1 Institute of Pharmacology, Medical School Hannover, 30623 Hannover, Germany
The first two authors contributed equally to this work
Correspondence to: M. Szamel; E-mail: Szamel.Marta{at}MH-Hannover.de
Transmitting editor: T. Hünig
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Abstract |
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Keywords: cytokine, protein kinase/phosphatase, signal transduction, T lymphocyte, transcription factor
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Introduction |
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Introducing neutralizing antibodies by electroporation, we have previously demonstrated that different PKC isoenzymes regulated either IL-2 synthesis or IL-2 receptor expression in human peripheral blood lymphocytes. These results suggested PKCß to be the major regulator of IL-2 synthesis (10,11). However, the molecular mechanisms whereby PKCß affected IL-2 gene expression could not be investigated in detail by this approach.
The IL-2 gene is activated by the concerted action of several transcription factors (12,13). Generation, activation and nuclear translocation of these transcription factors is the ultimate function of extracellular-regulated kinase (ERK), JNK and p38 mitogen-activated proteinkinase (MAPK) signal transduction cascades activated via TCR and CD28 co-receptor (14). As a consequence of TCR-induced elevation of intracellular calcium, the calciumcalmodulin-dependent phosphatase calcineurin additionally activates NF-AT (12,13). Furthermore, stimulation via TCRCD28 activates specific protein kinase cascade(s) leading to activation and nuclear translocation of NF-B (15).
The aim of this study was to investigate the influence of PKC and ß on the protein kinase cascades participating in regulation of the IL-2 gene in Jurkat T cells. PKC isoenzyme-specific phosphorothioate antisense oligonucleotides (ODN) were introduced into these cells by electroporation. Antisense technology provides a highly specific approach for functional studies of individual protein isoforms in signal transduction processes (16,17). To do so, the antisense oligonucleotides should be designed to hybridize with high affinity to mRNA sequences unique for a given protein, thereby inhibiting RNA translation and expression of this protein. Due to the long half-lives of some proteins requiring several days to significantly lower protein levels, it is advantageous to decrease sensitivity of antisense ODN to endogenous nucleases by chemical modifications, e.g. use of phosphorothioate analogs (16,17).
The results show that phosphorothioate-modified antisense ODN effectively inhibited new synthesis of PKC and ß protein. TCRCD28-induced JNK activation was specifically impaired in PKCß-depleted cells, whereas other signaling cascades, like Raf/mitogen-activated ERK kinase (MEK)/ERK or I
B kinase (IKK) catalyzed I
B phosphorylation were dependent on both PKC
and ß. A significant decrease in IL-2 gene transcription was observed and IL-2 secretion was completely abolished in PKCß-specific antisense ODN-treated cells. The results substantiate the decisive role of PKCß in regulation of IL-2 synthesis and secretion in the course of T cell activation.
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Methods |
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Human peripheral blood lymphocytes were separated by Ficoll gradient centrifugation from heparinized blood of healthy volunteers. Cells were washed with RPMI and stimulated for 15 min with 2.5 µg/ml OKT3 antibody and 1.25 µg/ml mouse anti-human CD28 antibody in serum-free RPMI medium for activation of the JNK protein kinase cascade or in RPMI supplemented with 5% FCS for 16 h for determination of IL-2 synthesis.
Introduction of PKC-specific antisense ODN by electroporation
Jurkat T cells were adjusted to a cell density of 2 x 107/ml in RPMI 1640 medium with 1% FCS (heat-inactivated at 65°C for 30 min). An aliquot of 8 x 106 cells was electroporated for 5 ms at 400 V (rectangle impulse) in the presence and absence of 10 nmol of a 20mer PKC (5'-GTTCTCGCTGGTGAGTTTCA-3')- and 13 nmol of a 15mer PKCß (5'-CGCAGCCGGGTC AGC-3')-specific phosphorothioate-modified antisense ODN (MWG Biotech, Ebersberg, Germany). After electroporation, cells were transferred into RPMI 1640 medium with 10% FCS (65°C heat inactivated) and adjusted to 2.5 x 105 cells/ml. After culturing for 48 h, cells were taken for stimulation in serum-free medium.
With minor modifications, Jurkat cells stably transfected with a plasmid containing the luciferase reporter gene together with either a single copy of the minimal IL-2 promoter (3) or four copies of the distal NF-AT-binding domain of the IL-2 promoter were also used for electroporation. Thereby, 8 x 106 cells at a cell density of 1 x 107/ml were electroporated with specific PKC (10 nmol) and PKCß (13 nmol) antisense ODN for 5 ms at 350 V, and cultured for 34 (IL-2 promoter activity) or 42 h (NF-AT-binding domain activity).
Flow cytometry
Jurkat cells were electroporated as described in the previous section with or without 10 nmol PKC or 13 nmol PKCß FITC-labeled antisense ODN or 50 µg/ml FITCdextran (Sigma, Deisenhofen, Germany). Immediately after electroporation, and 24 and 48 h later an aliquot of cells was washed twice in PBS, pH 7.4 with 0.1% BSA. From these samples, 105 events were acquired using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and analyzed with the Lysis II software.
Preparation of subcellular fractions
For preparation of subcellular fractions, cells were resuspended in a buffer consisting of 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 2 mM MgCl2, 1 mM DTT, 50 µg/ml leupeptin, 400 nM ocadaic acid and 1 mM sodium vanadate, pH 7.4. Cells were sonicated (50 W, 5 impulses) and centrifuged (10 min at 2500 g). For preparation of cytoplasmic fractions, the 2500 g supernatant was further centrifuged at 100,000 g for 30 min. The pellet (crude membrane fraction) was washed with the buffer described above.
Alternatively, nuclear-free lysates were obtained by incubating cells for 30 min on ice in a buffer containing 10 mM Tris, 30 mM sodium pyrophosphate, 50 mM NaCl, 5 mM NaF, 20 mM ß-glycerophosphate, 1 mM Na3VO4, 400 nM okadaic acid, 0.5 mM PMSF, 10 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 mM p-nitrophenylphosphate, 1 mM DTT and 1% Triton X-100, pH 7.4. Nuclei were removed by centrifugation for 15 min at 6000 g and 4°C.
Protein concentration of the subcellular fractions was determined using the bicincholinic acid assay and BSA as a standard.
Detection of phosphorylated protein kinases by immunoblot analysis
Cellular proteins were separated by SDSPAGE. Proteins were transferred onto nitrocellulose. For the detection of protein kinases, membranes were blocked in TBST (20 mM Tris, pH 7.6, 137 mM NaCl and 0.1% Tween 20) supplemented with 5% non-fat milk for 1 h at 20°C, thoroughly rinsed in TBST followed by incubation with the primary antibodies raised against PKC and ß, Raf-1, MEK-1 (Transduction Laboratories, Lexington, UK), phospho-Raf-1, phospho-MEK-1/2, pan-ERK, phospho-ERK-1/2, phospho-MAPK kinase (MKK)-4, phospho-JNK, p38 MAPK, phospho-p38 MAPK and phospho-I
B (NEB, Beverly, MA) in TBST over night at 4°C. The membranes were washed again and incubated for 1 h at room temperature with appropriate secondary antibodies conjugated to horseradish peroxidase. Finally, membranes were rinsed and developed using the enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL). In some cases blots were stripped and reprobed.
Determination of protein kinase activities
For in vitro determination of JNK activity, 50 µg of cytosolic protein in 2 x kinase buffer (100 mM Tris and 20 mM MgCl2, pH 7.6) was incubated with 1.5 µg human recombinant GST-c-jun (NEB, Beverly, MA) and 5 µM ATP as substrates for 20 min at 37°C. Phosphorylated GST-c-jun was obtained by GSH beads and separated by SDSPAGE. Phosphorylated c-jun was detected in immunoblots by specific antibodies (NEB).
Determination of luciferase activity
NF-AT-binding domain activity and IL-2 reporter gene activity were detected using 1 x 106 cells/ml of stably transfected Jurkat cells seeded in serum-free RPMI 1640 medium and stimulated for 6 (NF-AT-binding domain) and 14 h (IL-2 promoter activity) with or without OKT3 and anti-CD28 antibody in PKC and ß ODN-treated cells. Incubation was stopped by washing three times with ice-cold PBS, pH 7.4. Lysis of cells resulted from incubation in a buffer containing 91.5 mM K2HPO4, 8.5 mM KH2PO4, 0.5 mM PMSF, 2.5 µg/ml leupeptin, 0.7 µg/ml pepstatin and 0.2% Triton X-100 for 15 min at 4°C. Luciferase activity was determined in the supernatants gained after centrifugation for 15 min at 13,000 r.p.m. and 4°C. Finally, the resulting relative light units were normalized for protein concentration.
RNA isolation and RT-PCR
Jurkat T cells (5 x 106) were stimulated with OKT3 and anti-CD28 antibody for the times indicated in the experiments. Cells were then harvested and RNA isolated with a Qiagen RNA isolation kit according to the manufacturers instructions (Qiagen, Hilden, Germany).
For RT-PCR, 3 µg RNA was incubated with 500 ng poly(dT) (Amersham Pharmacia Biotech, Freiburg, Germany) as a primer, and dATP, dCTP, dGTP and dTTP, 10 nmol of each (Q-Biogene, Heidelberg, Germany) at 65°C for 5 min. Samples were chilled on ice and incubated for 2 min in the presence of 40 U RNase inhibitor (RNaseOut; Gibco, Karlsruhe, Germany) for 2 min at 37°C. Synthesis of cDNA was started by addition of 200 U DNA polymerase (M-MLV-RT; Gibco) in a buffer containing 50 mM Tris, 75 mM KCl, 3 mM MgCl2 and 10 mM DTT. Following incubation at 37°C for 50 min, the reaction was terminated at 95°C for 10 min.
The primers used were from MWG Biotech and from Stratagene (Amsterdam, Netherlands). IL-2 sense: 5'-ATG TAC AGG ATG CAA CTC CTG TCT T-3'; antisense: 5'-GTC AGT GTT GAG ATG ATG CTT TGA C-3' (457 kb PCR product). GAPDH sense: 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; antisense: 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (598 kb PCR product).
PCR was carried out in a buffer containing 10 mM TrisHCl, 50 mM KCl, 2 mM MgCl2 and 0.8% Nonidet P-40 with 2 µl cDNA in the presence of the specific primers (10 pmol of each), dATP, dCTP, dGTP and dTTP (10 nmol of each), and 1 U Taq polymerase (Q-Biogene) with the further specifications: denaturation at 94°C for 5 min, annealing at 60°C for 1 min, elongation at 72°C for 1 min and 30 cycles (within the linear range).
Agarose gel electrophoresis
PCR products were separated in a 1.7% agarose gel in 40 mM TrisCH3COOH, pH 8.0, 1 mM EDTA and 0.5 µg/ml ethidium bromide, and documented with the geldocumentation system from Bio-Rad (Hercules, CA).
Determination of IL-2 synthesis
IL-2-specific ELISA was from R & D Systems (Minneapolis, MN). Jurkat T cells (1 x 106/ml) were incubated in RPMI with 2% FCS, and stimulated with or without OKT3 and anti-CD28 antibody for 16 h. IL-2 concentration of the supernatants was determined by a specific ELISA according to the manufacturers instructions.
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Results |
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Phosphorylation and activation of protein kinases of the TCR-stimulated Raf/MEK/ERK cascade is effectively inhibited in PKC- and ß-specific antisense ODN-treated cells
Stimulation of cells with a combination of anti-CD3/CD28 antibodies resulted in enhanced phosphorylation and thus activation of Raf-1, MEK-1/2 as well as ERK-1/2. As shown in Fig. 2(A), electroporation itself had no influence on the amount of phosphorylated Raf-1; neither was hyperphosphorylation and subsequent activation of this protein kinase influenced. In sharp contrast, the amount of phosphorylated Raf-1 was reduced by >50% in PKC- and to 80% in PKCß-specific antisense ODN-treated cells compared to control, as determined by densitometric analysis.
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Both PKC and ß regulate IKK-catalyzed I
B phosphorylation
NF-B is one of the most important transcription factors regulating the IL-2 gene (8,9,12,15). In resting cells I
B is associated with and retards NF-
B in the cytoplasm in its inactive form. Upon activation via TCRCD28, I
B is phosphorylated by IKK, dissociates and NF-
B translocates to the nucleus (15,19). Figure 3 shows that stimulation of cells with anti-CD3/CD28 resulted in an enhanced phosphorylation of I
B
. Introduction of both PKC
- and ß-specific antisense ODN resulted in a marked reduction of IKK-catalyzed I
B
phosphorylation. In cells pretreated with the proteosome inhibitor Z-Leu-Leu-Phe-CHO, a more pronounced phosphorylation of I
B
was observed that was significantly inhibited in PKC
and ß ODN-treated cells.
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Strikingly similar effects of the PKC isotype-specific inhibitors were observed on peripheral blood lymphocytes. Gö 6976 exerted a marked inhibitory effect on activation of the JNK protein kinase cascade (Fig. 5C). TCR-induced enhanced phosphorylation of MKK-4 and p46 JNK was inhibited in a concentration-dependent manner; 100 nM of the inhibitor has been effective in significant inhibition of phosphorylation and activation of both enzymes. Ro 32-0432 and Gö 6850 exerted only marginal effects on activation of JNK (data not shown).
Activation of NF-AT and IL-2 gene expression requires PKCß in stimulated Jurkat T cells
As depicted in Table 1, different PKC-specific inhibitors exerted differential effects on activation of the IL-2 reporter gene expression. Ro 32-0432 exerted no significant effect on IL-2 gene expression. Gö 6976 exerted a marked inhibitory effect on IL-2 reporter gene activation. At the concentration used, Gö 6976 proved to be as effective as cyclosporin A in inhibiting IL-2 gene expression, suggesting that proper functioning of PKCß was necessary for onset of IL-2 gene expression (Table 1).
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Discussion |
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In several in vitro model systems, including transfected Jurkat T cells, PKC was considered to be the prototype for classical PKC and its influence on TCRCD28-induced signaling was investigated (3,5,6). Our data confirm the recent observations showing that regulation of the Raf/MEK/ERK protein kinase cascade, as well as activation of the transcription factor NF-
B, is a function of classical PKC (Figs 2 and 3), illustrating overlapping biological activities of PKC
and ß for these protein kinase cascades (25,26). Furthermore, both PKC did not influence activation of p38 MAPK, additionally proving the specificity of PKC antisense ODN used in these experiments (Fig. 4).
Regulation by PKC isoenzymes of TCR-induced NF-B signaling is well documented (2729). Our data indicate that besides PKC
, PKC
and ß also participate in activation of IKK-induced I
B phosphorylation. The decisive function of PKCß controlling I
B kinase activation has been shown very recently in response to BCR signaling (30).
The molecular mechanisms involved in the activation of the Raf/MEK/ERK protein kinase cascade are well established (31,32). Phosphorylation and thus activation of the Raf/MEK/ERK cascade was impaired in TCRCD28-stimulated Jurkat T cells depleted of PKC or ß. Both PKC
and ß have been suggested to regulate the Raf/MEK/ERK cascade in epidermal growth factor-stimulated Cos cells, although in a different manner (33). To our knowledge, there are no published data on the participation of PKCß in regulation of TCR-induced Raf-1 activation.
Several studies have provided evidence that the Raf/MEK/ERK protein kinase cascade is a necessary (but not sufficient) effector pathway for NF-AT activation. These conclusions, which mainly result from expression of dominant-negative or constitutive active p21Ras, Raf-1 or MEK-1 mutants, have created a paradigm that the Raf-1/MEK-1/ERK-2 pathway was one of the major routes for activation of IL-2 gene transcription in TCRCD3-stimulated cells (3436). Our results show that both PKC and ß interfered with this protein kinase cascade in a similar way. However, only the PKCß isoform seems to be specifically involved in activation of NF-AT binding, IL-2 gene expression and IL-2 secretion. This implies the contribution of other TCRCD28-regulated signal transduction pathways for regulation of IL-2 synthesis and secretion.
Regulation of TCRCD28 induced activation of the JNK cascade seemed to be a specific function of PKCß in our experimental system. Phosphorylation of MKK-4 and subsequent activation of JNK was significantly reduced in PKCß-depleted cells (Fig. 5A).
The function of JNK and regulation of their activity by PKC isoenzymes is a matter of discussion. While enhanced activity of JNK was an absolute necessity for regulation of IL-2 gene expression in T cell lines (5,6,21), a defect in JNK signaling was claimed to be involved in T cell differentiation, but not in T cell activation in vivo, i.e. in JNK-deficient animals (37,38). It has been demonstrated that activation of NFAT and subsequent regulation of cytokine gene expression was impaired in JNK-deficient mice (39). The importance of JNK in IL-2 gene expression has been further emphasized by showing that calcineurin and JNK synergized in induction of IL-2 synthesis (5,6,20 21). Furthermore, JNK-catalyzed phosphorylation of c-jun, as well as JNK-regulated elevation of c-jun synthesis, was a prerequisite of high-affinity NF-AT binding and subsequent cytokine synthesis in T lymphocytes (20,21,38,39). It is well documented that JNK participating in the re-phosphorylation of NF-AT in the nucleus regulates the duration (length) of NF-ATDNA interactions involved in cytokine gene expression (39). Furthermore, it was suggested that JNK might be responsible for stabilization of IL-2-specific mRNA in T cells (40). Since activation and functioning of JNK is critically involved in regulation of the NF-AT-binding site of the IL-2 promoter, our data indicate that PKCß-regulated JNK activation is required for efficient, high-affinity NF-AT binding and IL-2 gene expression in Jurkat T cells. Our data imply that PKCß operates upstream of MKK-4, leading to enhanced phosphorylation, and thus activation of MKK-4-catalyzed JNK and subsequent phosphorylation of c-jun, as well as high-affinity NF-AT binding (Figs 5 and 6).
Numerous reports emphasized the importance of the calcium-independent PKC isoform, PKC, in the regulation of JNK activation as well as in the generation of transcription factors participating in enhancing the activation of the IL-2 promoter in vivo and in vitro (68,41). The hypothesis that PKC
played a significant role in T lymphocyte activation, as indicated by numerous studies in cell lines, was recently confirmed in mice deficient in the expression of this enzyme (8). Peripheral T cells lacking PKC
failed to activate NF-
B and AP-1, and failed to express IL-2, in response to TCR stimulation. This revealed a critical function for this PKC family member in linking membrane-proximal activation cascades to transcriptional responses governing T cell activation (8).
The molecular interactions involved in the engagement of PKC have not been fully delineated. In T cell lines, PKC
-regulated activation of JNK was claimed to participate in IL-2 gene expression (57). In contrast, no change in JNK activation has been observed in T cells of PKC
-deficient animals, although the amount of IL-2 was significantly decreased (8). The results indicated that PKC
was an essential component activating the NF-
B signaling cascade and thus leading to expression of the IL-2 gene during T cell activation. However, stimulation with phorbol ester plus ionomycin resulted in restoration of IL-2 gene synthesis and cellular proliferation. These findings suggest the involvement of a conventional PKC which might compensate for the absence of PKC
.
If PKCß can fit into this function, one would expect enhanced amounts (activity) of PKCß in T cells of PKC-deficient animals. In fact, elevated expression of cPKC has been observed in those cells (42).
Participation of cPKC, after all that of PKCß, in regulation of T cell activation was also controversially discussed, depending on the experimental system used. On the one hand, TCR-induced cellular proliferation was not influenced in PKCß-deficient mice (43); on the other hand, several reports dealt with regulation by cPKC, including PKCß of signal transduction and cytokine gene expression in T cells and T cell lines (3,6,11,44).
At first glance our results contradict those of Long et al. (44) showing that PKCß was specifically implicated in secretion (but not transcription) of IL-2 in HUT 78 cells. IL-2 gene expression was inhibited by 50% in Jurkat cells; however, IL-2 secretion was completely abolished upon PKCß depletion, implying that PKCß must be specifically involved in both processes. Since T cells and T cell lines showed marked functional differences, it is not surprising that this heterogeneity was also reflected in differential expression of PKC genes and protein. Gene expression and function of PKC was claimed to be strictly cell-type specific (42,45,46).
While Jurkat T cells express high amounts of both PKC and ß, PKC
is the dominant PKC isoform in the HUT 78 cell line. It is obvious that IL-2 synthesis is differentially regulated in Jurkat and HUT 78 cells. Resembling TCRCD3 signaling, the phenotypically non-activated Jurkat T cell line requires phorbol myristate acetate plus ionomycin for IL-2 synthesis. In contrast, HUT 78, a phenotypically activated T cell line, secreted IL-2 in response to phorbol myristate acetate alone, suggesting that a calcium-independent PKC isoform might be involved in regulation of IL-2 synthesis (47).
Both depletion of Jurkat cells for PKCß and repression of its activity by an isotype-specific inhibitor resulted in a significant reduction of IL-2 gene expression in our experimental system. In accordance with our recent data and those of other groups, the PKCß-specific inhibitor Gö 6976 was effective in suppression of JNK activation and IL-2 synthesis in human peripheral blood lymphocytes (11,47,48). These results underline the importance of PKCß in regulation of the IL-2 gene (Figs 5 and 6, and Tables 1 and 2) (23). Our more recent results showing that overexpression of PKCß or its catalytic subunit leads to a remarkable elevation of IL-2 synthesis support the assumption of the involvement of PKCß in regulation of IL-2 gene expression (manuscript in preparation).
In summary, data presented in this paper confirm and extend our hypothesis on the decisive role of PKCß in the regulation of IL-2 synthesis in T lymphocytes. The results suggest that interference of PKCß with activation of the JNK cascade might be one of the important mechanisms in the regulation of high-affinity NF-AT binding, IL-2 synthesis and secretion. The fact that a complete inhibition of IL-2 synthesis and secretion was achieved in PKCß-specific antisense ODN-treated cells indicates a highly specific (and most important) function of the PKCß isoform in the gene expression and processing of this cytokine.
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Acknowledgements |
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Abbreviations |
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ERK-1p44 MAPK
ERK-2p42 MAPK
IKKIB kinase
JNKJun-N-terminal kinase
MAPKmitogen-activated protein kinase
MEKmitogen-activated ERK kinase
MKKMAPK kinase
ODNoligodesoxynucleotide
PKCprotein kinase C
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References |
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