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Originally published In Press as doi:10.1074/jbc.M709330200 on March 3, 2008

J. Biol. Chem., Vol. 283, Issue 18, 12512-12519, May 2, 2008
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Store-operated Ca2+ Influx Causes Ca2+ Release from the Intracellular Ca2+ Channels That Is Required for T Cell Activation*

Sepehr Dadsetan{ddagger}, Liudmila Zakharova{ddagger}, Tadeusz F. Molinski§, and Alla F. Fomina{ddagger}1

From the {ddagger}Department of Physiology and Membrane Biology, University of California, Davis, Davis, California 95616 and §Department of Chemistry and Biochemistry, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093

Received for publication, November 13, 2007 , and in revised form, January 17, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The precise control of many T cell functions relies on cytosolic Ca2+ dynamics that is shaped by the Ca2+ release from the intracellular store and extracellular Ca2+ influx. The Ca2+ influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca2+ concentration ([Ca2+]i) necessary for T cell activation, whereas the role of intracellular Ca2+ release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca2+]i elevation observed upon activation of the store-operated Ca2+ entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca2+-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca2+ release channels were activated in parallel with SOCE and contributed to global [Ca2+]i elevation. Pretreating cells with (-)-xestospongin C (10 µM) or ryanodine (400 µM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca2+]i elevation evoked by the passive store depletion or TCR ligation. Although the Ca2+ release from the IP3R can be activated by TCR stimulation, the Ca2+ release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca2+]i signaling in T cells, that is SOCE-dependent Ca2+ release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca2+-dependent functions of T lymphocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In T lymphocytes, sustained elevation in [Ca2+]i following activating signals, such as engagement of T cell receptor (TCR)2 with a foreign antigen, is required to drive the diverse transcription programs leading to T cell clonal expansion and secretion of effector cytokines necessary for coordination of the immune response (1-3). Diminished [Ca2+]i signaling results in impaired T cell activation and, consequently, development of severe immunodeficiency (4, 5).

It is established that TCR stimulation leads to production of numerous second messengers (6), including inositol 1,4,5-trisphosphate (IP3), which evokes Ca2+ release from the IP3R, located predominantly on the endoplasmic reticulum (ER) (7). Two IP3R isoforms (IP3R-2 and IP3R-3) are expressed in lymphocytes, thymocytes, and splenocytes, whereas Jurkat T cells express all three known IP3R isoforms (IP3R-1, IP3R-2, IP3R-3) (8, 9). Binding of IP3 to the IP3R releases Ca2+ from the IP3-sensitive store, followed by activation of plasmalemmal storeoperated Ca2+ (SOC) channels that allow Ca2+ entry (SOCE) across the plasma membrane (7, 10, 11). Because the volume of the ER is estimated to be only ~1% of the volume of the T cell cytoplasm, it is believed that SOCE is the only mechanism capable of maintaining a sustained [Ca2+]i elevation for hours and days as required for T cell activation (4, 5, 7, 12). Therefore, the role of Ca2+ release from the IP3R in T cells is believed to be limited to the generation of a signal that activates plasmalemmal SOC channels (7).

It was also shown that TCR activation stimulates production of cyclic adenosine 5'-diphosphate-ribose (cADPR) and/or nicotinic acid adenine dinucleotide phosphate, the endogenous agonists of RyR (13, 14). Jurkat T lymphocytes express type 3 RyR (13, 15), whereas the peripheral blood human T lymphocytes express type 1 and 2 ryanodine (Ry) receptors (RyR) (16). In contrast to the IP3-dependent pathway, the sequence of events leading to the RyR activation and Ca2+ release from the Ry-sensitive store in T cells is less clear. Because down-regulation of IP3R is sufficient to abolish sustained [Ca2+]i elevation and cytokine production following TCR stimulation (17), the RyR activation appears to be secondary to IP3R activation. Consistently, the formation of cADPR has been shown to be delayed compared with IP3 production following TCR engagement (13). Nevertheless, down-regulation of cADPR levels or type 3 RyR expression reduced the TCR-dependent [Ca2+]i signaling in Jurkat T cells (13, 18). In addition, RyR antagonists ruthenium red and dantrolene inhibited murine T cell proliferation and IL-2 production following TCR stimulation (19). Thus, the RyR appears to play a major role in regulating the [Ca2+]i dynamics and, therefore, T cell functions. However, the exact mechanisms by which RyR regulates [Ca2+]i signaling in T cells, and its functional connections with the IP3R and/or plasmalemmal SOC channels, are not established.

It is known that following store depletion the SOCE supplies Ca2+ for the store replenishing from the cytosol via sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) (20). Consequently, store replenishing exerts negative feedback inhibition of SOC channels and termination of SOCE (21). However, the store replenishing via SERCA can be accomplished only if the intracellular Ca2+ release channels (IP3R and RyR) are inactivated. Elevation in [Ca2+]i and/or conformational changes of plasmalemmal Ca2+ channels may stimulate Ca2+ release from the IP3R and/or RyR either by direct interactions or via Ca2+-induced Ca2+ release (CICR) mechanism (22). Although it is well established that in some cell types, such as skeletal and cardiac muscles, the Ca2+ release from the intracellular store plays a crucial role in generation of [Ca2+]i signals and is controlled by activation of plasmalemmal Ca2+ channels, the effect of SOC channel activation on intracellular Ca2+ release channels in T cells has not been elucidated. Recently, the activation of CICR by Ca2+ influx via SOC channels was demonstrated in parotid acinar cells (23), although the specific type of intracellular channels that support the CICR and the functional significance of the CICR in non-excitable cells were not established.

In this study we explored whether Ca2+ release from the IP3R and/or RyR is triggered by activation of plasmalemmal SOC channels and whether it is essential for regulation of Ca2+-dependent T cell functions. The study was performed on Jurkat T cells (human leukemia T cell line) that express TCR and produce IL-2 in response to stimulation with mitogenic lectins (24). We found that Ca2+ release from both IP3R and RyR was activated in parallel with SOCE and significantly amplified [Ca2+]i signaling in T lymphocytes. Accordingly, inhibition of IP3R or RyR reduced [Ca2+]i elevation following TCR cross-linking and down-regulated proliferation and IL-2 production, the major functions of activated T cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Chemicals—Human acute T cell leukemia line Jurkat E6-1 (ATCC, Manassas, VA) was maintained in cell culture medium containing RPMI 1640 medium (Lonza/Bio-Whittaker, Basel, Switzerland), 10% FBS (Omega Scientific, Tarzana, CA), 2% L-glutamine, 2% vitamin solution, 1% RPMI amino acid solution, and 0.01% β-mercaptoethanol. Cells were kept in suspension in 5% CO2 at 37 °C and passaged every 2 days. Unless indicated, all chemicals were from Sigma-Aldrich; ionomycin and dantrolene were from Calbiochem; fura-2/AM, pluronic F-127, and ryanodine were from Invitrogen. (-)-Xestospongin C was isolated from the Xestospongia species as described previously (25) and repurified by crystallization before use. Stock solutions of (-)-xestospongin C (1 mM) were prepared in methanol. Stock solutions of ryanodine (10 mM) or dantrolene (10 mM) were prepared in DMSO.

Before experiments, cells were plated on poly-L-lysinecoated glass-bottom clambers and then loaded with 1 µM fura-2/AM and pluronic F-127 for 5 min in modified Tyrode solution containing (in mM): 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 D-glucose, pH 7.3. After washing, cells were incubated for an additional 30 min at 37 °C. Some cells were incubated in modified Tyrode solution containing 10 µM (-)-XeC, or 400 µM Ry, or vehicle (methanol or DMSO) alone for 30 min at 37 °C. In nominally Ca2+-free solution, Ca2+ was omitted. In some experiments La3+ was added to modified Tyrode solution and/or nominally Ca2+-free solution.

Calcium Imaging—Fluorescence images were acquired from adherent cells using a SenSys CCD camera (Roper Scientific, Tucson, AZ) and a x40 oil immersion Zeiss objective on a Zeiss Axiovert 200 inverted microscope (Thornwood, NY). A Lambda DG-4 filter changer (Sutter Instrument, Novato, CA) was used for switching between 340 and 380 nm excitation wavelengths. All experiments were performed at room temperature. Solution exchange was performed via a gravity-driven perfusion system. Solution exchange in the recording chamber was completed within 5 s. The vehicle-containing solutions (methanol or DMSO) were applied in place of the drugs in all control experiments. Data acquisition was performed using MetaFluor v7.0 software (Universal Imaging, Downingtown, PA). [Ca2+]i values were estimated from fura-2 calibration as described previously (26). Kd was taken as 248 nM (27). Unless otherwise indicated, in each experiment changes in [Ca2+]i were recorded from 15-20 cells and then averaged.

Manganese Quench of Fura-2 Fluorescence—Because Mn2+ readily passes through SOC channels (28) and is unlikely to be transported from the cytosol (29, 30), the rate of fura-2 quenching in the presence of Mn2+ was used as a measure of divalent cation influx into the bulk cytoplasm in intact cells. The extracellular solution used in all Mn2+ quench experiments contained (in mM): 130 NaCl, 5.6 KCl, 1 MgCl2, 0.3 CaCl2, 0.5 MnCl2, 10 HEPES, 10 D-glucose, pH 7.3. Changes in Ca2+-independent fluorescence due to Mn2+ quench were determined using the method described by Zhou and Neher (31). Briefly, background-corrected fura-2 fluorescence excited at 340 nm (F340) was plotted against background-corrected fura-2 fluorescence excited at 380 nm (F380) during the initial 10 s following application of 30 µM cyclopiazonic acid (CPA). The slope of the F340 versus F380 relationship equals the "isocoefficient" {alpha}. The fluorescence value, Fi, which is unaffected by changes in [Ca2+]i, was determined as Fi = F340 + {alpha}F380. The rate of Fi decline is proportional to the rate of Mn2+ entry into the cytosol and was used as an indicator of plasma membrane Ca2+ permeability.

Proliferation Assay—Cell division track assay was conducted using the CFSE proliferation kit (Molecular Probes). Stock solution of CFSE (4 mM) was prepared in DMSO. Cells were washed, resuspended in phosphate-buffered saline (PBS) containing 4 µM CFSE at density 1 x 106 cells/ml, and incubated at 37 °C for 10 min. Labeling was quenched by adding 5x volume of RPMI 1640 culture medium containing 10% FBS. After washing three times with RPMI 1640 + 10% FBS, cells were placed into FBS-free cell culture medium to abolish cell division and kept overnight in 5% CO2 at 37 °C. After overnight incubation, the CFSE-labeled cells were pelleted down and a fraction of them (~50 x 103) were fixed with 1% paraformaldehyde in PBS and analyzed by flow cytometry to establish the CFSE fluorescence profile of undivided cells. The remaining CFSE-labeled cells were resuspended in cell culture medium supplemented with 10% FBS at a density 0.05 x 106 cells/ml and seeded into 24-well tissue culture plate (1 ml/well). Some cells were preincubated for 30 min at 37 °C with (-)-XeC (10 µM), or Ry (400 µM), or dantrolene (30 µM) prior to transfer into FBS-containing cell culture medium. Drugs were also added to the same cells on each consecutive day of culturing. The vehicle-containing solutions (methanol or DMSO) were applied in place of the drugs in all control experiments. After 72 h in culture, cells were harvested, washed with PBS, and fixed with 1% paraformaldehyde in PBS. Fluorescence intensity was measured at 488 nm using FACScan flow cytometer and CellQuest software (BD Biosciences). The number of cell divisions was calculated using FlowJo software (Tree Star Inc., Ashland, OR).


Figure 1
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  		FIGURE 1.
Sequential CPA application reveals an inverse relationship between global [Ca2+]i and store refilling. A and B, time courses of [Ca2+]i changes recorded from the individual cells (thin gray traces) in a typical experiment. Thick black traces are the averages of 20 traces recorded from the individual cells in the same experiment. A, extracellular Ca2+ was omitted as indicated, and then CPA (30 µM) was applied in nominally Ca2+-free extracellular solution for 10 min. After washing, CPA was applied a second time as indicated. B, following the first CPA application, a modified Tyrode solution containing 2 mM Ca2+ was readded to the bath for 10 min as indicated. Extracellular Ca2+ was then removed and CPA was reapplied a second time. Note that application of ionomycin (Iono,1 µM) following the second CPA application in A and B generated only a small elevation in [Ca2+]i. C, [Ca2+]i traces recorded from two individual cells (dotted and solid lines) pooled from the experiment shown in B. Brackets indicate areas under the [Ca2+]i traces that were integrated to estimate store content during sequential CPA application (CPA1 and CPA2) and global [Ca2+]i elevation during Ca2+ readdition ([Ca2+]SOCE). Values of [Ca2+]i prior to the CPA or extracellular Ca2+ readdition were subtracted before integration. D, the relationship between the ratio of CPA2 to CPA1 and the [Ca2+]SOCE. Data are from six experiments; each point represents measurements from an individual cell. The straight line is a linear regression fit of the experimental data (r =-0.71).

 
IL-2 Production Assay—Jurkat T cells were washed three times with PBS, resuspended in FBS-free cell culture medium at a density of 0.8 x 106 cells/ml, seeded into 96-well tissue culture plates (0.4 ml/well), and then stimulated with 100 µM mitogenic lectin phytohemeagglutinin P (PHA). Some cells were preincubated for 30 min at 37 °C with (-)-XeC (10 µM), or Ry (400 µM), or dantrolene (30 µM) prior to stimulation with PHA. After 24 h of incubation, cell culture supernatants were collected and contents of IL-2 were determined by enzyme-linked immunosorbent assay using Quantikine kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Data Analysis—Image analysis was performed using MetaFluor v7.0 software (Universal Imaging). Further measurements and statistical analysis were performed using Origin 7 software (OriginLab, Northampton, MA). All data are presented as the means ± S.E. of the mean; n = number of experiments. Slopes were determined using a linear fit of experimental data to minimize the sum of the squared differences between the data and the fitted line. Statistical differences between means were accepted as statistically significant at p < 0.05, using Student's paired or unpaired t test or Wilcoxon nonparametric test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Store Refilling Inversely Correlates with Levels of Global [Ca2+]i—To explore the contribution of Ca2+ release from the internal store to the global [Ca2+]i dynamics, we employed fura-2 Ca2+ indicator and CPA, a reversible SERCA blocker, that allowed us to assess the [Ca2+]i dynamics during SOCE activation and the store content in the individual T cells in the same experiment. As expected, when CPA (30 µM) was applied in Ca2+-free solution, a transient elevation in [Ca2+]i due to Ca2+ release via "leak" channels was observed (Fig. 1A). A second application of CPA following wash in Ca2+-free solution did not produce detectable [Ca2+]i responses, indicating that the CPA-sensitive store was empty. Ionomycin, a Ca2+ ionophore, applied at the end of the experiment produced only a small elevation in global [Ca2+]i, demonstrating that CPA depleted most of the intracellular Ca2+ store.


Figure 2
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  		FIGURE 2.
(-)-Xestospongin C or ryanodine reduce global [Ca2+]i elevation during Ca2+ readdition and facilitate store refilling. A and B, average [Ca2+]i time courses showing the absence of acute effects of (-)-XeC (A;10 µM, red trace) or Ry (B; 400 µM, blue trace) applied in nominally Ca2+-free extracellular solution on [Ca2+]i. Vehicles (methanol (MeOH) or DMSO) alone were applied instead of drugs in control experiments (black traces). Extracellular Ca2+ was omitted and (-)-XeC, Ry, or CPA were applied as indicated. Each trace is an average of 20 traces recorded from individual cells in a typical experiment (n = 5). C and D, time courses of Mn2+ quench of fura-2 fluorescence (Fi) in untreated cells (black traces, Control) and cells preincubated for 30 min with 10 µM (-)-XeC (C; red traces) or 400 µM Ry (D; blue traces). Base-line time courses of Mn2+ quench of fura-2 fluorescence were recorded in the absence of CPA (upper traces). To record Mn2+ influx via SOC channels, 30 µM CPA was applied 10 min prior to Mn2+ to deplete the store (lower traces). Each trace is an average of 20 traces recorded from individual cells in a typical experiment (n = 5). E and F, average [Ca2+]i time courses recorded from untreated cells (black traces, Control) or cells preincubated with (-)-XeC (E; red trace) or Ry (F; blue trace). Extracellular Ca2+ was omitted prior to the recordings. CPA and 2 mM Ca2+-containing extracellular solution were applied as indicated. Each trace is an average of 20 traces recorded from the individual cells in a typical experiment. G and H, summarized data from experiments performed as shown in E and F. [Ca2+]SOCE and CPA2/CPA1 ratios were determined as described in Fig. 1. G, average [Ca2+]SOCE in untreated cells (open bars; Control) and in cells preincubated with (-)-XeC (hatched bars) or Ry (gray bars). H, average CPA2/CPA1 ratio in untreated cells (open bars; Control) and in cells preincubated with (-)-XeC (hatched bars) or Ry (gray bars). Stars indicate that differences between means are statistically significant (p <0.01, independent t test). n, number of experiments; error bars, S.E. of the means.

 
Readdition of extracellular Ca2+ following the first CPA application produced a global elevation in [Ca2+]i, presumably due to Ca2+ entry via SOC channels (Fig. 1B). After removing extracellular Ca2+, the second application of CPA evoked [Ca2+]i transients, indicating that the store was allowed to be refilled during extracellular Ca2+ readdition. Although on average the amplitude of the [Ca2+]i transient produced by the second CPA application was smaller than that produced by the first CPA application, the individual cells exhibited a variety of [Ca2+]i responses to the second application of CPA (Fig. 1, B and C). We found that cells that displayed a large elevation in [Ca2+]i upon Ca2+ readdition exhibited a small response to the second CPA application (Fig. 1C). In contrast, in cells that displayed small or no [Ca2+]i transient in the presence of extracellular Ca2+, both CPA applications evoked similar [Ca2+]i transients (Fig. 1C). Ionomycin, applied following the second CPA application, produced similar responses in both cases, indicating that Ca2+ retention within the store may not account for the absence of Ca2+ transient upon a second CPA application.

Integration of [Ca2+]i transients evoked by SERCA blockers has been shown to provide an accurate estimate of releasable store content (32). We took the ratio of background-subtracted integrated [Ca2+]i transients induced by the second CPA application (CPA2) to the first one (CPA1) (Fig. 1C) as the measure of the extent of store refilling. The [Ca2+]i transients recorded during Ca2+ readdition that were presumably induced by SOCE were also integrated and denoted as [Ca2+]SOCE. When CPA2/CPA1 ratios were plotted against [Ca2+]SOCE measured in the individual cells, an inverse relationship between the extent of store refilling and global [Ca2+]i elevation following Ca2+ readdition was revealed (Fig. 1D).


Figure 3
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  		FIGURE 3.
(-)-Xestospongin C and ryanodine down-regulate Ca2+ responses to TCR agonist. A and B, average [Ca2+]i responses to 20 µM PHA and 30 µM CPA, applied as indicated, in cells preincubated with 10 µM (-)-XeC (A; red trace), or 400 µM Ry (B; blue trace), or vehicle alone (black traces, Control). Extracellular Ca2+ was omitted prior to the recordings. PHA or CPA were applied as indicated. C and D, average [Ca2+]i responses to 20 µM PHA, applied as indicated in nominally Ca2+-free extracellular solution, and readdition of 0.5 mM extracellular Ca2+ in cells preincubated with 10 µM (-)-XeC (C; red trace), or 400 µM Ry (D; blue trace), or vehicle alone (black traces, Control). Each trace represents an average of 20 traces recorded from the individual cells. E-H, summarized data from experiments performed as shown in A-D. E, average integrated [Ca2+]i transients evoked by PHA ([Ca2+]PHA) in cells preincubated with (-)-XeC (hatched bars), or Ry (gray bars), or vehicle alone (open bars; Control). F, average integrated [Ca2+]i transients evoked by CPA ([Ca2+]CPA) applied following PHA in cells preincubated with (-)-XeC (hatched bars), or Ry (gray bars), or vehicles alone (open bars; Control). Brackets in A-D indicate the areas that were background-subtracted and integrated to obtain [Ca2+]PHA and [Ca2+]CPA values. G, average baseline-subtracted peak values of [Ca2+]i transients, shown in C and D, evoked by 0.5 mM Ca2+ readdition in cells preincubated with (-)-XeC (hatched bars), or Ry (gray bars), or vehicle alone (open bars; Control). *, indicates that differences between means are statistically significant at p <0.01 (independent t test); **, indicates that differences between means are statistically significant at p <0.05 (independent t test). n, number of experiments; error bars, S.E. of the means.

 
The store refilling in the absence of the global [Ca2+]i elevation was described previously and was attributed to colocalization of the plasmalemmal Ca2+ channels with SERCA (33-36). The absence of store refilling in cells with high levels of [Ca2+]i implied that in these cells intracellular Ca2+ release channels are active and may contribute into [Ca2+]i elevation following Ca2+ readdition.

Inhibition of IP3R or RyR Reduces Global [Ca2+]i Elevation during SOCE Activation and Facilitates Store Refilling—To explore the contribution of Ca2+ release from IP3R and/or RyR into the [Ca2+]i elevation following SOCE activation in T cells, we employed (-)-XeC and Ry that specifically block IP3R and RyR, respectively (37, 38). When applied extracellularly, neither (-)-XeC (10 µM) nor Ry (400 µM) affected basal [Ca2+]i levels or CPA responses (Fig. 2, A and B). Furthermore, pretreatment with either (-)-XeC or Ry did not affect the rate of the storeoperated divalent cation influx assessed by the Mn2+ quench of fura-2 fluorescence (Fig. 2, C and D). When applied extracellularly, Mn2+ produced a slow base-line quench of fura-2 fluorescence that was significantly accelerated following CPA application due to the activation of the store-operated Mn2+ influx via SOC channels. Preincubation with either (-)-XeC or Ry did not significantly affect rates of Mn2+ quench in the absence or presence of CPA. These experiments indicate that neither (-)-XeC nor Ry release Ca2+ from the CPA-sensitive store or directly affect SOCE.

In addition, the sequential store depletion experiments revealed that [Ca2+]i transients evoked by the first application of CPA were similar in shape and magnitude in both untreated cells (Control) and cells preincubated with either (-)-XeC or Ry (Fig. 2, E and F). However, the [Ca2+]i transients during Ca2+ readdition were smaller in (-)-XeC- or Ry-treated cells compared with the control. Given that neither (-)-XeC nor Ry antagonize SOCE (Fig. 2, C and D), the inhibition of Ca2+ release from IP3R or RyR should account for the reduction in [Ca2+]i elevation during Ca2+ readdition in cells preincubated with (-)-XeC or Ry. Consistently, CPA application following Ca2+ readdition produced larger [Ca2+]i transients and therefore increased the CPA2/CPA1 ratio in (-)-XeC- or Ry-treated cells compared with untreated cells (Fig. 2, E-H). Thus, these results demonstrate that activation of Ca2+ release from IP3R or RyR contributes significantly to global [Ca2+]i elevation upon SOCE activation.


Figure 4
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  		FIGURE 4.
(-)-Xestospongin C, ryanodine, or dantrolene inhibit T cell proliferation and IL-2 production. A, the fluorescence profile of CFSE-loaded cells incubated for 72 h in the presence of 10 µM (-)-XeC (top panel; red line), or 400 µM Ry (middle panel; blue line), or 30 µM dantrolene (lower panel; green line), or vehicle alone (Control; black lines in all panels). The dashed lines show the fluorescence profiles of CFSE-loaded cells incubated for overnight in FBS-deprived medium (undivided cell population at time 0). The brackets and numbers above the brackets indicate an estimated fraction of undivided cells in control cell populations and cell populations incubated with (-)-XeC, or Ry, or dantrolene (Da). Representative data from five experiments performed with each blocker are shown. B, IL-2 content in cell culture supernatants. Cells were incubated for 24 h in cell culture medium supplemented with 20 µM PHA alone or in combination with (-)-XeC (10 µM), or Ry (400 µM), or dantrolene (Da;30 µM), as indicated with (+), or vehicle alone, as indicated with (-). The (-)-XeC, or Ry, or dantrolene were applied 30 min prior to PHA. * indicates that differences between means are statistically significant at p < 0.01 (independent t test, n = 6). Error bars, S.E. of the means.

 
Inhibition of IP3R or RyR Down-regulates [Ca2+]i Signaling following TCR Stimulation—We next explored whether Ca2+ release from IP3R or RyR plays a role in sustained Ca2+ elevation at physiologically relevant conditions, such as following TCR stimulation. In the absence of extracellular Ca2+, a TCR agonist PHA evoked [Ca2+]i responses that were significantly reduced in cells preincubated with (-)-XeC (Fig. 3, A and B). On average, (-)-XeC reduced integrated [Ca2+]i responses to PHA by ~40% compared with those in untreated cells (Fig. 3, A and E). The same experiments revealed that [Ca2+]i transients in response to CPA, applied following PHA application to completely deplete the store, were up-regulated by ~30% in (-)-XeC-treated cells (Fig. 3, A and F), which is consistent with (-)-XeC inhibition of IP3R. Application of RyR agonist caffeine (20 mM) produced low amplitude [Ca2+]i oscillations in the absence of extracellular Ca2+, which were essentially (by 90%) inhibited in cells preincubated with 400 µM Ry (n = 4, not shown). Despite inhibition of [Ca2+]i responses to caffeine, preincubation with Ry did not significantly affect the PHA- or CPA-induced [Ca2+]i transients (Fig. 3, B, E, and F). These results indicate that TCR cross-linking with PHA readily mobilizes Ca2+ from the IP3-, but not Ry-, sensitive stores.

When Ca2+ was readded following TCR stimulation to allow SOCE, the amplitudes of [Ca2+]i transients were significantly lower in both (-)-XeC- or Ry-pretreated cells than those in the untreated cells (Fig. 3, C and D). On average, preincubation with (-)-XeC or Ry reduced the maximal amplitudes of [Ca2+]i transients during Ca2+ readdition by ~44 and 42%, respectively (Fig. 3G), indicating that at physiological conditions Ca2+ release from IP3R and/or RyR is activated in parallel with SOCE and contributes significantly to [Ca2+]i elevation.

Inhibition of IP3R or RyR Down-regulates T Cell Proliferation and IL-2 Production—To establish the importance of Ca2+ release from IP3R and/or RyR for T cell functions, we investigated the effects of IP3R or RyR inhibitors on cell proliferation and IL-2 production. We employed the CFSE fluorescent dye, which can be readily loaded into the cytoplasm and then equally distributed between cells after each cell division, resulting in a progressive decrease in CFSE fluorescence in cells that have undergone multiple rounds of divisions (39). Because Jurkat T cells proliferate instantly without stimulation, we first incubated the CFSE-labeled cells in FBS-free medium to inhibit cell divisions. The proliferation was then induced by transferring cells into complete cell culture medium. Some cells were preincubated with (-)-XeC-, or Ry, or dantrolene, a clinically relevant RyR inhibitor (40, 41), prior to the induction of proliferation. The analysis of CFSE fluorescence intensity in individual cells following 72 h of incubation revealed that IP3R or RyR inhibitors significantly (p < 0.05, Wilcoxon directional test, n = 5) reduced the proliferative activity of Jurkat T cells (Fig. 4A). The fractions of undivided cells constituted 55, 60, and 54% in cell cultures incubated with (-)-XeC, Ry, or dantrolene, respectively, compared with 38% in the untreated cell population. In addition, the peaks of CFSE fluorescence profiles of cells exposed to IP3R or RyR inhibitors were shifted to the right, indicating slower cell cycle progression (Fig. 4A). We also found that preincubation with (-)-XeC-, or Ry, or dantrolene significantly inhibited PHA-induced IL-2 production (Fig. 4B). These data indicate that Ca2+ release from IP3R or RyR is a powerful regulator of Jurkat T cell proliferation and TCR-mediated IL-2 production.


Figure 5
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  		FIGURE 5.
Hypothetical scheme of Ca2+ dynamics in T cells upon TCR activation. Engagement of TCR with the antigen (A) triggers Ca2+ release from IP3R and possibly from RyR, leading to a transient or localized store depletion, which in turn activates SOC channels in the plasma membrane (PM). Activation of SOCE via SOC channels may elevate [Ca2+]i in the vicinity of IP3R and/or RyR, which activates CICR from both IP3R and RyR, further elevating [Ca2+]i levels. Alternatively, SOC channels may directly interact with IP3R and/or RyR to trigger Ca2+ release (not shown). Because store is continuously refilled with extracellular Ca2+ at the junctional sites of the ER with the plasma membrane, the Ca2+ release from IP3R and/or RyR is active as long as SOC channels are activated and extracellular Ca2+ is present. The Ca2+ release from IP3R and/or RyR depletes the store, which keeps SOC channels open. The Ca2+ release from IP3R and/or RyR and Ca2+ influx via SOC channels act synergistically to sustain [Ca2+]i elevation necessary for activation of transcription factors. The location of IP3R, RyR, SOC channel, and SERCA on the diagram is for convenience of the presentation only.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is that in T lymphocytes activation of the plasmalemmal SOC channels directly stimulates Ca2+ release from both IP3R and RyR, which significantly amplifies [Ca2+]i signaling at the expense of store refilling (Fig. 5). These findings underscore the role of intracellular Ca2+ store in maintaining the sustained [Ca2+]i elevation, which was previously solely attributed to the extracellular Ca2+ influx via SOC channels (4, 5, 7, 12).

We found that consistent with a number of previous studies performed on other cell types (33-36), intracellular store in T cells may be completely refilled without global [Ca2+]i elevation (Fig. 1C). This indicates that in T cells SOC channels may colocalize with the SERCA, which readily pumps Ca2+ from the vicinity of SOC channels into the store lumen, so that Ca2+ do not appear in the cytosol. The areas of close apposition of the ER and the plasma membrane were found in T cells (42-44) and may provide the structural platforms for direct Ca2+ entry into the store (Fig. 5).

We have shown that (-)-XeC or Ry specifically block Ca2+ release from the IP3R or RyR, demonstrating that signaling through the respective intracellular Ca2+ release channel may be selectively abolished in T cells. Using these blockers we established that in the presence of the extracellular Ca2+ and activated SOC channels, the Ca2+ release from either IP3R or RyR may account for at least 25-45% of global [Ca2+]i elevation. Thus, in combination, the Ca2+ release from IP3R and RyR may account for >70% of the sustained [Ca2+]i elevation following TCR stimulation. These numbers could be substantially underestimated as neither (-)-XeC nor Ry completely blocked the [Ca2+]i responses to IP3R or RyR agonists. These data are consistent with a previous report demonstrating that in parotid acinar cells maximal Ca2+ influx via SOC channels accounts for only a small fraction of the agonist-induced [Ca2+]i elevation in the presence of the extracellular Ca2+ compared with the Ca2+ release from the store (23). Thus, similarly to other cell types, plasmalemmal Ca2+ channels in T cells may serve for direct store refilling and for activation of intracellular Ca2+ channels but by themselves contribute little to [Ca2+]i elevation necessary for T cell activation.

Interestingly, activation of SOC channels in T cells was sufficient to trigger Ca2+ release from both IP3R and RyR without stimulation of the surface receptors (Fig. 2, E-H), and, therefore, in the absence of the endogenous agonists. Moreover, in our study activation of TCR with PHA did not activate Ca2+ release from the RyR (Fig. 3B), although up-regulation of the endogenous RyR agonist cADPR has been previously observed following TCR stimulation (13). This discrepancy may be due to the fact that formation of cADPR depends on the type of stimulus. Apparently, in our study PHA failed to produce robust elevation in cADPR and/or nicotinic acid adenine dinucleotide phosphate levels and consequent Ca2+ release from the RyR in the absence of extracellular Ca2+ influx. Nevertheless, SOC channel activation in the presence of extracellular Ca2+ was sufficient to trigger the Ca2+ release from the RyR, indicating that this is a primary mechanism for RyR activation in T cells. The production of RyR endogenous agonists cADPR and/or nicotinic acid adenine dinucleotide phosphate at stronger TCR stimulation may facilitate the opening of RyR to further sustain Ca2+ release.

Thus, taken together, our data demonstrated that activation of SOC channels directly stimulates Ca2+ release from both IP3R and RyR, which is a major determinant for [Ca2+]i elevation following TCR stimulation. We further speculate that the long-lasting store depletion via IP3R and/or RyR may be necessary to prevent inactivation of plasmalemmal SOC channels (Fig. 5). Our findings are essential because they emphasize the role of intracellular Ca2+ release channels in shaping [Ca2+]i signaling in T cells and identify IP3R and RyR as novel potential targets for immunosuppression. This is especially important in view of the absence of specific SOC channel blockers and availability of a number of the IP3R and RyR blockers, one of which (dantrolene) is clinically approved (40, 41).

Our study demonstrated that pharmacological inhibition of IP3R or RyR exerts profound effects on major functions of human T cells such as proliferation and IL-2 production (Fig. 4). These findings are compatible with previous reports that down-regulation of IP3R expression in Jurkat T cells (17) and RyR inhibition with Ry or dantrolene in murine T cells (19) suppressed TCR-mediated proliferation and IL-2 production. Thus, pharmacological blockers of IP3R or RyR may be used as powerful tools for immunosuppression.

The important question that emerged from this study is the nature of the relationship between Ca2+ release channels in the ER and plasmalemmal SOC channels. The subplasmalemmal localization of IP3R and RyR in Jurkat T cells (45) and the dependence of Ca2+ release via RyR and/or IP3R on SOC channel activation imply that the IP3R and/or RyR may be coupled to the plasmalemmal SOC channels. In muscle, interaction between the RyR and plasmalemmal Ca2+ channels occurs in highly specialized junctional regions where the sarcoplasmic reticulum is located in close apposition to the plasma membrane (46). In T cells, the functional SOC channels were found at the junctional sites of the ER with the plasma membrane (43, 44), implying that functional interactions between the SOC channels and IP3R and/or RyR may take place. We speculate that similarly to muscle cells (47), the interaction between SOC channels and IP3R and/or RyR may be mediated either by conformational changes of SOC channel that do not require extracellular Ca2+ entry or via CICR mechanism (Fig. 5). The existence of the physical coupling between SOC channels and IP3R and/or RyR in T cells, and whether SOC channel activation may trigger Ca2+ release from the intracellular channels in the absence of Ca2+ entry, remains to be determined.


   FOOTNOTES
 
* This work was supported by American Heart Association Grant-in-aid 0755086Y (to A. F. F.) and by Philip Morris USA Inc. and Philip Morris International (to A. F. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: 4138 Tupper Hall, One Shields Ave., Davis, CA 95616. Tel.: 530-754-4454; E-mail: affomina{at}ucdavis.edu.

2 The abbreviations used are: TCR, T cell receptor; [Ca2+]i, cytosolic Ca2+ concentration; SOCE, store-operated Ca2+ entry; CPA, cyclopiazonic acid; SERCA, sarco-endoplasmic reticulum Ca2+-ATPase; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Ry, ryanodine; RyR, Ry receptor; (-)-XeC, (-)-xestospongin C; cADPR, cyclic adenosine 5'-diphosphate-ribose; ER, endoplasmic reticulum; CICR, Ca2+-induced Ca2+ release; PHA, phytohaemagglutinin P; IL-2, interleukin-2; FBS, fetal bovine serum; PBS, phosphate-buffered saline; CFSE, carboxy fluoroscein succinimidyl ester. Back


   ACKNOWLEDGMENTS
 
We thank Pirooz Parsa for valuable technical assistance. We also thank Dr. Isaac Pessah, Dr. Michael Toney, and Dr. Irvin Segel for fruitful discussion of data and Anna Shyrokova and Erica Whitney for critical reading and comments on the manuscript. This investigation was conducted in part in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR17348-01 from the National Center for Research Resources, National Institutes of Health.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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