A Role for Protein Kinase Cbeta I in the Regulation of Ca2+ Entry in Jurkat T Cells*

(Received for publication, December 30, 1996, and in revised form, March 24, 1997)

Doris M. Haverstick Dagger §, Michael Dicus Dagger , Moira S. Resnick , Julianne J. Sando and Lloyd S. Gray Dagger

From the Departments of Dagger  Pathology and  Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

T cell activation leading to cytokine production and cellular proliferation involves a regulated increase and subsequent decrease in the intracellular concentration of Ca2+ ([Ca2+]i). While much is understood about agonist-induced increases in [Ca2+]i, less is known about down-regulation of this pathway. Understanding the mechanism of this down-regulation is critical to the prevention of cell death that can be the consequence of a sustained elevation in [Ca2+]i. Protein kinase C (PKC), activated by the diacylglycerol produced as a consequence of T cell receptor engagement, has long been presumed to be involved in this down-regulation, although the precise mechanism is not wholly clear. In this report we demonstrate that activation of PKC by phorbol esters slightly decreases the rate of Ca2+ efflux from the cytosol of Jurkat T cells following stimulation through the T cell receptor or stimulation in a receptor-independent manner by thapsigargin. On the other hand, phorbol ester treatment dramatically reduces the rate of Ca2+ influx following stimulation. Phorbol ester treatment is without an effect on Ca2+ influx in a different T cell line, HSB. Down-regulation of PKCbeta I expression by 18-h phorbol ester treatment is associated with a loss of the response to acute phorbol ester treatment in Jurkat cells, suggesting that PKCbeta I may be the isozyme responsible for the effects on Ca2+ influx. Electroporation of an anti-PKCbeta I antibody, but not antibodies against PKCalpha or PKCgamma , led to an increase in the rate of Ca2+ influx following stimulation. Taken together, these data suggest that PKCbeta I may be a component of the down-regulation of increases in [Ca2+]i associated with Jurkat T cell activation.


INTRODUCTION

Activation of T lymphocytes via stimulation through the T cell receptor for antigen (TCR)1 leading to proliferation, cytokine production, or effector function requires a regulated increase and subsequent decrease in the intracellular concentration of Ca2+ ([Ca2+]i). The biochemical events associated with activation of T lymphocytes and leading to Ca2+ entry have been extensively studied (1). Engagement of the TCR leads to the activation of phospholipase C, which subsequently cleaves phosphatidylinositol bisphosphate into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3, binding to an intracellular receptor, induces the release of Ca2+ from an intracellular storage depot (2). In a receptor-independent manner, the Ca2+/ATPase inhibitor thapsigargin (3) causes an uncompensated leak of Ca2+ from this internal storage pool. In T lymphocytes, as well as in most electrically nonexcitable cells, this depletion of the intracellular storage pool induces the opening of the plasma membrane Ca2+ entry pathway and permits influx of extracellular Ca2+ (1). Previously, we have provided evidence that the pathway between release of intracellular Ca2+ and influx of extracellular Ca2+ is mediated by Ca2+-activated calmodulin (4), which permits Ca2+ entry carried by a current we have called IT (5).

The DAG produced subsequent to TCR stimulation is the naturally occurring activator of a family of serine-threonine kinases known collectively as protein kinase C (PKC). It has been proposed that DAG, via activation of PKC, represents the down-regulatory arm of Ca2+ signaling initiated by IP3 generation (for example, see Ref. 6). Four points in Ca2+ signaling have been implicated as targets for PKC action. First is a PKC-mediated phosphorylation of the gamma  subunit of the TCR·CD3 complex and subsequent down-regulation of this complex at the surface of the cell (7, 8). Second is the PKC-dependent phosphorylation of phospholipase C, with a consequent reduction in phospholipase C activity and IP3 generation (9). Third is the phosphorylation of the plasma membrane Ca2+/ATPase, leading to its activation and a facilitation of efflux of Ca2+ (10). Depending upon the methodology used and the cell type examined, activation of PKC can (11) or cannot (12) be shown to affect the rate of Ca2+ efflux following stimulation-induced increases in [Ca2+]i. The fourth potential target for a PKC effect is on the rate of Ca2+ influx itself, as suggested for HPB-ALL cells (13). In this report, we show that in Jurkat T cells the rate of Ca2+ influx is reduced by approximately 50% following activation of PKC.

The PKC family consists of at least 11 isozymes that vary in their requirements for lipid and Ca2+ for activation and are differentially expressed in various cell types (reviewed in Refs. 14 and 15). The study of the functions of this family of isozymes in cellular regulation has been facilitated by the use of phorbol esters, such as phorbol myristate acetate (PMA), which activate PKC, although with little specificity for any individual isozyme (14). In the past, the lack of isozyme-specific inhibitors (16) has made assignment of a PKC effect to a specific isozyme difficult. However, the recent development of some isozyme-specific inhibitors (17) and the use of technology such as RNA aptamers to block transcription of individual isozymes (18) is beginning to allow for the assignment of specific functions within a given cell type to a specific isozyme. For example, the development of a specific isozyme inhibitor has implicated PKCbeta directly as a mediator of the vascular dysfunctions seen in diabetic rats (17).

In T lymphocytes, expression of the various isozymes of PKC depends upon the cell lineage and the state of differentiation of the cell line (for example, see Ref. 19). The state of differentiation also confers upon T lymphocytes varying degrees of susceptibility to apoptosis induced by increases in [Ca2+]i (20). While apoptosis is used by the immune system to regulate the expression of specific subsets of T lymphocytes during development, disregulation of this process can lead to immune suppression (21). To further define the role of PKC in T cell activation, we have examined the expression of PKC isozymes in Jurkat T lymphocytes and begun the determination of which isozyme(s) may function in the normal down-regulation of antigen- or agonist-induced increases in [Ca2+]i.


MATERIALS AND METHODS

Cell Lines

Jurkat E6.1 and HSB cells were purchased from ATCC (Rockville, MD). Cells were maintained in RPMI 1640 (BioWhitaker, Walkersville, MD) containing 5% fetal bovine serum (Hyclone, Logan, UT), SerXtend (Irvine Scientific, Santa Ana, CA), and glutamine (BioWhitaker, Walkersville, MD) at 37 °C in a CO2 incubator.

Reagents

Indo-1 and BAPTA were purchased from Molecular Probes (Eugene, OR). Anti-PKC isoform-specific antibodies and the peptides used for immunization were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Unless otherwise indicated, all other reagents were purchased from Sigma.

Intracellular [Ca2+]i Measurements

Cells at a concentration of 4 × 106/ml were incubated with 1 µM indo-1/AM for 1 h at 37 °C in culture medium. Cells were washed three times in buffer A (10 mM HEPES, pH 7.4, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 140 mM NaCl, 0.1% glucose, 1% fetal bovine serum) and suspended at a final concentration of 1 × 106/ml in buffer A. The [Ca2+]i was determined from the fluorescence ratio (398/480 nm) with excitation at 360 nm in an SLM 8100 spectrofluorometer (SLM/Aminco, Urbana, IL) in the T format as described previously (4, 22). Calibration was conducted as described previously (22, 23) using the equation developed for an earlier generation of Ca2+ indicator dyes (24, 25).

Western Blotting

Cells were lysed in SDS-polyacrylamide gel electrophoresis sample buffer and 50 µl, representing 5 × 105 cells, were applied to a 10% acrylamide gel. Proteins were then transferred to blotting membrane and probed for isoform expression using enhanced chemiluminescence (ECL) detection (Amersham Corp.) according to the manufacturer's directions.

Electroporation of Antibodies

The protocol for electroporation was based on that published by Lukas et al. (26). Briefly, cells were washed three times in buffer B (10 mM PO4, pH 7.4, 150 mM NaCl) and suspended to a final concentration of 2 × 108/ml. Cells (50 µl) were mixed with 50 µl of antibody at 100 µg/ml in buffer B plus 0.1% NaN3 and 0.2% gelatin (as supplied by Santa Cruz Biotechnology) and placed in the electroporation chamber. For those experiments using antibody plus peptide, 50 µl of buffer B or 50 µl of peptides (200 µg/ml in buffer B plus 0.1% NaN3 and 200 µg/ml bovine serum albumin as supplied by Santa Cruz Biotechnology) was mixed with the anti-PKC antibodies and incubated on ice for 30 min prior to the addition of 50 µl of cells to the electroporation chamber. Electroporation (CellPorator, Life Technologies, Inc., Bethesda, MD) was carried out with the following settings: high resistance, 270 V, and 800 microfarads. These settings were chosen based on testing of multiple settings and balancing percentage of cell survival with antibody incorporation (determined by Western blotting and immunofluorescence; data not shown). Cells were then removed from the electroporation chamber, suspended to 2 × 106/ml in culture medium, and placed in a CO2 incubator for a period of recovery (6-18 h) prior to use. Controls include cells mixed with antibody but not electroporated or cells electroporated in the absence of antibody.

Immunofluorescence

Cells subjected to the electroporation procedure and prepared for use in experiments to measure changes in [Ca2+]i were allowed to settle on poly-L-lysine-coated microscope slides. The cells were lightly permeabilized by incubation with 9% buffered formalin and washed three times in buffer B. Anti-PKC antibody was visualized by incubating the slide with fluorescein isothiocyanate-labeled goat anti-rabbit antibody. For the images presented in Fig. 8, all fluorescence fields were photographed using T-Max 400 film (Eastman Kodak Co., Rochester, NY) using a 16-s exposure time. Phase contrast images were taken of the same fields immediately thereafter using a 3-s exposure time.


Fig. 8. Immunofluorescent visualization of antibody uptake following electroporation. Cells were prepared and antibody visualized as indicated under "Materials and Methods." For each panel, the upper part represents the field taken using phase contrast optics, and the lower part shows the same field taken under fluorescence conditions. A, Jurkat cells mixed with anti-PKCbeta I antibody but not electroporated. B, Jurkat cells mixed with anti-PKCbeta I antibody and electroporated under the conditions listed under "Materials and Methods." C, Jurkat cells mixed with anti-PKCbeta I antibody that was previously incubated with the peptide used for immunization and then electroporated. Each panel is representative of over 50 fields examined in each of three experiments.
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Statistics

All statistical analyses were performed using GraphPad Prism or Microsoft Excel.


RESULTS

Effects of Phorbol Ester on Agonist-induced Changes in [Ca2+]i

In most electrically nonexcitable cells, the Ca2+ entry pathway can be opened by depletion of the intracellular Ca2+ storage depot (1). In T cells, the Ca2+ entry pathway can be opened in a receptor-dependent or -independent manner, using, respectively, an activating monoclonal antibody to the T cell receptor (OKT3) or thapsigargin (4). The effects of activation of PKC by the phorbol ester PMA on these two methods of opening the Ca2+ entry pathway are shown in Fig. 1. The addition of PMA prior to stimulation of Jurkat cells with thapsigargin results in a concentration-dependent decrease in the maximum [Ca2+]i reached (Fig. 1A). Fig. 1B demonstrates that the inhibitory effect of PMA also occurs once the Ca2+ entry pathway has been opened. The addition of PMA at a time when the storage pool has refilled but the Ca2+ entry pathway is still open (4) results in a concentration-dependent reduction of [Ca2+]i. Similar results were observed when the Ca2+ entry pathway was opened in a receptor-dependent manner using the anti-CD3-specific antibody OKT3 (Fig. 1, C and D). Previously, it has been reported that such inhibitory effects of PMA are not seen in all T lymphocytes (19). Therefore, a different T cell line, HSB, was examined as well. This human T cell line has lost expression of the T cell receptor for antigen; thus, the Ca2+ entry pathway can only be opened by thapsigargin treatment. As shown in Fig. 2, there was no effect of up to 3 µM PMA on the changes in [Ca2+]i induced by thapsigargin in this cell line.


Fig. 1. Increases in [Ca2+]i in Jurkat cells are reduced in the presence of phorbol ester. Changes in [Ca2+]i were monitored spectrofluorometrically as outlined under "Materials and Methods." A, Jurkat cells were stimulated with 100 nM thapsigargin at 90 s in the absence (solid line) or presence of the indicated concentrations of PMA added 60 s prior to thapsigargin. B, Jurkat cells were stimulated with 100 nM thapsigargin at 30 s. PMA (0-100 nM) was added at 150 s. C, cells were stimulated with 1 µg/ml of anti-CD3 antibody (OKT3) in the absence (solid line) or presence of the indicated concentration of PMA added 30 s prior. D, cells were stimulated at 60 s with OKT3. PMA (0-100 nM) was added at 150 s. Traces are means of three determinations and are representative of five experiments.
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Fig. 2. Increases in [Ca2+]i in HSB cells are not affected by phorbol ester treatment. Changes in [Ca2+]i in HSB cells were monitored as outlined under "Materials and Methods." A, 100 nM thapsigargin was added to HSB cells in the absence (solid line) or presence (dotted line) of 3 µM PMA added 30 s prior. B, HSB cells were stimulated with 100 nM thapsigargin at 30 s, and 3 µM PMA was added at 150 s (dotted line). Traces are means of three determinations and are representative of five experiments.
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It has been proposed that the decreased magnitude of agonist-stimulated changes in [Ca2+]i after PMA pretreatment is due to a PKC-dependent phosphorylation and inactivation of phospholipase C (9). Such a phosphorylation would result in decreased production of IP3 with a corresponding reduction in the amount of Ca2+ released from the intracellular storage depot. To measure the amount of Ca2+ released from the intracellular storage pool without interference from Ca2+ influx, extracellular Ca2+ was chelated with EGTA prior to stimulation. Consistent with inhibition of phospholipase C, prior PMA treatment results in a reduction in the amount of Ca2+ released from the intracellular storage pool following receptor stimulation in Jurkat cells (Fig. 3A). However, the same inhibition of Ca2+ release is seen with thapsigargin treatment (Fig. 3C). Since thapsigargin increases [Ca2+]i without the involvement of the T cell receptor, it seems unlikely that this latter effect is due to inhibition of phospholipase C. Whatever the site of PKC action is, no significant effect on [Ca2+]i was observed when PMA was added after stimulation of Ca2+ release in the absence of Ca2+ influx (Fig. 3, B and D).


Fig. 3. Effect of PMA on release of Ca2+ from the intracellular storage pool. Jurkat cells were monitored for changes in [Ca2+]i as outlined under "Materials and Methods." The influx of extracellular Ca2+ induced by either OKT3 (A and B) or thapsigargin (C and D) treatment was blocked by chelation of extracellular Ca2+ with EGTA (5 mM) added 30 s prior to stimulation. A, solid line, increase in [Ca2+]i induced by 1 µg/ml of OKT3 in the absence of PMA; dashed lines, increase in [Ca2+]i in the presence of the indicated concentration of PMA added at 30 s. B, the same experiment as in A, except that PMA was added at 150 s. C, the same experiment as in A, except the increase in [Ca2+]i was induced by 100 nM thapsigargin added at 90 s. D, the same experiment as in C, except that PMA was added at 150 s. Traces are means of three determinations and representative of three experiments.
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Phorbol Ester Treatment Specifically Affects Ca2+ Influx in Jurkat Cells

It has been proposed that activation of PKC results in activation of the plasma membrane Ca2+ pump, such that there is a more rapid efflux of intracellular Ca2+ following PMA treatment (10). Additionally, it is possible that activation of PKC inhibits Ca2+ influx. To examine which of these mechanisms might be active in Jurkat cells, it was necessary to measure influx and efflux separately. To measure Ca2+ pump activity, cells were stimulated to open the Ca2+ entry pathway, and then extracellular Ca2+ was rapidly chelated by the addition of 3 mM BAPTA to the extracellular medium (Fig. 4A). At this point, the rate at which Ca2+ returns to base line is a reflection of the activity of the Ca2+ pump, since there is no Ca2+ entry. Because the rate of pump activity is dependent primarily upon the [Ca2+]i (27), a calculation of the exponential rate of decay was used to provide an estimate of pump activity; i.e. calculation of the t1/2 for the return of Ca2+ to basal levels can be used to compare efflux under various experimental conditions (Fig. 4B).


Fig. 4. Measurement of Ca2+ influx and Ca2+ efflux in Jurkat cells. Jurkat cells were incubated with indo-1 and monitored for changes in [Ca2+]i as outlined under "Materials and Methods." A, thapsigargin (100 nM) was added at 30 s, and the change in [Ca2+]i was monitored until 250 s, at which time 3 mM BAPTA was added to the cuvette to chelate extracellular Ca2+. After [Ca2+]i had returned to base line, 3 mM CaCl2 was added to the cuvette (350 s). The solid line represents the experiment conducted in the absence of prior PMA treatment; the dashed line represents the experiment conducted with 100 nM PMA added at zero time. B, calculation of the rate of Ca2+ efflux. The rate of the return of [Ca2+]i to base line was measured using the [Ca2+]i values from 250 to 290 s. The solid line represents the [Ca2+]i measurements, and the dashed line represents the curve calculated to fit these data. C, calculation of the rate of Ca2+ influx. The rate of rise in [Ca2+]i, measured from 350 to 375 s, was calculated using linear regression analysis of the data. The solid line represents the [Ca2+]i measurements; the dashed line represents the line calculated to fit these data. This figure is representative of the experiments used to generate the data of Fig. 5.
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Unlike a calcium channel blocker such as Ni2+ (4), chelation of extracellular Ca2+ with BAPTA does not close the Ca2+ entry pathway. Therefore, readdition of extracellular Ca2+ to the cell suspension (Fig. 4A) allows for the measurement of entry specifically during the first 10-15 s after readdition of Ca2+ (27). Because the activity of the plasma membrane pump is governed by [Ca2+]i, during this time when [Ca2+]i is low, pump activity will be greatly reduced. Therefore, a straight line function can be applied, and the rate of Ca2+ influx in nmol/s can be determined and compared under the various experimental conditions (Fig. 4C).

Influx and efflux rates in the presence and absence of PMA were calculated from several independent experiments (Fig. 5). PMA caused a slight, but reproducible, decrease in the rate (increased t1/2) of Ca2+ efflux, measured during the 30 s following the addition of BAPTA in Jurkat cells (Fig. 5B). In paired experiments, this difference ranged from a 13 to 23% change but was not, overall, statistically significant. However, PMA caused a pronounced, statistically significant decrease in the rate of Ca2+ influx measured in Jurkat cells during the 15 s following readdition of extracellular Ca2+. This reduction ranged from 37 to 56% when thapsigargin was used and up to 71% when cells were stimulated through the T cell receptor with OKT3 (Fig. 5A).


Fig. 5. Influx and efflux of Ca2+ in Jurkat and HSB cells. Jurkat cells were stimulated with 100 nM thapsigargin (thap) or 1 µg/ml of OKT3 without (open bars) or with (hatched bars) a prior addition of 100 nM PMA. HSB cells were stimulated with 100 nM thapsigargin without (open bars) or with (hatched bars) a prior addition of 100 nM PMA. Influx of intracellular Ca2+ (A) and efflux of extracellular Ca2+ (B) were calculated as described under "Results" and in the legend to Fig. 4.
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Because the release of Ca2+ from the internal stores is reduced in the presence of PMA (Fig. 3), it is possible that this reduction in the rate of influx is merely a consequence of reduced release from internal stores. However, when the rate of influx was measured in cells stimulated with a concentration of OKT3 that resulted in a 50% reduction in the amount of Ca2+ released from internal stores (0.1 µg/ml of OKT3, data not shown), the rate of influx was not different from the rate of influx in the presence of the maximally effective concentration of 1 µg/ml of OKT3 (83-102 nmol/s with 1 µg/ml OKT3 versus 98-114 nmol/s using 0.1 µg/ml OKT3, n = 3).

As a control for the calculations of influx and efflux, the same experiment was performed using HSB cells, which show no response to PMA on thapsigargin-induced changes in [Ca2+]i. No differences in Ca2+ influx or efflux were observed between cells that were treated with 100 nM PMA and those that were not (Fig. 5, right bars). For HSB cells, influx rates in the presence of PMA varied from 82 to 105% of control, and efflux rates varied from 93 to 110% of control.

In many cell types, treatment with PMA for 12-18 h ablates the effects of acute PMA addition; under most circumstances, this is due to extensive proteolytic degradation of PKC (28). To determine whether this same phenomenon occurred in Jurkat cells, cells were treated for 18 h with 250 nM PMA, and the effects of acute PMA addition on stimulated changes in [Ca2+]i were examined. Fig. 6 demonstrates that the rapid return to basal levels that occurs following acute PMA addition (Fig. 6A) was lost in cells that had been treated overnight with PMA (Fig. 6B). Similarly, when influx and efflux were measured as in Fig. 4, the effect of PMA on the rate of Ca2+ influx was completely lost in cells that had been treated for 18 h with PMA prior to stimulation (Fig. 6C).


Fig. 6. The effect of PMA treatment on agonist-induced changes in [Ca2+]i in cells treated for 18 h with PMA. A, Jurkat cells, incubated for 18 h with vehicle (0.01% ethanol), were monitored for changes in [Ca2+]i following OKT3 treatment at 30 s. PMA (100 nM) was added at 130 s. B, the same experiment as in A, except that cells had been incubated with 250 nM PMA for 18 h, and 3 µM PMA was added at 130 s. Traces are means of three determinations and representative of four experiments. C, influx (right bars) and efflux (left bars) of Ca2+ were measured in the absence (open bars) and presence (hatched bars) of the prior addition of 100 nM PMA in Jurkat cells that had been treated for 18 h with 250 nM PMA prior to use.
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Involvement of PKCbeta I in the Effects of PMA on Ca2+ Levels

As outlined above, PKC is a family of serine-threonine kinases, and although the isozymes appear to be differentially sensitive to PMA within any single cell type, there is little consistency among various cell types (29, 30). To determine whether the lack of phorbol ester-induced inhibition of Ca2+ influx in HSB cells was due to the absence of a certain PKC isozyme, isozyme expression in Jurkat and HSB cells was compared. As shown in Table I, although there were some differences in the level of expression of a specific isozyme (for example, PKCalpha ), there were no clear cut examples of an isozyme present in Jurkat cells but absent in HSB.

Table I. PKC isozyme expression by Jurkat and HSB cells

Cells were lysed and proteins were separated by polyacrylamide gel electrophoresis as outlined under "Materials and Methods." Proteins were transferred to blotting membrane, incubated with isozyme-specific anti-PKC antibodies, and visualized with ECL reagents. Relative signal intensity (greater to lesser) is designated by +++, ++, and +. - indicates no antibody binding detected.

Isozyme-specific antibody Jurkat HSB

 alpha +++ +
 beta I + +
 beta II ++ +++
 eta + +
 theta + +
 zeta + +
 delta  -  -
 epsilon ± +
 gamma ± +

Because the effect of acute PMA addition was ablated by 18 h of PMA exposure, the Western blot analyses were repeated in Jurkat and HSB cells that had been incubated for 18 h with 250 nM PMA. Expression of PKCalpha , -beta II, and -zeta were unchanged following this treatment (data not shown). However, PKCbeta I expression was markedly reduced following PMA treatment in Jurkat cells but only minimally affected in HSB cells (Fig. 7). This lack of an effect of PMA in HSB cells is similar to that observed by others (31, 32).


Fig. 7. Down-regulation of PKCbeta I expression with overnight PMA treatment. Cells were lysed with SDS-polyacrylamide gel electrophoresis sample buffer, and proteins were resolved on a 10% acrylamide gel; proteins were transferred and blotted for PKCbeta I expression as described under "Materials and Methods." Left arrows, molecular weight markers; lane A, Jurkat cells incubated for 18 h with vehicle (0.01% ethanol); lane B, Jurkat cells incubated for 18 h with 250 nM PMA; lane C, HSB cells incubated overnight with vehicle (0.01% ethanol); lane D, HSB cells incubated for 18 h with 250 nM PMA; right arrow, PKCbeta I marker.
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Since isozyme-specific inhibitors (16) are not commercially available, the effect of electroporation of isozyme-specific antibodies on Ca2+ entry was examined to garner additional support for a role for PKCbeta I in inhibition of Ca2+ entry. Jurkat cells were electroporated in the presence of isozyme-specific antibodies as outlined under "Materials and Methods." As shown in Fig. 8, cells mixed with antibody but not electroporated showed no incorporation of antibody (Fig. 8A). However, electroporation resulted in a reasonably uniform incorporation of antibody, as visualized by immunofluorescence (Fig. 8B). Prior incubation of antibody with the peptide used for immunization did not affect the incorporation of antibody (Fig. 8C).

The effects of the incorporation of antibody on agonist-induced changes in [Ca2+]i were examined next. Jurkat cells that had been electroporated in the presence of anti-PKCbeta I showed a reduced rate of return of [Ca2+]i to base line compared with cells that had been electroporated in the absence of antibody or those electroporated in the presence of anti-PKCalpha antibody (Fig. 9A). In five such experiments, electroporation in the presence of anti-PKCbeta I reduced this rate of decrease in [Ca2+]i by 40-45%, while electroporation in the presence of anti-PKCalpha or anti-PKCgamma had no significant effect on this rate (Fig. 9B). Furthermore, the effects of anti-PKCbeta I antibody could be blocked by a 2-fold mass excess of the peptide used to generate this antibody, but not the peptide used to generate the anti-PKCalpha antibody (Fig. 9B).


Fig. 9. Effect of electroporation of anti-PKC antibodies on changes in [Ca2+]i induced by receptor stimulation. Jurkat cells were electroporated in the presence of anti-PKCbeta I, anti-PKCalpha , or anti-PKCgamma antibody and in the presence of anti-PKCbeta I antibody plus the peptide used for immunization (beta ) or an irrelevant peptide (alpha ) as described under "Materials and Methods." Following incubation with indo-1, the cells were monitored for changes in Ca2+ following the addition of OKT3. A, representative experiment using cells electroporated in the absence of antibody (solid line), anti-PKCalpha (short dashes) or anti-PKCbeta I (long dashes) antibody. B, summary of the experiments. The rate of decline in [Ca2+]i was measured during the 60 s following the peak [Ca2+]i reached. Rates were compared with those in cells electroporated in the absence of antibody (100%). Data are shown as means ± S.D. for 15 experiments under each antibody condition in the absence of peptide and for 4 experiments in the presence of peptides.
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To determine if this reduction in rate was due to reduced efflux or enhanced influx, both were measured in cells electroporated with the isozyme-specific antibodies using the protocol described for the experiment of Fig. 4. The rate of efflux of Ca2+ from cells electroporated in the presence of anti-PKCbeta I was very similar to the rate observed in cells electroporated in the absence of antibody (Fig. 10B) or in cells electroporated in the presence of anti-PKCalpha (data not shown). Inclusion of peptides for PKCalpha or -beta caused a small but statistically insignificant increase in the efflux time (decrease in rate). There was a slight reduction in the magnitude of the PMA effect in cells electroporated with either anti-PKC antibody, compared with cells electroporated without antibody. However, this effect could not be assigned to a specific isozyme of PKC.


Fig. 10. Influx and efflux of Ca2+ in Jurkat cells electroporated in the presence of anti-PKC antibodies. Jurkat cells were electroporated in the absence or presence of the indicated anti-PKC isozyme-specific antibody without or with immunization peptides as indicated. Influx (A) and efflux (B) of Ca2+ were measured following stimulation with 1 µg/ml of OKT3 as outlined in Fig. 4 and under "Results." Data represent means of triplicate determinations in four experiments.
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The effects of electroporation were more striking when the influx of Ca2+ was examined. In individual experiments, a 25-40% increase in the rate of Ca2+ influx was observed in cells electroporated in the presence of anti-PKCbeta I compared with a less than 5% decrease in Ca2+ influx in cells electroporated in the presence of anti-PKCalpha or anti-PKCgamma (Fig. 10A). Additionally, the effect of PMA treatment on Ca2+ influx was lost in cells electroporated in the presence of anti-PKCbeta I, while the PMA effect was still seen in cells electroporated with anti-PKCalpha or anti-PKCgamma . As with the effects of anti-PKCbeta I antibody shown in Fig. 9, the effects of anti-PKCbeta I on Ca2+ influx could be blocked by a 2-fold mass excess of the peptide used for immunization but were not blocked by the peptide used for the production of anti-PKCalpha antibody (Fig. 10A).


DISCUSSION

Control of intracellular free Ca2+ concentration is a critical component of cellular homeostasis for all cell types. Increases in [Ca2+]i subsequent to receptor stimulation are necessary for the regulated progression of most cells through the cell cycle (33), for appropriate expression of gene products such as cytokines in T lymphocytes (1), and for some T cell effector functions, such as perforin-dependent cell killing by cytolytic T lymphocytes (34). Equally necessary is the regulated decrease with a return to prestimulation calcium levels subsequent to any increase. The consequence of a sustained increase in [Ca2+]i is generally cell death, often due to induction of apoptosis (21).

Changes in [Ca2+]i subsequent to engagement of the T cell receptor for antigen are initiated by release of Ca2+ from an internal storage depot mediated by the IP3 generated by receptor-induced activation of phospholipase C. This release of stored Ca2+ is followed by an influx of extracellular Ca2+. This process has been called by a number of names in electrically nonexcitable cells, such as capacitative calcium entry and the storage-operated calcium entry pathway (1). Regardless of the nomenclature, most current models for this component of cellular signaling indicate a feed forward mechanism.

In general, PKC activation, mediated by the other product of phospholipase C, DAG, has been associated with down-regulation of this pathway, although the data are not wholly consistent. The discrepancies may be due to differences in methodologies employed as much as to differences in cell lines examined. For example, in mixed populations of peripheral blood lymphocytes, phorbol ester treatment has been shown to have either an inhibitory (12) or augmenting (8) effect on receptor-mediated changes in [Ca2+]i. These opposing results may be due to the relative state of differentiation of the cells as at least one study has implied (19). The data are more consistent when cell lines are examined. When changes in [Ca2+]i are examined in Jurkat cells using fluorescent dyes, stimulation through the TCR with prior activation of PKC is associated with a reduction in the magnitude of increases in [Ca2+]i (this report and Refs. 8, 11, and 35). These results are in contrast to stimulation of Jurkat cells with a mitogenic lectin, such as concanavalin A, where there appear to be no effects of PKC activation on the subsequent changes in [Ca2+]i (35).

In addition to controversy over the effects of PKC activation, multiple targets of PKC in the Ca2+ entry pathway have been proposed. While in both a murine T lymphoma line (9) and in neutrophils (7, 36) phosphorylation and down-regulation of receptors by PKC has been implicated, this observation cannot explain the effect of PMA on receptor-independent changes in [Ca2+]i. Similarly, a reduction in IP3 generated due to a PKC-dependent phosphorylation of phospholipase C (9) fails to explain the observed reduction in release of Ca2+ from internal stores following thapsigargin treatment (Fig. 3).

The effects of PKC activation on Ca2+ influx versus efflux also have been examined. In peripheral blood lymphocytes, no effect on efflux following PMA treatment was seen when a straight line function was applied to the decrease in [Ca2+]i following chelation of extracellular Ca2+ (12). However, in Jurkat cells, activation of PKC was associated with enhanced efflux of Ca2+ when a nonlinear, biexponential calculation was used (11). Efflux of Ca2+ has been associated with a pump mechanism, most likely the plasma membrane Ca2+/ATPase (27). The activity of the pump has been shown to be dependent upon the intracellular concentration of Ca2+ (27), which we believe makes this application of a nonlinear function more appropriate. The observation of a PMA effect on Ca2+ efflux is confirmed by the data in this report, although the magnitude of the change was slightly less in the current study than observed in the previous one (11), perhaps due to the different curve fitting method used in this study.

There is more consistency when the effects of PKC activation on Ca2+ influx are examined. In nonlymphoid cells, PKC activation was associated with inhibition of Ca2+ influx when the current induced by Ca2+ influx was measured (37). In another study, a current others (38), but not ourselves (5), have implicated as the mediator of storage pool depletion-induced Ca2+ entry was reduced in the presence of activated PKC (38). Consistent with these observations, we observed a 30-50% inhibition of the rate of Ca2+ influx in Jurkat cells following PKC activation with PMA (Fig. 5).

Some of the discrepancies reported among various cell types may be due to differences in the expression of individual isozymes of PKC. Differences in the Ca2+ and lipid requirements for activation of the various isozymes suggest that specific functions are associated with each. Isozyme-specific functions in T cells were recently elegantly demonstrated in a study examining a subclone of Jurkat cells transfected with constitutively active expression vectors for different isozymes (39). Regulation of some transcription factors was enhanced in cells that overexpressed PKCepsilon but not PKCzeta (39).

Several observations in the present study support a role for PKCbeta I in inhibition of Ca2+ influx. First, although PKC isozyme expression did not significantly differ between Jurkat cells that respond to PMA and HSB cells that did not, PKCbeta I was down-regulated by long term PMA treatment only in Jurkat cells (Fig. 8). Second, this long term PMA treatment abolished the PMA-induced inhibition of Ca2+ influx (Fig. 7). Third, electroporation of an antibody to PKCbeta I, but not an antibody to PKCalpha or PKCgamma , reduced the rate of Ca2+ influx and abolished the PMA effect (Fig. 10).

The presence of PKCbeta I in HSB cells seems difficult to reconcile with the failure of these cells to exhibit PMA-induced down-regulation of [Ca2+]i if this isozyme is responsible for the effect. However, the failure of PMA to down-regulate this isozyme in HSB cells (Fig. 7) suggests that PMA may fail also to activate the isozyme in these cells. This is consistent with previous reports suggesting that HSB cells fail to respond to PMA (31, 32). Other possible explanations for the failure of these cell to down-regulate [Ca2+]i despite the presence of PKCbeta I include a defective enzyme and altered or missing downstream substrates.

Collectively, the results of these and other studies provide support for a closed loop pathway initiated by engagement of the TCR in human lymphocytes. Following TCR ligation and activation of phospholipase C, IP3 induces the release of Ca2+ from the intracellular stores. This released Ca2+ activates calmodulin, which initiates the influx of extracellular Ca2+ (23), carried by a current we have named IT (5). This feed forward portion of the pathway is down-regulated by DAG-activated PKCbeta I, which inhibits Ca2+ influx. Whether or not this inhibition is by an action on IT is currently under investigation.


FOOTNOTES

*   This work was supported by the Department of Pathology (University of Virginia) and National Institutes of Health Grant GM31184 (to J. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Dept. of Pathology, University of Virginia, Health Sciences Center, Box 214, Charlottesville, VA 22908. Tel.: 804-924-9202; Fax: 804-924-8060; E-mail: dmh2t{at}virginia.edu.
1   The abbreviations used are: TCR, T cell receptor for antigen; [Ca2+]i, intracellular concentration of Ca2+; DAG, diacyl glycerol; IP3, inositol trisphosphate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

ACKNOWLEDGEMENTS

We thank Paul Jung for excellent technical work examining Ca2+ influx and efflux and Chien Ying Lee for assistance with Western blots.


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