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The Journal of Immunology, 1999, 163: 5813-5819.
Copyright © 1999 by The American Association of Immunologists

Protein Kinase C{theta} Cooperates with Calcineurin to Induce Fas Ligand Expression During Activation-Induced T Cell Death1

Martin Villalba2,*, Shailaja Kasibhatla3,{dagger}, Laurent Genestier4,{dagger}, Artin Mahboubi{dagger}, Douglas R. Green{dagger} and Amnon Altman2,*

Divisions of * Cell Biology and {dagger} Cellular Immunolgy, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKC{theta}, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKC{theta} mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKC{theta} mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKC{theta} synergized with calcineurin to provide a potent stimulus for FasL promoter activation. Full activation of the promoter required its binding sites for the transcription factors NF-AT, AP-1, and NF-{kappa}B. The biological significance of these findings is implicated by the finding that rottlerin, a selective PKC{theta} inhibitor, blocked FasL induction by anti-CD3 or PMA plus ionomycin stimulation and, consequently, protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from activation-induced cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Triggering of the Ag-specific TCR induces lymphocyte activation, cytokine secretion, and proliferation. To maintain T cell homeostasis, activated lymphocytes are removed by apoptosis once the Ag has been cleared (1). This form of apoptosis is known as activation-induced cell death (AICD),5 and it involves the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells (2, 3, 4, 5, 6). Once FasL is expressed on the T cell surface, it induces T cell apoptosis through activation of its receptor Fas (6, 7). However, the precise mechanisms that regulate FasL expression are poorly understood.

Recent studies focused on characterization of the structure and properties of the FasL gene promoter. This promoter possesses potential binding sites for several transcription factors including the "housekeeping" promoter elements, NF-AT, AP-1, and NF-{kappa}B (8). Two NF-AT-binding sites are necessary for proper FasL induction in activated T cells (9, 10), and a role for NF-{kappa}B in FasL expression after T cell activation has been shown (11, 12). Stress-induced FasL expression also requires the activation of NF-{kappa}B and AP-1 (8, 13). In addition, the FasL promoter also contains a new response element (RE), designated RE-3 (14), and a mitogen-activated protein kinase/extracellular signal-related kinase (ERK) kinase-1 (MEKK1) RE (13). Taken together, these findings suggest that FasL induction is mediated by the coordinated action of several transcription factors.

In addition to TCR ligation, the combination of phorbol ester plus Ca2+ ionophore, which mimicks the two physiologic signals required for T cell activation and IL-2 production, can also induce FasL expression (14, 15) and AICD (16). Phorbol esters mediate their pleiotropic effects mainly via the activation of protein kinase C (PKC), suggesting that some PKC isoform(s) regulates FasL expression. In contrast, phosphatase 2B or calcineurin (Cn) represents an important target of Ca2+ signals (17), and its TCR-induced enzymatic activation leads to dephosphorylation of NF-AT and its translocation to the nucleus (18), where it combines with AP-1 (19, 20) and other transcription factors to stimulate transcription of the IL-2 gene.

In the present work, we have investigated the potential involvement of PKC{theta} in the pathway that induces FasL expression following TCR/CD3 stimulation. We focused on this particular isoform because it is selectively expressed in T cells (21), where it colocalizes with the TCR complex to the contact site between Ag-specific T cells and APCs (22, 23). Moreover, PKC{theta} selectively cooperates with Cn in the activation of JNK and induction of the IL-2 gene (24, 25). We report that PKC{theta} plays an important role in regulating FasL expression and AICD and, furthermore, that it cooperates with Cn in these processes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and chemicals

Polyclonal Abs against the PKC isoenzymes {theta} and {zeta} were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mAbs against PKC{alpha} and PKC{epsilon} were obtained from Transduction Laboratories (Lexington, KY). The anti-CD3 mAb, OKT3, was purified from culture supernatants of the corresponding hybridomas by protein A-Sepharose chromatography. The anti-hemagglutinin mAb 12CA5 was obtained from Boehringer Mannheim (Indianapolis, IN), Rottlerin and Gö6976 were obtained from Calbiochem (La Jolla, CA). All other products were obtained from Sigma (St. Louis, MO).

Plasmids

The cDNAs encoding human wild-type PKC{theta} and PKC{alpha}, the constitutively active mutants of human PKC{theta} (A148E) and PKC{alpha} (A25E), rat PKC{epsilon} (A159E), or mouse PKC{zeta} (A119E), and the dominant-negative (kinase-inactive) mutant of PKC{theta} (K409R) were cloned in the eukaryotic expression vector pEFneo. An hemagglutinin-tagged, constitutively active Cn A mutant (CaM-AI; Ref. 25) cDNA was cloned in pSR{alpha}. The FasL reporter constructs have been previously described (8, 12). As control for transfection efficiencies, a ß-galactosidase (ß-gal) expression plasmid in the pEF vector was used.

Cell culture and transfection

SV40 large T Ag (TAg)-transfected human leukemic Jurkat T cells (Jurkat-TAg), Jurkat-CE cells (which are highly sensitive to AICD), and the T cell hybridoma A1.1 were grown in RPMI 1640 medium (Life Technologies), supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, pH 7.2, 1x MEM nonessential amino acid solution (Life Technologies), and 100 U/ml of each penicillin G and streptomycin. Cells in a logarithmic growth phase were transfected with the indicated amounts of plasmid DNAs by electroporation as previously described (26, 27). In each experiment, cells in each group were transfected with the same total amount of DNA by supplementing expression vector DNA with the proper amounts of the corresponding empty vector. In some experiments, the cells were pretreated with PKC inhibitors as indicated.

Luciferase and ß-gal assays

Transfected cells were harvested after 2 days, washed twice with PBS, and lysed in 100 µl of lysis buffer (100 mM KPO4, pH 7.8, 1 mM DTT, 0.5% Triton X-100) for 10 min at room temperature. The lysates were then centrifuged (15,000 x g, 5 min at 4°C). For the luciferase assay, 50 µl of the supernatants were mixed with 100 µl of assay buffer (17.5 mM glycilglycine, pH 7.8, 10 mM MgCl2, 5 mM ATP, 0.135 mM coenzyme A, 0.235 mM luciferin), and the luciferase activity was determined in a luminometer. Luciferase activity in each group was normalized to the activity of a cotransfected ß-gal reporter plasmid. For the ß-gal assay, 20 µl of the supernatants were incubated at 37°C in 150 µl of assay buffer containing 60 mM Na2HCO, 80 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 10 mM DTT, and 60 µg o-nitrophenyl ß-D-galactopyranoside until a yellow color developed. Absorbance was measured at 400 nm in a spectophotometer. The results were expressed as arbitrary luciferase units per arbitrary ß-gal units. All experiments were performed in duplicate and were repeated several times with similar results.

Induction and assessment of apoptosis

Human Jurkat-CE leukemic cells or murine A1.1 hybridoma cells were induced to undergo apoptosis by treatment with a combination of PMA (100 ng/ml) plus ionomycin (1 µg/ml), or with anti-human (OKT3) or anti-mouse (2C11) CD3 mAbs (28). For TCR stimulation, 96-well plates were precoated with optimal Ab concentrations in 50 mM Tris-HCl, pH 9.0. Apoptosis of Jurkat-CE cells was assessed by staining the cells using the annexin-V-FLUOS staining kit (Boehringer Mannheim) and analyzing them on a FACScan analyzer following the manufacturer’s instructions. In A1.1 cells, AICD was determined by propidium iodide staining (28).

In vitro PKC kinase assay

Sixty nanograms of purified PKC{theta} (29) and PKC{alpha} (Panvera, Madison, WI) proteins were incubated for 5 min at 30°C with gentle shaking in a reaction mixture containing 10 µg of the synthetic peptide substrate RFARKGSLRQKNVY homologous to the pseudosubstrate sequence of PKC{alpha}, 100 ng L-{alpha}-phosphatidyl-L-serine, 20 ng diacylglycerol, 10 µCi [{gamma}-32P]ATP, 20 mM HEPES, pH 7.5, 10 mM MgCl2, and 0.1 mM ATP. In additon, the PKC{alpha} and PKC{theta} kinase reactions contained 0.1 mM CaCl2 or 0.1 mM EGTA, respectively (30). Samples were placed on ice to stop the reaction, collected on phophocellulose units (Pierce, Rockford, IL), and processed following the instructions of the manufacturer. The amount of radioactivity incorporated into the synthetic peptide substrate was determined in a scintillation counter.

FasL expression analysis by RT-PCR

In experiments performed with transfected cells, 20 x 106 Jurkat-TAg cells were transfected with 5 µg of the appropriate plasmid. After 2 days, total RNA was extracted and subjected to RT-PCR analysis of FasL and actin expression as previously described (8). In experiments performed with Jurkat-CE cells, 10 x 106 cells were incubated for 30 min at room temperature with 30 µM of rottlerin and then stimulated for 4 h with PMA (100 ng/ml) plus ionomycin (1 µg/ml) or with anti-human (OKT3) CD3 mAb (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKC{theta} plays an important role in FasL expression

To determine whether a particular PKC isoform is preferentially involved in FasL expression, we cotransfected Jurkat-TAg cells with a plasmid carrying the FasL promoter cloned upstream of a luciferase reporter gene (8) plus constitutively active mutants of several PKC isoforms representing Ca2+-dependent ({alpha}), -independent ({epsilon}, {theta}), or atypical ({zeta}) PKC isoforms. These mutants have been previously characterized (30, 31, 32). The constitutively active PKC{theta} mutant ({theta}-A/E) induced a ~5-fold activation of the FasL reporter, whereas similar mutants of the other PKC isoforms caused a much weaker stimulation that did not exceed 2-fold (for PKC{alpha}-A/E) (Fig. 1GoA). Under the same conditions, and in agreement with a recent report (25), the various PKC isoforms tested were capable of stimulating the kinase activity of an ERK2 reporter plasmid to a similar degree (data not shown), indicating that they were functional in the transfected cells. The mean ± SEM of eight experiments was 103 ± 25-fold and 332 ± 53-fold induction for pEF- or PKC{theta}-A/E-transfected cells, respectively. Wild-type PKC{theta} (or PKC{alpha}) was unable to induce FasL promoter activation, suggesting that PKC{theta} activation is required for FasL promoter stimulation. Immunoblotting with the corresponding PKC-specific Abs confirmed the proper overexpression of the transfected PKC enzymes in all groups (Fig. 1GoB). RT-PCR analysis showed that transient overexpression of PKC{theta}-A/E, but not PKC{alpha}-A/E, also induced expression of the endogenous FasL (Fig. 1GoC, top), although both PKC isoforms were similarly overexpressed (Fig. 1GoC, bottom), ruling out the possibility that the differential induction of FasL mRNA reflects lower expression of PKC{alpha}.



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  		FIGURE 1. PKC{theta} selectively up-regulates FasL expression. A, A total of 10 x 106 Jurkat-TAg cells were transfected with 5 µg each of FasL-Luc reporter and pEF-ß-gal plasmids together with 5 µg of the indicated wild-type (wt) or constitutively active (A/E) PKC mutants. Luciferase and ß-gal activities were determined after 48 h as described in Materials and Methods. B, Aliquots of cell lysates from the different groups of the transfected cells (1 x 106) were immunoblotted with the indicated Abs. C, PKC{theta} induces the expression of endogenous FasL. A total of 10 x 106 Jurkat-TAg cells were transfected with 5 µg of constitutively active PKC{theta} or PKC{alpha} mutants. Two days later, an RT-PCR was performed to assess the expression of FasL- and a control (actin)-specific transcripts (two upper panels). Aliquots of cell lysates (1 x 106 cells) were immunoblotted with PKC{theta}- or PKC{alpha}-specific Abs to confirm the overexpression of the transfected PKC plasmids (two lower panels).

 
Next, we used two independent approaches to address the question whether functional PKC{theta} is necessary for FasL promoter activation induced by an anti-CD3 Ab or the combination of PMA plus ionomycin. First, we determined the effect of selectively inhibiting cellular activity of PKC{theta} on FasL promoter induction. Because no specific PKC{theta} inhibitor was described to date, we addressed the possibility that rottlerin, which was previously found to inhibit the activity of PKC{delta} (33) also inhibits PKC{theta}. This was prompted by the high degree of similarity between PKC{theta} and PKC{delta} (21). The effect of rottlerin was tested on the in vitro enzymatic activity of purified PKC{theta} or PKC{alpha}, the latter being a representative of the Ca2+-dependent PKC subfamily. As a control, the effect of another compound, Gö6976, which is a selective inhibitor of Ca2+-dependent PKC enzymes (34), was also determined. Fig. 2GoA shows that, as expected, Gö6976 blocked the enzymatic activity of PKC{alpha}, but had no effect on the activity of PKC{theta} (Fig. 2GoA, left). Conversely, rottlerin inhibited the activity of PKC{theta} by nearly 50%, but had only a marginal effect on PKC{alpha} (Fig. 2GoA, right). Importantly, the various Jurkat cell sublines used (see below) express PKC{theta} but not PKC{delta}; conversely, and as a specificity control, COS cells expressed PKC{delta} but not PKC{theta} (Fig. 2GoB).



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  		FIGURE 2. Functional PKC{theta} is required for FasL induction. A, Rottlerin inhibits PKC{theta} activity. Purified PKC{theta} and {alpha} in the respective kinase buffers were incubated with a peptide substrate in the presence of the indicated concentrations of the PKC inhibitors Gö6976 (left) or rottlerin (right), and the amount of radioactivity incorporated into the peptide substrate was quantitated. Complete (100%) activity (59,000 and 485,000 cpm for PKC{theta} or PKC{alpha}, respectively) represents the amount of radioactivity incorporated in the absence of inhibitor after subtracting the background activity in control reactions lacking PKC. B, Expression of PKC{theta} or PKC{delta} in different Jurkat cell lines and COS cells. Lysates of the two cell lines were separated by SDS-PAGE and immunoblotted with PKC{theta}- or PKC{delta}-specific Abs as indicated. C, Rottlerin blocks FasL promoter induction by constitutively active PKC{theta} or PMA plus ionomycin stimulation. A total of 10 x 106 Jurkat-TAg cells were transfected with 5 µg FasL-Luc and pEF-ß-gal reporter plasmids together with 5 µg of constitutively active (A/E) or dominant-negative (K/R) PKC{theta} mutants. The cells were treated for the final 6 h of culture with rottlerin (30 µM) or Gö6976 (0.5 µM). Normalized luciferase activity was determined after 48 h of culture in unstimulated cells (left) or in cells stimulated for the final 6 h of culture with 100 ng/ml PMA plus 1 µg/ml ionomycin (right).

 
Having established the relative selectivity of rottlerin for PKC{theta}, we examined whether this drug can inhibit activation of the FasL promoter in intact T cells induced by transiently overexpressed PKC{theta} or activating stimuli. Jurkat-TAg cells were transfected with constitutively active (A/E) PKC{theta}, and the luciferase activity of a cotransfected FasL reporter was analyzed in rottlerin-pretreated vs untreated cells. As an additional control, some cells were pretreated with Gö6976. In unstimulated cells, rottlerin reduced FasL promoter activity by 90% in empty vector-transfected cells, and completely blocked the PKC{theta}-A/E-induced activity (Fig. 2GoC, left). In that regard, the effect of rottlerin was very similar to that of the PKC{theta}-K/R mutant (see below). In contrast, Gö6976 failed to inhibit FasL promoter activity and, in fact, appeared to augment it. In other experiments, we found that rottlerin also blocked PKC{theta}-, but not ionomycin-induced NF-AT activation (data no shown). When the cells were stimulated with PMA plus ionomycin, rottlerin again blocked FasL promoter activation in empty vector- or PKC{theta}-A/E-transfected cells, whereas Gö6976 was markedly less effective (Fig. 2GoC, right). Similar results were obtained when Jurkat-TAg cells were stimulated with an anti-CD3 Ab (data no shown).

In a second approach, Jurkat-TAg cells were cotransfected with dominant-negative (K/R) PKC{theta} mutant and the FasL reporter plasmid, and luciferase activity was determined in resting or stimulated cells. Dominant-negative PKC{theta} profoundly inhibited the basal (Fig. 2GoC, left) as well as PMA plus ionomycin-induced (Fig. 2GoC, right) or anti-CD3-induced (data not shown) FasL promoter activity. The level of inhibition reached 80–95%. Taken together, these findings strongly suggest that PKC{theta} is required for FasL activation.

PKC{theta} cooperates with Cn to induce FasL expression

Recent studies demonstrated that PKC{theta} cooperates with Cn to activate JNK, NF-AT, and the IL-2 promoter in T cells (24, 25). Therefore, the cooperative induction of FasL by the combination of PMA and Ca2+ ionophore led us to determine whether Cn represents a potential Ca2+ target in this system as well. Jurkat-TAg cells were transfected with various combinations of constitutively active (A/E) mutants of PKC{theta}, PKC{alpha}, and/or Cn (CaM-AI), and the luciferase activity of a cotransfected FasL reporter was determined (Fig. 3Go). Transient overexpression of PKC{theta} or CaM-AI, but not PKC{alpha}, led to a limited activation of the FasL promoter. However, when PKC{theta} and CaM-AI were combined, a synergistic activation of the promoter was observed (Fig. 3GoA). The mean values ± SEM of six independent experiments were 103 ± 26-fold and 2,740 ± 507-fold induction for the empty vector- or PKC{theta} plus CaM-AI-transfected groups, respectively. PKC{alpha} also cooperated with CaM-AI to stimulate the promoter’s activity, but this effect was 4.5-fold lower than that induced by PKC{theta} plus CaM-AI. Both PKC{theta} and PKC{alpha}, as well as CaM-AI, were properly overexpressed in the cells (Fig. 3GoB). This result indicates that there is considerable selectivity to the cooperation of PKC{theta} with CaM-AI in the activation of the FasL promoter.



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  		FIGURE 3. PKC{theta} cooperates with Cn to activate the FasL promoter. A, A total of 10 x 106 Jurkat-TAg cells were transfected with 5 µg each of FasL-Luc and pEF-ß-gal reporter plasmids together with 5 µg of the indicated combinations of constitutively active PKC{theta}, PKC{alpha}, and/or Cn (CaM-AI) mutants. Luciferase and ß-gal activities were determined after 48 h as described in Materials and Methods. B, Aliquots of cell lysates (1 x 106 cells) from the corresponding transfection groups were immunoblotted with PKC{theta}-, PKC{alpha}-, or CaM-AI (hemagglutinin epitope tag)-specific Abs.

 
The role of distinct binding sites in FasL promoter induction

To investigate in more detail the transcription factor(s) involved in FasL expression, cells were transfected with constitutively active PKC{theta} and/or Cn mutants together with various FasL reporter constructs. The latter consisted of the wild-type promoter or mutations in its distal NF-AT-binding site ({Delta}NF-AT), or in the binding sites for AP-1 ({Delta}AP-1) or NF-{kappa}B ({Delta}NF-{kappa}B), respectively. These binding sites were previously found to be involved in FasL expression (8). The results (Fig. 4GoA) show that maximal FasL promoter activation by PKC{theta} plus CaM-AI in unstimulated T cells required all three binding sites, because mutation of any binding site resulted in at least 68% decrease in promoter activity. Mutation of the NF-AT-binding site had the most deleterious effect. This was also evident when the suboptimal induction of the FasL promoter by either PKC{theta} or CaM-AI alone was assessed. Thus, mutating the NF-AT-binding site completely blocked FasL promoter activity induced by CaM-AI, in agreement with previous reports (9, 10) and also had a more severe effect on the PKC{theta}-mediated promoter activity when compared with mutations in the AP-1- or NF-{kappa}B-binding sites. This is consistent with our finding that PKC{theta} can stimulate the activity of an NF-AT reporter derived from the IL-2 promoter in T cells (data not shown).



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  		FIGURE 4. The role of different binding sites in the FasL promoter in PKC{theta}- and/or Cn-induced promoter activation. Ten x 106 Jurkat-TAg cells were transfected with 5 µg of pEF-ß-gal and the indicated FasL wild-type (wt) or mutated reporter plasmids together with 5 µg of constitutively active PKC{theta} and/or Cn (CaM-AI) mutants. Luciferase and ß-gal activities were determined after 48 h. The cells were either left unstimulated (A) or stimulated during the final 6 h of culture with 100 ng/ml PMA plus 1 µg/ml ionomycin (B).

 
Similar results were obtained when FasL promoter activity was assessed in cells stimulated with PMA plus ionomycin (Fig. 4GoB). The finding that this stimulation did not increase the activity of the FasL promoter above the level observed in unstimulated cells (compare Fig. 4Go, B and A) strongly suggests that PKC{theta} and Cn are the predominant, if not exclusive, targets for the action of PMA and ionomycin, respectively, on FasL. It is noteworthy that the combination of constitutively active PKC{theta} and Cn was by far more effective than PMA plus ionomycin stimulation in stimulating the activity of the FasL promoter (Fig. 4Go, A vs B). This may reflect the possibility that the less selective stimulation by PMA and ionomycin induces signals that negatively regulate FasL expression, e.g., the down-regulation and degradation of PKC by PMA or the induction of negative regulatory effectors such as Ca2+/calmodulin-dependent protein kinase II (35) by ionomycin. This explanation is consistent with our finding that constitutively active PKC{theta} is much more potent than PMA in inducing an NF-{kappa}B reporter activity in T cells (N. Coudronniere and A. Altman, manuscript in preparation).

Rottlerin inhibits AICD

FasL expression leads to apoptosis in cells that also express Fas, a process termed AICD (2, 3, 4, 5, 6). Therefore, to determine whether PKC{theta} plays a physiologically relevant role in regulating AICD, we investigated whether the inhibition of cellular PKC{theta} activity by rottlerin can reduce AICD. First, we determined that rottlerin abolishes FasL expression induced by an anti-CD3 mAb or PMA plus ionomycin in Jurkat CE cells (Fig. 5GoA). As a control, the level of actin mRNA was not affected by this pretreatment, suggesting that rottlerin specifically inhibits FasL expression and, moreover, that this inhibition is not due to drug toxicity.



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  		FIGURE 5. Rottlerin blocks AICD. A, A total of 10 x 106 Jurkat-CE cells were preincubated for 20 min with 30 µM rottlerin and stimulated for 4 h with an anti-CD3 Ab (OKT3; 1 µg/ml) or PMA (100 ng/ml) plus ionomycin (1 µg/ml). The cells were harvested, RNA was extracted, and FasL (top) or actin (bottom) mRNA expression was analyzed by RT-PCR. B, A total of 1 x 106 Jurkat-CE cells were preincubated for 20 min with 30 µM rottlerin or 0.5 µM Gö6976 and stimulated for 6 h with an anti-CD3 Ab (OKT3; 1 µg/ml) or PMA (100 ng/ml) plus ionomycin (1 µg/ml). The cells were harvested, and the percentage of apoptotic cells was determined. C, A total of 1 x 105 A1.1 cells were preincubated for 20 min with different rottlerin concentrations (or in its absence) and then stimulated for 6 h with an anti-mouse CD3 mAb (2C11; 1 µg/ml). The cell were harvested and analyzed for surviving cells using the propidium iodide staining protocol (28 ). Results represent the mean ± SEM of one representative experiment performed in triplicate.

 
When Jurkat-CE T cells were treated with an anti-CD3 or PMA plus ionomycin for 6 h, ~15% of the cells underwent apoptosis compared with only 3% spontaneous apoptosis in unstimulated and vehicle-treated cells, or 6–7% apoptosis in unstimulated cells pretreated with rottlerin or Gö6976 (Fig. 5GoB). Rottlerin inhibited AICD by ~50% in both anti-CD3- or PMA plus ionomycin-stimulated cells. In contrast, Gö6976 was completely ineffective in protecting the T cells from AICD. Under the same conditions, rottlerin was ineffective in protecting cells from apoptosis induced by an anti-Fas mAb, indicating that it mediates AICD protection by inhibiting a step preceding the binding of FasL to its receptor, Fas, and supporting the notion that it inhibits FasL expression. Furthermore, the failure to inhibit anti-Fas-induced apoptosis indicates, again, that rottlerin is not toxic to the cells in these 6 h cultures. However, due to the toxicity of rottlerin for Jurkat T cells under prolonged culture conditions (~20 h), we could not evaluate its effects on AICD in longer-term cultures where AICD reached a level of ~50%.

Because of the relatively low level of AICD in this system, we decided to use the mouse T cell hybridoma A1.1. These cells were characterized in AICD studies, and undergo extensive apoptosis when stimulated with an anti-CD3 Ab for a relatively short time period (2). Pretreatment with rottlerin concentrations of up to 30 µM reduced cell survival (i.e., increased cell death) by only ~10%, i.e., from a basal level of ~90% to ~80% (Fig. 5GoC). When stimulated for 6 h with an anti-CD3 Ab, the majority of the cells (~65%) underwent apoptosis. However, pretreatment with rottlerin protected the cells from AICD in a dose-dependent manner, with 50% protection achieved by 2–3 µM rottlerin. Complete protection was observed when the cells were pretreated with 10–30 µM rottlerin (Fig. 5GoC).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we demonstrate that PKC{theta} and Cn are important components of the TCR-induced pathway that leads to FasL expression. Previous studies demonstrated that the combination of phorbol ester and Ca2+ ionophore can replace and mimic the physiological FasL-inducing signal provided by TCR ligation (14, 15) and induce AICD (16). Furthermore, these and other studies have linked Cn, and its principal cellular target, NF-AT, to this pathway, demonstrating that Cn represents the target for the Ca2+ signal (9, 10, 28). However, the identity of the target for the other inducing signal provided by phorbol ester has remained elusive. Because phorbol esters mediate their pleiotropic cellular effects predominantly via the activation of conventional (cPKC: {alpha}, ß, and {gamma}) or novel (nPKC: {delta}, {epsilon}, {eta}, {theta}, and µ) PKC enzymes, we sought to determine whether a particular phorbol ester-responsive PKC isoform is selectively involved in the positive regulation of FasL expression. For this analysis, we chose four T cell-expressed isoforms that represent the three PKC subfamilies, i.e., PKC{alpha} (cPKC), PKC{theta} and PKC{epsilon} (nPKC), and PKC{zeta}, a member of the atypical PKC subfamily (aPKC).

Our findings demonstrate that PKC{theta} activation preferentially plays an important, albeit perhaps not exclusive, role in up-regulation of FasL expression following TCR ligation. This conclusion is based on the following results. First, constitutively active PKC{theta} caused a marked activation of the FasL promoter, whereas other PKC isoforms were much less effective or ineffective (Fig. 1Go). Second, PKC{theta} activity is necessary for FasL expression because transient overexpression of wild-type PKC{theta} in resting cells failed to induce the FasL promoter (Fig. 1GoA). Third, dominant-negative PKC{theta} effectively inhibited FasL promoter activation induced by PMA plus ionomycin treatment (Fig. 2GoC) or TCR/CD3 ligation (data not shown). Finally, rottlerin, a compound that inhibits the activity of PKC{theta} (as well as PKC{delta}), but not Ca2+-dependent PKCs such as PKC{alpha} (Ref. 33 ; Fig. 2GoA), blocked PKC{theta}- or PMA plus ionomycin-induced FasL promoter activation (Figs. 2GoC and 5A), and protected two types of T cells from AICD (Fig. 5Go, B and C). The absence of detectable PKC{delta} in Jurkat T cells (Fig. 2GoB) strongly suggests that PKC{theta} is the predominant target for this inhibitory effect of rottlerin.

Although these results demonstrate that PKC{theta} is a critical rate-limiting factor in the induction of FasL expression, they do not rule out the possibility that other T cell-expressed PKC isoforms also play a role in FasL expression, either as components of a PKC{theta}-mediated pathway or as parts of PKC{theta}-independent contributory pathways. This may account for the weak activation of the FasL promoter by PKC{alpha} either alone (Fig. 1GoA) or in combination with Cn (Fig. 3GoA) and for the partial inhibition of PKC{theta}-A/E-induced FasL promoter activity by Gö6976 in PMA plus ionomycin-stimulated cells (Fig. 2GoC, right). In this regard, PKC{alpha} was recently found to up-regulate the expression of PKC{delta} (36), an isoform that is closely related to PKC{theta} (21). This raises the possibility of a cross-talk between PKC{theta} and PKC{alpha}.

The second novel finding emerging from the current study is that PKC{theta} synergizes with Cn to activate the FasL promoter. This result, as well as the selective role of PKC{theta} in FasL induction, are in general agreement with recent findings by Villunger et al. (32). The synergy between PKC{theta} and Cn is consistent with the findings that, first, phorbol ester and Ca2+ ionophore also synergize to induce FasL expression (14, 15) and, second, cyclosporin A or FK506 (3, 28, 37) and rottlerin (Fig. 2GoB), which selectively interfere with the activity of Cn or PKC{theta}, respectively, can each block TCR- or PMA plus ionomycin-induced FasL expression in different T cell lines. The findings that inhibition of either Cn or PKC{theta} is sufficient to inhibit FasL expression suggests that the combination of these two signaling elements is required for optimal induction.

The concentration of rottlerin required for half-maximal protection from AICD (2–3 µM; Fig. 5GoC) was ~100-fold lower than that which caused a 50% inhibition of the in vitro PKC{theta} kinase activity (~300 µM; Fig. 2GoA). One possible explanation is that rottlerin is taken up by the cells in a highly efficient manner and, thus, accumulates intracellularly to a higher concentration than in the culture medium. A similar situation has been observed with another PKC inhibitor, bisindolylmaleimide, in an analysis of apoptosis in cerebellar granule cells (38).

Several recent studies demonstrated a unique role for PKC{theta} in intracellular signaling pathways induced by TCR engagement. For example, PKC{theta} specifically translocates to the contact site between T cells and APCs during Ag-specific interactions (22, 23) and, together with the TCR complex, forms the central core of the newly described supramolecular activation cluster (23). Second, PKC{theta} specifically activates the JNK/AP-1 pathway (24, 25, 30) and, furthermore, similar to our finding herein (Fig. 3Go), it cooperates with Cn to activate JNK and the IL-2 promoter in T cells (25). Finally, it was very recently suggested that PKC{theta} may be involved in the negative selection of thymocytes (39). The current study demonstrates that FasL and the physiological process of AICD represent novel targets of activated PKC{theta} in T cells.

A dual role for PKC{theta} in signaling pathways that lead to either T cell proliferation or apoptosis is not surprising. Proliferative and apoptotic signals can coexist and mediate crosstalk in the same pathway (40), with the final result depending on how and when these signals are integrated. For example, several studies demonstrated that induction of apoptosis by the JNK pathway is enabled or enhanced when the activation of the anti-apoptotic MEK/ERK pathway is blocked (41, 42). Similarly, c-Myc (43, 44) or Ras (40, 45, 46, 47) are known to generate signals that can lead to either proliferation or apoptosis, depending on the input of additional signals provided in stimulus- and cell type-specific manners.

The upstream and downstream signaling elements that are linked to PKC{theta} in the TCR-initiated signaling pathway leading to FasL expression and AICD are not well understood, but several relevant clues emerge from recent studies. Thus, it has recently been shown that the recruitment of the Zap-70 tyrosine kinase to the TCR-associated phospho-{zeta} chain following TCR engagement (48) is necessary for FasL expression, but not for IL-2 production in apoptosis-defective cells (49, 50), suggesting a bifurcation of TCR signals at this level. The related kinase, Syk, can induce JNK activation (51), suggesting that the effects of Zap-70 and/or Syk on FasL expression and T cell apoptosis may be mediated by JNK. In contrast, PKC{theta} functions as a selective JNK activator in T cells (24, 25, 52). Therefore, Zap-70/Syk and PKC{theta} could be linked in a physiological pathway leading to FasL expression. One possibility is that following their activation, these tyrosine kinases phosphorylate and activate PLC{gamma}1, which leads to diacylglycerol production and subsequent activation of PKC enzymes, including PKC{theta}.

PKC{theta} and PKC{delta} were found to be involved in the induction of apoptosis in different cell types (38, 53, 54, 55, 56, 57), but the molecular basis for their action is poorly understood. These data do not necessarily mean that PKC{theta} and {delta} are proapoptotic but, rather, may reflect the possibility that these enzymes are involved in a signal transduction pathway, such as the induction of FasL expression demonstrated here, that can lead to apoptosis under certain circumstances.

In summary, we have shown that PKC{theta} activation is necessary for FasL expression following TCR engagement or PMA plus ionomycin treatment. PKC{theta} cooperates with Cn to induce FasL expression, and this cooperation appears to depend on the integrity of the different transcription factor-binding sites in the FasL promoter. Nonetheless, the presence of functional PKC{theta} is necessary for AICD.


   Acknowledgments
 
We thank Drs. Gottfried Baier and Michael Karin for generous gifts of plasmids.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant CA35299. This is publication number 285 from the La Jolla Institute for Allergy and Immunology. Back

2 Address correspondence and reprint requests to Drs. Amnon Altman or Martin Villalba, Division of Cell Biology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: Back

3 Current address: Cytovia, San Diego, CA 92121. Back

4 Current address: Institut National de la Santé et de la Recherche Médicale Unité 80, Hopital E. Herriot, 69437 Lyon Cedex 03, France. Back

5 Abbreviations used in this paper: AICD, activation-induced cell death; FasL, Fas ligand; RE, response element; PKC, protein kinase C; Cn, calcineurin; JNK, c-Jun N-terminal kinase; ß-gal, ß-galactosidase; ERK, extracellular signal-related kinase; MEKK, mitogen-activated protein kinase/ERK kinase; TAg, SV40 large TAg. Back

Received for publication July 13, 1999. Accepted for publication September 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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