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*
Institute of Molecular Pharmacology and
Transplantation Laboratory, Medical School, Hannover, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PKC-) and PKC-
, peaking at 10 min, whereas
PKC-ß, -
, and -
were translocated with a delay of >90 min and
remained activated for up to 2 h. To characterize specific
functions of distinct PKC isoenzymes, Abs against different PKC
isoenzymes were introduced by means of electropermeabilization.
Neutralization of PKC- and - resulted in the complete inhibition
of IL-2R expression, whereas anti-PKC-ß, -, and - Abs
inhibited IL-2 synthesis. Extensive control experiments have shown that
neither electropermeabilization nor control Ig influenced PKC activity
and cellular functions. Our data thus clearly show that specific PKC
isoenzymes regulate different cellular functions in stimulated human
lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, -ß1, -ß2, and -
are dependent on calcium for
activation, whereas PKC-, -, -
, and - are not. A third
group of PKC isoenzymes (PKC-
, -
, and -
) structurally belongs
to the PKC family, but, atypically, is not activated by phorbol esters
or DAGs (3, 4). A fourth group of enzymes has been described recently
that binds DAG/phorbol esters and has structural homologies with the
PKC family, but has unusual catalytic domains (5). Particular PKC
isoenzymes differ in their substrate specificity in vitro, suggesting
that different PKC isoforms may have distinct cellular functions,
reflecting their substrate preferences in vivo. Since the late seventies, the involvement PKCs in the course of T cell activation has been well established (6, 7). T lymphocyte activation represents a highly pleiotropic set of cellular responses that includes cell cycle entry and release of cytokines. Stimulation of T cells results in the transcription of several genes and expression of a variety of molecules, including cytokines and their specific receptors, such as IL-2 and its receptor.
PKCs regulate T cell activation genes via control of several
transcription factors (1, 2, 8). T cells express multiple isotypes of
PKC, including PKC-, -ß1, -, -, -, -, and -;
however, the role of different PKC isotypes in the regulation of
cellular signaling and gene transcription is not definitely
established. Several conclusions regarding the role of PKC in T cell
activation were based on experiments that examined the effects of
phorbol esters or synthetic DAGs, which simultaneously activated
multiple isotypes of PKC (8, 9, 10).
A significant approach to define the specific functions of PKC isoenzymes is to investigate functional changes in cells transfected with mutated constitutively active or inactive PKCs, respectively (11, 12, 13, 14, 15, 16, 17, 18). Such PKC mutants have been used to examine directly the functional effects of individual PKC isotypes in various cell lines (19, 20, 21, 22). However, recent data have shown significant differences in molecular mechanisms of signal transduction between T cell lines and normal resting T lymphocytes (23, 24).
Accordingly, to examine the role of PKC in T cell activation we have
analyzed the consequences of neutralization of PKC-, -ß, -,
-, -, and -, as representatives for the three major
subdivisions of the PKC family, by introduction of specific Abs into
PBL. Of the many diverse PKC responses that could be explored,
regulation of PKC-responsive IL-2 synthesis and surface expression of
IL-2R were investigated. The results show that stimulation of T
lymphocytes via the TCR/CD3 complex leads to activation and
translocation of PKC isoenzymes with differential kinetics. The data
show that PKC- and - are the main regulators of IL-2R expression,
while PKC-ß, PKC-, and, to a minor extent, PKC- participate in
the regulation of IL-2 synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemicals if not otherwise indicated were obtained from Sigma Chemical Co. (St. Louis, MO).
Antibodies
Mouse mAb, BMA 031 (IgG2b), raised against monomorphic
determinants of the TCR was a gift from Dr. R. Kurrle (Behringwerke,
Germany). OKT3 raised against the -chain of the TCR/CD3 complex was
purified from CD3 hybridoma (American Type Culture Collection,
Rockville, MD) supernatants by means of affinity chromatography on
protein A-agarose. The Abs against protein kinases C were from
different sources: anti-PKC-, raised against the epitope
corresponding to amino acids 651 to 672 mapping at the carboxyl
terminus of cPKC-, anti-PKC-ß raised against an epitope
corresponding to amino acids 656 to 671 mapping at the carboxyl
terminus of cPKC-ß, anti-PKC- against an epitope corresponding
to amino acids 657 to 673 at the carboxyl terminus of nPKC-,
anti-PKC- against an epitope corresponding to amino acids 722 to
736 mapping at the carboxyl terminus of nPKC-, and anti-PKC-
against an epitope corresponding to amino acids 573 to 592 mapping at
the carboxyl terminus of nPKC- were purchased from Dianova,
(Hamburg, Germany). Anti-PKC- raised against residues 21 to 217 of
the regulatory domain of nPKC- was purchased from Transduction
Laboratories (Lexington, Kentucky). Anti-pan-PKC Abs were from
Seikagaku Corp (Kyoto, Japan). For immunoblotting, mAbs against PKC
isoenzymes from Transduction Laboratories were used.
Cells and cell culture
PBL were isolated by Ficoll gradient centrifugation. Mononuclear cells were washed with RPMI 1640 and taken up in RPMI 1640 supplemented with 20% FCS. After adherence depletion overnight in plastic dishes, the cell suspension contained <1% of monocytes as assessed with an FITC-conjugated Ab against CD14 (Dianova) by flow cytometric analysis. Cells were cultured in RPMI 1640 supplemented with 10% FCS, 100 U of penicillin, 100 µg/ml streptomycin, and 2 mM glutamine in flat-bottom microtiter plates with 4 x 105 cells/well.
Lymphocytes were stimulated with BMA 031 (5 µg/ml), OKT3 (5 µg/ml), or PMA (10 ng/ml), respectively, as indicated in the experiments. Cells were incubated for 68 h (if not otherwise stated), then 0.5 µCi of [3H]TdR (20 Ci/mmol; Amersham, Arlington Heights, IL) was added for an additional 4 h. Cells were harvested with an automatic cell harvester, and incorporation of [3H]TdR into DNA was determined by liquid scintillation counting.
Introduction of anti-PKC Abs by electroporation into human lymphocytes
Electropermeabilization was performed by the capacitor discharge
method, as described previously (25), in a Bio-Rad Gene Pulser
(Bio-Rad, Richmond, CA) at 20°C with slight modifications. Briefly,
human lymphocytes (2 x 106/ml) in
permeabilization buffer containing 10 mM PIPES (pH 7.4), 0.5 mM
MgCl2, 120 mM NaCl, 10 mM EGTA, and 8.2 mM
CaCl2 were processed to electroporation in the presence of
Abs raised against different PKCs at a concentration of 2 µg/ml. As
control Ab, 2 µg/ml rabbit Ig (IgG1; Dianova) was used. Except when
indicated otherwise, permeabilization was achieved by using a
capacitance of 500 µF at 300 V current, which decreased to
1/e (= 
) in 8.5 ms (see also Table I
). After electropermeabilization,
lymphocytes were allowed to recover in RPMI/50% FCS for 1 h at
37°C. Cells were then washed, and viability was routinely tested by
trypan blue exclusion test. Alternatively, propidium iodide
incorporation was determined by incubation of 105 cells
with 50 ng/ml propidium iodide for 20 min at 4°C. Propidium
iodide-positive cells were analyzed by flow cytometry. Under the
conditions used, >90% of cells proved to be viable following
electropermeabilization.
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Lymphocytes (5 x 106) were electropermeabilized in the presence of 2 µg/ml phycoerythrin (PE)-conjugated IgG2a (Dianova) as described above. Cells were then incubated in RPMI/50% FCS for 1 h. After washing with PBS containing 0.5% BSA, lymphocytes were incubated with 5 µg/ml BMA 031 for 30 min. Cells were then washed and incubated with dichlorotriazinylaminofluorescein (DTAF)-conjugated goat anti-mouse Ig (F(ab')2) fragment (Dianova) for an additional 30 min and extensively washed with PBS/0.1% BSA. Flow cytometric analysis was conducted with a FACS (Becton Dickinson, Mountain View, CA).
Determination of IL-2 synthesis
Human lymphocytes were electropermeabilized, then stimulated with the mAb BMA 031 (5 µg/ml) or OKT3 (5 µg/ml), respectively, as indicated in the experiments, at a cell density of 2 x 106/ml in RPMI medium supplemented with 2% FCS for 20 h. Supernatants were harvested, and the concentration of IL-2 was quantitated by means of a specific ELISA with a sensitivity of 15 pg/ml. The Abs against human IL-2 were obtained from Genzyme (Cambridge, MA).
Determination of IL-2R (CD25)
Lymphocytes (5 x 106) were stimulated with BMA 031 for 48 h in 2 ml of medium RPMI supplemented with 2% FCS. Cells were washed with PBS containing 0.5% BSA, then taken up in 20 µl of intraglobin (10 mg/ml). After incubation with 2 µg of PE-labeled anti-CD25 Abs (Dianova) for 30 min at 4°C, cells were washed three times in PBS containing 0.1% BSA. CD25-positive cells were determined by flow cytometric analysis.
RNA extraction and PCR-assisted analysis of mRNA
Human lymphocytes (2 x 107) were stimulated with 5 µg/ml BMA 031 for the times indicated in the experiments. Total RNA was then isolated on RNeasy spin columns (Qiagen, Hilden, Germany) and transcribed into cDNA using reverse transcriptase from Moloney murine leukemia virus (Stratagene, La Jolla, CA). Amplification of cDNA was conducted with 2.5 U of Taq polymerase (Stratagene) in a Gene Amp PCR System 9600 (Perkin-Elmer/Cetus, Norwalk, CT) by 30 cycles. Three temperature cycles consisted of denaturation at 94°C (55 s), annealing at 55°C (60 s), and elongation at 72°C (60 s). The following primers were used: IL-2, 5'-GAATGGAATTAATAATTACAAGAATCCC-3'; antisense primer, 5'-TGTTTCAGATCCCTTTAGTTCCAG-3'; tubulin, 5'-TTCCCTGGCCAGCTGCAAAGCTGCAGCTGACCTAGCTCGCAAG-3'; and antisense primer, 5'-CATGCCCTCGCCAGCTGTGTACCAGTGAGCTAAGCTGAAGGC-3'.
PCR products were separated in 2% agarose gels and visualized by staining with ethidium bromide.
Binding of [125I]IL-2 to surface receptors
IL-2 binding was conducted as described previously (26). Briefly, lymphocytes were stimulated with OKT3 for 24 h. Cells were then washed and incubated with 0.1 µCi 125I-labeled IL-2 (sp. act., 600 Ci/mmol; Amersham) and different concentrations of human recombinant IL-2 ranging from 20 pmol to 200 nmol at 4°C for 1 h. Free and bound radioactivity was separated by centrifugation through silicon oil as described previously (26). The number of high affinity receptors was calculated by Scatchard analysis with a computer program.
Determination of PKC activity
Determination of PKC activity in permeabilized cells. PKC activity was determined in permeabilized cells with the PKC-specific substrate, peptide GS (Bachem, Torrance, CA), as described previously (27). An essential component of this assay is the translocation of PKC to cellular membranes, since without translocation, PKC is rapidly lost from streptolysin O-permeabilized cells (27). Cells were washed before permeabilization with intracellular buffer, containing 120 mM KCl, 5 mM MgCl2, 5 mM PIPES (pH 7.4), 12.5 mM EGTA, and 8.17 mM CaCl2, resulting in final concentrations of 3.5 mM free Mg2+ and 150 nM free Ca2+, respectively. Cells were permeabilized with streptolysin-O (0.05 U/ml), and phosphorylation with 50 µM [32P]ATP (500 cpm/pmol) was conducted in a total volume of 250 µl at 37°C 5 min. The reaction was stopped by the addition of 100-µl portions of 25% (w/v) TCA in 2 M acetic acid. After being left for at least 10 min on ice, samples were centrifuged, and aliquots were spotted on P81 ion exchange chromatography paper (Whatman, Clifton, NJ), which was then washed three times in 30% (w/v) acetic acid containing 1% H3PO4. Assays were performed in duplicate, and a single tube containing no peptide was included for each pair of duplicate assays to estimate background phosphorylation of basic cell components that were not precipitated by TCA and that adhered to P81 paper. The values of such blanks were <10% of the values determined in the presence of peptide GS. Specific PKC activity is expressed as the difference between the values measured in the presence and those measured in the absence of the PKC-specific inhibitor, bis-indolylmaleimide (100 nM), in nanomoles of 32P transferred per minute per 2 x 106 cells.
Determination of PKC activity in subcellular fractions. Cytoplasmic PKC was measured in aliquots of the cytosolic fraction immediately after isolation, without further purification, in a reaction mixture containing 40 mM HEPES, 10 mM MgCl2, 0.4 mM EGTA, 400 µM peptide GS, and 60 µM [32P]ATP (500 cpm/pmol) with or without 2 mM CaCl2, 200 µg/ml phosphatidylserine, and 20 µg/ml 1,2-diolein. Lipids were dispersed by sonification. The reaction was started by mixing 100 µl of reaction medium with 10 µg of sample protein in isolation buffer. After an incubation for 3 min at 37°C, the reaction was stopped by the addition of 100-µl portions of 25% (w/v) TCA in 2 M acetic acid. Aliquots were spotted on P81 ion exchange chromatography paper, which was then washed three times in 30% (w/v) acetic acid containing 1% H3PO4. The enzyme activity was calculated as the difference between the values measured in the presence and those measured in the absence of phospholipids and calcium in nanomoles of 32P transferred per minute per mg protein of the sample. As peptide substrates do not reflect the calcium dependency shown for polypeptide substrates (27), the specific activity of both classical and calcium-independent PKC isoforms could be determined by this assay.
Isolation of subcellular fractions
Lymphocytes (5 x 106 cells/ml) were stimulated in serum-free medium RPMI with BMA 031 (5 µg/ml) and/or PMA (100 ng/ml) for different lengths of time as indicated in the experiments. Cells were then washed and resuspended in lysis buffer (20 mM Tricine (pH 7.4), 1 mM EGTA, 1 mM EDTA, 5 mM dithioerythritol, and 50 µg/ml leupeptin) and disrupted by nitrogen cavitation. Cytosolic and plasma membrane fractions were separated by removing nuclei and large granules (mitochondria, lysosomes) by centrifugation at 12,000 x g for 20 min, followed by centrifugation at 100,000 x g for 1 h. The membrane pellet was resuspended in lysis buffer as previously described (28). As resting lymphocytes contain a relatively small amount of endoplasmic reticulum, our membrane preparation can be considered a crude plasma membrane fraction containing <10% contamination by endoplasmic reticulum (29). Protein was determined by a method described previously (30).
Plasma membranes of stimulated lymphocytes were solubilized with 2 mg of 3-[(3-cholaminopropyl)dimethylammonio]-1 propanesulfonate/mg protein for 30 min at 4°C. Solubilized proteins were separated from nonsoluble material by centrifugation at 150,000 x g for 1 h as previously described (31).
SDS-PAGE and immunoblotting
Membrane protein (40 µg) was processed by SDS-PAGE on 10% polyacrylamide gels. Electroblotting of proteins to Immobilon membranes (Millipore Corp., Bedford, MA) was conducted at a constant current of 200 mA for 2 h. Blots were then incubated with mAbs against different PKC isoforms for 4 h. To visualize PKC subspecies, goat anti-mouse IgG and enhanced chemiluminescence reagent (Amersham) were used according to the manufacturer’s instructions.
It should be mentioned that all Abs used for electropermeabilization
and neutralization of different PKCs were tested for their
cross-reactivity by immunoblotting. While anti-PKC- and - did
not show any cross-reactivity, some, but not significant,
cross-reactivity was detectable between PKC-ß and -, but not
PKC-, suggesting that anti-PKC Abs recognized their
corresponding Ags, i.e., distinct PKC isoenzymes, with high
specificity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Figure 1,
stimulation of human PBL with BMA 031 via the TCR/CD3 complex led to a
bimodal activation of PKC activity. PKC activity was enhanced in the
plasma membrane between 5 and 10 min following stimulation, then it
declined to control levels after 30 min. After 90 to 120 min of
stimulation, a second wave of PKC activation was detected.
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As shown in Figure 2, different PKC
isoenzymes were translocated to the plasma membrane upon BMA 031
treatment in the early and late phases of T cell stimulation,
corresponding to the first and second waves of PKC activation (see Fig. 1
). PKC- and PKC- isoenzymes were translocated rapidly, i.e.,
within 10 min of stimulation. PKC-ß, -, and - isoenzymes were
activated and translocated only after 90 min of stimulation. PKC-
was not activated via the TCR/CD3 complex (data not shown). It should
be emphasized that upon activation with PMA, all conventional and
calcium-independent PKC isoenzymes were activated and translocated
(Fig. 2). As expected, PKC- was not activated by PMA either (data
not shown).
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To investigate regulation of activation by different PKC
isoenzymes, Abs against different PKC isoforms were introduced by means
of electropermeabilization into human T lymphocytes. To test optimal
experimental conditions, cells were electropermeabilized, and cell
viability was determined under different experimental conditions. As
shown in Table I, at a capacitance of 960 µF, the majority of cells
were dead, as measured by propidium iodide staining. At 500 µF, a
significant decrease in 1/e = (i.e., decay time of current)
was observed; consequently, cell viability proved to be about 90%.
Upon decreasing voltage and capacitance, cell viability reached >90%.
Preliminary experiments, however, have shown that only trace amounts of
macromolecules could be introduced into the cells below 500 µF (28).
Thus, in the additional experiments cells were electroporated at 500
µF and 300 V at 20°C.
As shown in Figure 3, under these
experimental conditions the majority of cells carrying TCR (i.e., T
lymphocytes) were loaded with PE-conjugated Ig (lower
panels). Nonspecific adsorption of IgG to the cell surface
was controlled by incubating lymphocytes with DTAF- or PE-labeled Igs
(upper panels).
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depicts the effects of different
anti-PKC Abs on the specific PKC activity. In isolated cytoplasmic
fractions, a pan-PKC Ab at a concentration of 2 µg/ml completely
suppressed PKC activity in the cytosol.
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). After introducing specific Abs raised against different PKC isoenzymes into intact lymphocytes, the sp. act. of the cytoplasmic PKC was decreased to a similar extent as in the cytosol upon direct addition of the respective Abs. It should be noticed that neither electropermeabilization itself nor the introduction of a control Ig influenced PKC activity significantly, as measured by phosphorylation of peptide GS. These results indicated that sufficient amounts of anti-PKC Abs have been introduced into the cells to neutralize and thus to inhibit selective PKC activities.
Inhibition by anti-PKC Abs of lymphocyte proliferation
Introduction of PKC-specific Abs into human lymphocytes resulted
in a significant inhibition of TCR/CD3-mediated cellular proliferation
(Table III). Extensive control
experiments have shown that neither electroporation nor introduction of
control Ig influenced cellular proliferation. BMA 031 or
OKT3-stimulated TdR incorporation was influenced by different Abs in a
different way. While the inhibitory effects of anti-PKC-ß, -,
and - Abs were reversible upon the addition of 200 pg/ml human IL-2,
inhibition of BMA 031- or OKT3-stimulated TdR incorporation by Abs
against PKC- and - could not be reversed by exogenous human IL-2.
These results suggested that PKC-ß and - might be the major PKC
isoenzymes involved in the regulation of IL-2 synthesis, while PKC-
and - might be responsible for the regulation of high affinity IL-2R
in stimulated human lymphocytes.
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and - Abs of IL-2R expression
In fact, introduction of anti-PKC- and - Abs into human
lymphocytes completely suppressed BMA 031-induced elevated expression
of IL-2R as determined by two independent methods. BMA 031-induced
up-regulation of CD25 was detected with PE-labeled anti-CD25 Abs by
flow cytometry. While neutralization of PKC- and - suppressed
CD25 expression, no change in the number of IL-2R (CD25) was detectable
in cells treated with Abs against other PKC isoenzymes (i.e., PKC-ß,
-, and -) or with control IgG (Fig. 4).
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and - Abs
significantly suppressed specific IL-2 binding, anti-PKC-ß, -,
and - had no effect on the number and affinity of IL-2R (Table IV).
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As shown in Table III, TCR/CD3-stimulated IL-2 synthesis was
significantly inhibited upon introduction of anti-PKC-ß, -,
and, to a lesser extent, - Abs. Neither introduction of Abs against
other PKC isoenzymes (i.e., PKC- and -) nor control IgG had any
influence on IL-2 synthesis of OKT3-stimulated human lymphocytes.
Electropermeabilization itself was also without effect on the amount of
IL-2 secreted by stimulated lymphocytes.
To determine whether PKC isoenzymes involved in the regulation of IL-2
synthesis exerted their effects via different signaling mechanisms,
anti-PKC-ß and - Abs were introduced simultaneously into the
cells, and IL-2 synthesis was determined following TCR stimulation. As
shown in Table III, combination of the Abs did not result in additional
suppression of IL-2 synthesis, suggesting that at least PKC-ß and
- might interfere with cellular signaling at an identical
site.
As PKC-ß proved to be the major PKC isoform involved in the
regulation of IL-2 gene expression, kinetics of PKC-ß activation and
translocation were correlated with IL-2 gene expression. As shown in
Figure 5, activation of PKC-ß preceded
expression of IL-2 mRNA, as detected by RT-PCR. While an enhanced
amount of PKC-ß protein was translocated to the plasma membrane
between 90 min and 2 h of stimulation, only trace amounts of IL-2
mRNA were detectable after 2 h of stimulation, while IL-2 gene
expression was markedly enhanced after 4 h of activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we show for the first time that in easily obtainable physiologic cells, i.e., in resting human PBL, the expressions of two different genes, namely IL-2 and IL-2R, are regulated by different PKC isoenzymes.
As in resting, nonproliferating cells, the majority of molecular
biologic methods are hardly applicable, new methods had to be
developed. Reversible permeabilization of cells by means of
electroporation proved to be the most reliable method to introduce
specific Abs into human lymphocytes. The conditions used allowed uptake
of Abs without significant loss of cell viability. At least 80% of T
lymphocytes contained Abs that neutralized their respective PKC
isoenzymes, allowing functional analysis of PKCs. Thus, human T
lymphocytes can effectively be loaded with anti-PKC Abs by means of
electropermeabilization under the conditions used (Table I). Control
experiments indicated that (= 1/e, decay time of current) was of
special importance for reversible electropermeabilization and for
viability of cells (Table I).
Pan-PKC Abs inhibited PKC activity nearly completely when added
to isolated cytosol. PKC activity in the cytosol of
electropermeabilized cells after introduction of pan-PKC Abs was
inhibited in a similar extent (Table II). These results together with
the data showing that the majority of T cells were stained with
PE-labeled control Ig showed that the amounts of Abs entering T
lymphocytes were sufficient to neutralize their respective PKC
activities. These results were further supported by the finding that
elevation of the Ab concentration did not result in further inhibition
of cytoplasmic PKC activity either by direct addition or by
electropermeabilization (data not shown). It should be emphasized that
>95% of cells labeled with PE-coupled Ig were, in fact, T lymphocytes
(Fig. 3).
IL-2 synthesis and IL-2R expression were regulated by different PKC
isoenzymes upon stimulation of normal human lymphocytes. Introduction
of anti-PKC- and - Abs resulted in a nearly complete
suppression of IL-2R expression as measured by three different,
independent methods (Fig. 4 and Tables III and IV). The fact that
neutralization of both PKC- and - resulted in complete inhibition
of TCR/CD3-induced up-regulation of CD25 suggested that these PKC
isoforms might regulate differential and independent signal
transduction pathways that converge at the level of the IL-2R gene.
This finding was supported recently by very elegant experiments by
Monks et al. (34) showing immediate activation and specific involvement
of PKC- in Ag-stimulated T lymphocytes.
Neutralization by specific Abs of PKC-ß, -, and - led to
inhibition of IL-2 synthesis, although to different extents. As Abs
raised against PKC-ß inhibited IL-2 synthesis and secretion up to
70%, the results clearly show that PKC-ß was the most predominant
isoform involved in the regulation of IL-2 synthesis and secretion
(Table III). Detailed analysis of the kinetics of PKC-ß translocation
to the plasma membrane and the onset of IL-2 gene expression, as shown
in Figure 4, clearly demonstrates that under the experimental
conditions used, activation of PKC-ß preceded enhancement of
transcription of IL-2 mRNA. It should be emphasized that there are
marked differences in the activation of PKCs and IL-2 synthesis in T
cell lines and resting T cells. While in Jurkat T cells PKC-ß (and
other PKC isoenzymes) were activated via TCR/CD3 within 5 to 10 min,
PKC-ß (- and -) in PBL were activated with a significant delay,
i.e., between 90 and 120 min of stimulation (our manuscript in
preparation) (Figs. 1 and 4). Thus, activation kinetics of PKC-ß, the
putative main regulator of IL-2 synthesis, correlated well with that of
IL-2 gene expression, as TCR/CD3-stimulated IL-2 mRNA expression in PBL
was not observed before 2 h, reaching maximal levels as late as
4 h (Fig. 4). Identical results concerning IL-2 gene expression
were reported recently (35).
There are several possibilities explaining why Abs against PKC-ß,
-, or - inhibited IL-2 synthesis to different degrees. Although
our control experiments suggested that the single Abs inhibited their
specific corresponding PKC isotype after introducing them into the
cells, we cannot rule out the possibility that differential degrees of
inhibition by different anti-PKC Abs of IL-2 synthesis reflected
different avidities to their Ags, i.e., to single PKC isoforms.
Similarly, one should consider the possibility that some PKC was not
reached by the Abs (because of compartmentalization) and thus remained
active in Ab-treated cells. Furthermore, different PKCs have different
half-lives; new synthesis and posttranslational phosphorylation of
distinct isotypes might also be different, thus resulting in
differences in the amounts and activities of special isoforms (3, 4).
PKC-dependent regulation of IL-2 gene expression is well documented (36), and the results of this study show that PKC-ß especially was of great importance for the induction of IL-2 synthesis and secretion. IL-2 gene expression is, however, regulated by complex signal transduction pathways (2, 37, 38). Besides PKCs, the role of p21ras and subsequent activation of the cytoplasmic mitogen-activated protein kinase cascade is a well documented mechanism in the regulation of different transcription factors regulating IL-2 gene expression (39, 40, 41). On the other hand, it was suggested that signals from PKC and p21ras might converge to control the activity of the protein kinase, Raf-1. Calcium-dependent processes, especially dephosphorylation of transcription factor(s) by calcineurin, also seem to be critically involved in the regulation of cytokine gene expression (42, 43). More recent data indicated that Jun-N-terminal kinase(s), especially JNK1, might participate in the regulation and nuclear binding of specific transcription factors (44, 45). Thus, IL-2 synthesis might be a net effect of several regulatory mechanisms.
The fact that anti-PKC-ß Abs resulted in a more than 70%
inhibition of IL-2 synthesis strongly suggests, however, that
PKC-ß-regulated signaling pathways are of great importance for the
synthesis and secretion of this cytokine. Recent observations in Jurkat
T cells suggested that PKC- and - might regulate parallel
signaling pathways, and the point of convergence would be in the
nucleus at the level of transcription factors in the course of T cell
activation (19). Our results indicate that PKC- was one of the major
PKC isoforms regulating IL-2R expression, while PKC- participated in
the regulation of IL-2 synthesis.
The functional role of PKC- is controversially discussed in the
literature. PKC- has been shown to be involved in the activation of
important transcription factors and was claimed to regulate cellular
proliferation (33, 46, 47). On the other hand, overexpression of
PKC- had no influence on the activation of transcription factors
regulating IL-2 gene expression, nor was the expression of early
activation markers (CD69) influenced by the PKC isoform (19). In our
hands in resting T lymphocytes PKC- was not regulated by the TCR/CD3
complex; accordingly, induction of anti-PKC- Abs was without any
effect on IL-2 synthesis and receptor expression (data not shown).
Thus, PKC- seems to have different functions in different cells.
The present results confirm and extend recent observations showing that
long-lasting activation of PKC(s) was a prerequisite of IL-2 synthesis,
while for up-regulation of IL-2R, short term activation of PKC(s) was
sufficient (48, 49). In human PBL, the different PKC isoenzymes were
differentially regulated upon stimulation via the TCR; while PKC-
and - were translocated fast and transiently, with a peak at about
10 min, long-lasting activation of the ß, , and isoforms was
observed, with a delay following TCR stimulation. Accordingly,
inhibition of short term activation of PKC- and - resulted in
nearly complete suppression of IL-2R.
The involvement of different PKC isoenzymes in the induction of IL-2
synthesis or IL-2R expression was also suggested by the effects of
specific inhibitors. Thus, cholera toxin, which blocked degradation of
phosphatidylinositol bisphosphate, the so-called "Pl response,"
prevented activation of PKC- and subsequently IL-2R expression
without affecting IL-2 synthesis (50). On the other hand, cyclosporin
A, which is known to suppress IL-2 synthesis but not the expression of
its high affinity receptors, specifically inhibited activation of
PKC-ß, but left activation of PKC- unchanged (8, 51). The results
of this study strengthen the idea that PKC-ß might be critically
involved in the induction of IL-2 synthesis.
Taken together, the results clearly show that different PKC isoenzymes may have selective functions in the course of TCR-induced signal transduction and thus in the regulation of cellular functions in human lymphocytes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marta Szamel, Institute of Molecular Pharmacology, Medical School Hannover, 30623 Hannover, Germany.
3 Abbreviations used in this paper: DAG, 1,2-diacylglycerol; PKC, protein kinase C; PE, phycoerythrin; DTAF, dichlorotriazinylaminofluorescein; nPKC, novel (calcium-independent) PKC.
Received for publication August 4, 1997. Accepted for publication November 13, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J. Clin. Invest. 95:1906.
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indicates the control; • indicates stimulated.
