦IB-construct (Fig. 4C). To ensure that the IB␣ mutant inhibited NFB activity, ␦IB␣ was cotransfected with 3ϫ B-TATA, and a luciferase reporter assay was performed similar to that described above. Indeed, cotransfection of ␦IB␣ strongly inhibited the ability of TPA to activate the NFB reporter (Fig. 4D). Together these experiments strongly suggested that NFB plays a significant role in TPA-mediated repression of Fas-induced apoptosis.
TPA Activates NFB through Protein Kinase C-It has recently been suggested (37, 38) that phorbol esters elicit some of their biological effects through PKC-independent pathways. We therefore assessed whether TPA activated NFB through a PKC-dependent or -independent pathway. For this purpose, we employed two functionally unrelated and highly potent inhibitors of PKC, calphostin C and bisindolylmaleimide I, which specifically bind to the regulatory and catalytic regions of PKC, respectively (39,40). As shown in Fig. 5A, both PKC-inhibitors strongly inhibited TPA-induced activation of the NFB reporter, indicating that TPA activates NFB through a PKC-dependent pathway in Jurkat cells.
NFB and MEK/ERK Are Independent Signaling Pathways-Although we had shown that both the NFB and the MEK/ERK pathways were involved in repression of Fas-induced apoptosis, we could not exclude the possibility that this could be due to ERK and NFB being part of the same antiapoptotic signaling pathway instead of being parallel pathways that both contribute to the inhibition of apoptosis. To resolve this issue, we assessed whether inhibition of the MEK/ERK pathway would affect NFB activity and, conversely, whether inhibition of NFB would affect the activity of AP-1, a transcription factor that is known to be dependent on ERK for its maximal activity (41). As shown in Fig. 4A, whereas BAY11-7082 strongly inhibited NFB activity in a dose-dependent manner, the MEK-inhibitor U0126 instead slightly augmented TPA-induced NFB activation. To determine AP-1 transactivating activity, a transient transfection reporter assay with an AP-1-driven luciferase reporter construct was performed. For optimal activation of AP-1, the cells were cotransfected with an expression vector encoding a constitutively active form of MEKK (pFC-MEKK), and luciferase activities were measured after treatment with BAY11-7082 or U0126 for 5 h. U0126 inhibited MEKK-induced AP-1 activity in a dose-dependent manner (Fig. 5B). However, as anticipated, U0126 did not completely inhibit AP-1 activity, because U0126 does not inhibit MEKK-induced activation of c-Jun N-terminal kinase (JNK), a major contributor to AP-1 activity (41). Importantly, BAY11-7082 did not inhibit AP-1 activity (Fig. 5B). Furthermore, BAY11-7082 had no effect on TPA-stimulated phosphorylation of ERK (data not shown). These data indicate that ERK and NFB are components of parallel, and not sequentially activated, signaling pathways and that they therefore contribute to the anti-apoptotic effect of TPA independently of each other.

Simultaneous Inhibition of MEK and NFB Completely Abolishes TPA-mediated Suppression of Fas-induced Apoptosis-
We had shown that inhibition of neither ERK nor NFB alone was able to completely reverse TPA-mediated suppression of Fas-induced apoptosis. We therefore investigated whether the anti-apoptotic effect of TPA could be a result of cooperative actions between ERK and NFB. Simultaneous inhibition of MEK and NFB was enforced by combined treatment with U0126 and BAY11-7082, and TPA-mediated repression of Fas-induced apoptosis was analyzed. Whereas U0126 or BAY11-7082 alone only partly reversed TPA-mediated suppression of CH11-induced apoptosis, the combination of the two inhibitors completely abolished the anti-apoptotic effect of TPA (Fig. 6A). U0126 was also tested in combination with inhibitors of PI3K (LY294002 and wortmannin) or p38/MAPK (SB202190), but none of these inhibitors potentiated the effect of U0126 on TPA-mediated suppression of Fas-induced cell death (data not shown).

ERK and NFB Inhibit Apoptosis Upstream of Mitochondrial Membrane Depolarization in a Cooperative
Manner-Fas-induced apoptosis in Jurkat and other type II cells is dependent on the mitochondria (3, 6, 7). One of the major pro-apoptotic mitochondrial events is the disruption of the mitochondrial membrane potential, ⌬⌿ m (42,43). ⌬⌿ m can be monitored by the use of cationic dyes, among which JC-1 is the most specific for monitoring mitochondrial versus plasma membrane potential (44). In cells that contain mitochondria with a normal ⌬⌿ m , JC-1 forms red fluorescent aggregates. A decrease in ⌬⌿ m , however, leads to the disintegration of these aggregates concomitantly with an accumulation of green fluorescent JC-1 monomers. ⌬⌿ m was analyzed by flow cytometry as shown in Fig. 6C. Cells stained with JC-1 that displayed decreased red fluorescence concomitant with increased green fluorescence were classified as cells containing mitochondria with depolarized membranes. By analyzing the effects of U0126 and BAY11-7082 on TPA-mediated inhibition of Fas-induced depolarization of the mitochondrial membrane (Fig. 6, B and C), we observed that the effects were strikingly similar to those detected when apoptosis was analyzed by morphological criteria (Fig. 6A). Thus, whereas treatment with either U0126 or BAY11-7082 alone only partially reversed TPA-mediated suppression of Fas-induced loss of ⌬⌿ m , the presence of both inhibitors simultaneously almost completely abrogated the TPA effect (Fig. 6B). This suggests that the anti-apoptotic effects of ERK and NFB are upstream of the mitochondrial membrane depolarization event. Based on our results, a model is depicted (Fig. 7) of how we believe TPA protects Jurkat cells from Fas-induced apoptosis. DISCUSSION Although much studied, the mechanisms that regulate Fasinduced apoptosis in T cells are incompletely understood. Progress during recent years has implicated several different signal transduction pathways in protection against Fas-induced apoptosis in T cells (13-15, 21, 24 -26). However, in all these studies the anti-apoptotic signaling pathways have only been explored individually. In the present study we showed that in Jurkat T cells the disruption of one signaling pathway alone was never sufficient to fully abolish the strong repression of Fas-induced apoptosis mediated by TPA. We therefore hypothesized that the anti-apoptotic effect of TPA could only be explained by the involvement of at least two distinct and cooperative signaling pathways. Indeed, we report here that simultaneous inhibition of ERK and NFB was both necessary and sufficient to fully prevent TPA-mediated suppression of Fas-induced apoptosis in Jurkat cells.
Other signaling transduction pathways, including the PI3/ Akt and p38/MAPK pathways (13,25,26), have been implicated in repression of Fas-induced cell death in T cells. However, from our results, neither of these pathways seems to be involved in either TPA-or ConA-mediated protection from Fasinduced apoptosis in Jurkat cells. First of all, two widely used TPA inhibits Fas-receptor-stimulated depolarization of the mitochondrial membrane potential and apoptosis through a parallel activation of two anti-apoptotic pathways, i.e. those that involve NFB and ERK, respectively. Therefore, the simultaneous inhibition of both NFB and ERK is necessary to completely abolish TPA-mediated suppression of Fas-induced apoptosis.
inhibitors of PI3K, LY294002 and wortmannin, had no effect on either TPA-or ConA-mediated suppression of Fas-induced apoptosis. Second, in agreement with a recent report (20), TPA failed to activate Akt as determined by phospho-Akt Western blot analysis and an in vitro Akt kinase assay (data not shown). Third, we showed that TPA-and ConA-mediated suppression of Fas-induced apoptosis were unaffected by the p38/MAPKinhibitor, SB202190. This is also in line with a previous report (15) in which another widely used p38/MAPKinhibitor, SB203580, did not influence ConA-mediated protection from Fas-induced apoptosis in Jurkat cells. In addition to the PI3K/Akt and p38/MAPK pathway, the JNK/SAPK pathway has been implicated in protection from Fas-induced apoptosis in some cell types (45,46). However, in agreement with a recent study (20), we found that JNK activity was unaffected by TPA in Jurkat cells (data not shown). Thus, neither the PI3K/Akt pathway nor the stress-activated MAP kinases, p38/MAPK and JNK, seem to play any significant role in TPA-mediated suppression of Fas-induced apoptosis in Jurkat T cells.
It was not clear to us why there have been conflicting reports regarding the involvement of the MEK/ERK pathway in TPAmediated protection from Fas-induced apoptosis in Jurkat cells (15,16,21,22,27). One possible reason could be that previous studies have been based on the use of only PD98059 as a MEK inhibitor. We, and others (31), have experienced that PD98059 can be difficult to work with because of its very limited solubility in aqueous solution. When PD98059 was not properly solubilized (i.e. crystals were formed in the medium) it had no effect on TPA-mediated suppression of Fas-induced apoptosis (data not shown). We therefore used two structurally unrelated MEK-inhibitors (PD98059 and U0126) to analyze the effect on apoptosis. Moreover, we assessed apoptosis by three different methods, based on morphological (cell size and granularity), biochemical (PARP cleavage), and molecular (DNA fragmentation) criteria, respectively. Because we obtained essentially the same results with both inhibitors on all three apoptotic criteria, we concluded that the MEK/ERK pathway is indeed required for TPA-mediated suppression of Fas-induced apoptosis in Jurkat cells.
When utilizing small, cell-permeable inhibitors, there is a general problem of potential unspecific effects. However, we strongly believe that the inhibitor-induced effects we observed in the present study were due to specific interference with the activities of the targeted proteins. First, in a recent study, U0126, PD98059, wortmannin, and SB202190 were all found to be among the most specific of commonly used protein kinase inhibitors (47). Second, we demonstrated the specificity of the effects of U0126 and BAY11-7082 by the excellent correlations between inhibitor-induced repression of ERK and NFB activities, respectively, and their abilities to antagonize the anti-apoptotic effect of TPA. Finally, we directly assessed the ability of U0126 to inhibit NFB activity and, conversely, that of BAY11-7082 to inhibit AP-1 activity. Indeed, we did not find any unspecific inhibitory effects; if any, U0126 and BAY11-7082 rather slightly increased the activities of NFB and AP-1, respectively, which in fact may be due to an unleashing of the competition of AP-1 and NFB for a common co-factor, such as p300 (48).
In conclusion, we propose a model where TPA-mediated suppression of Fas-induced apoptosis in Jurkat T cells is dependent on the activation and cooperative action of both ERK and NFB (Fig. 7). In this model, the anti-apoptotic effects of ERK and NFB are suggested to lie upstream of the mitochondrial membrane disruption event. It will be important to identify the downstream targets that are responsible for the anti-apoptotic effects of ERK and NFB to further understand how Fas-induced apoptosis is regulated in human T cells.