(Received for publication, February 11, 1997, and in revised form, February 25, 1997)
From the U348 INSERM, Institut Fédératif de Recherche Circulation Lariboisière, Hôpital Lariboisière, 8, rue Guy Patin, 75475 Paris Cedex 10, France
Calcium mobilization from intracellular storage organelles is a key component of the second messenger system inducing cell activation. Calcium transport ATPases associated with intracellular calcium storage organelles play a major role in controlling this process by accumulating calcium from the cytosol into intracellular calcium pools. In this study the modulation of the expression of the sarco-endoplasmic reticulum calcium transport ATPase (SERCA) isoenzymes has been studied in lymphocytes undergoing phorbol myristate acetate and ionomycin-induced activation. In several T lymphocyte cell lines a combined treatment by the two drugs resulted in an approximately 90% decrease of the expression of the calcium pump isoform recognized by the PLIM430 isoform-specific antibody, whereas the expression of the SERCA 2b isoform was increased approximately 2-fold. Phorbol ester or ionomycin applied separately was ineffective. In Jurkat T cells the down-modulation of expression of the SERCA isoform recognized by the PLIM430 antibody appeared concomitantly with the induction of interleukin-2 expression and could be inhibited by the immunosuppressant drug cyclosporine-A. These data indicate that T cell activation induces a selective and cyclosporine-A-sensitive modulation of the expression of the SERCA calcium pump isoforms. This reflects a profound reorganization of the calcium homeostasis of T cells undergoing activation and may open new avenues in the understanding of the plasticity of the calcium homeostasis of differentiating cells and in the pharmacological modulation of lymphocyte function.
Calcium as a second messenger is a key component of the cellular
signaling network controlling lymphocyte function (Refs. 1-3 and
references therein). Activation of the T cell receptor complex and
associated coreceptors by antigen presenting cells leads to the
formation of two second messengers, diacylglycerol and inositol
1,4,5-trisphosphate (IP3).1 The
formation of diacylglycerol results in the activation of various
cellular protein kinase C isoenzymes, and the binding of
IP3 to its receptor induces the release of calcium into the cytosol from intracellular calcium storage organelles (1-3). Calcium
mobilization from the endoplasmic reticulum also provokes a calcium
influx across the plasma membrane (4-8). These events lead to the
activation of several inducible transcription factors such as NFB,
NFAT, or AP-1 (9). The induction of the transcription of
activation-associated genes, e.g. the gene coding for IL-2 (9, 10), or the
chain of the IL-2 receptor (11) leads to a profound
reorganization of the structure and function of the cell, resulting in
an activated phenotype.
Endoplasmic reticulum associated calcium pumps (SERCA enzymes) accumulate calcium ions from the cytosol by ATP-dependent active transport into endoplasmic reticulum-associated calcium storage organelles (8). Because the increase of cytosolic calcium concentration, as well as the depletion of intracellular calcium pools, generate several activatory signals (5-8), SERCA activity, by refilling intracellular calcium pools, represents an important control mechanism of cell activation.
The expression of SERCA isoenzymes is tissue-specific and developmentally regulated (12-16). Previously, we have shown that in human cell lines of hemopoietic origin, isoform-specific anti-SERCA monoclonal antibodies can detect two distinct enzyme species that are coexpressed in the same cells (17, 18). The IID8 antibody recognizes the SERCA 2b isoform at 100 kDa, whereas the PLIM430 antibody, obtained by immunizing with purified platelet internal membrane preparations (19), reacts with a distinct, 97-kDa pump species, temporarily designed as SERCAPLIM430 (17, 18). The biochemical characteristics and the intracellular localization of these two pump species are different (17-21), suggesting that they play functionally distinct roles within the same cell. To better elucidate the functional specialization of the coexpressed calcium pump isoforms, in the present work we investigated the modulation of the expression of these enzymes during in vitro lymphocyte activation, a process where significant changes of cell function, structure, and signaling occur.
Ionomycin and the IID8 anti-SERCA 2 monoclonal
antibody were purchased from BioMol (Plymouth Meeting, PA). PMA and
avidin-horseradish peroxidase conjugate were from Sigma. Cyclosporine-A
was purchased from Calbiochem. Biotin-conjugated anti-recombinant human
IL-2 receptor chain antibody and the IL-2 immunoassay kit
(Quantikine ELISA) were from R & D Systems Europe Ltd. and were used
according to the manufacturer's instructions. Peroxidase-conjugated as
well as fluorescein-conjugated anti-mouse IgG antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
The Jurkat-derived JurE6-1 clone (22), as well as the Molt-4 and the CCRF-CEM cell lines were obtained from ATCC (Rockville, MD). Cells were grown in RPMI 1640 medium with Glutamax-I supplemented with 10% heat-inactivated fetal calf serum and 2 mM glutamine (Life Technologies, Inc.) in a humidified cell culture incubator at 37 °C in an atmosphere of 95% air and 5% CO2. Glutamax-I and glutamine were used in combination to compensate for the increased metabolic requirements of the cells due to the experimentally induced increase of calcium permeability.
Cell TreatmentsExponentially growing cells were harvested by centrifugation, resuspended in fresh complete medium at a density of 2 × 105 cells/ml and placed in 9-cm-diameter Petri dishes. Cells were then placed for 1 h in the cell culture incubator before stimulation. Drugs were then added from concentrated stock solutions in Me2So. The concentration of Me2So did not exceed 0.1%, was included in control experiments, and did not interfere with the assays. Cyclosporine-A was added 1 h prior to PMA or ionomycin treatment.
Following the treatments for the time periods indicated on the figures, cell counts and viabilities were determined by the trypan blue exclusion method, and the cells were harvested by centrifugation. The supernatant was saved for IL-2 determination, and the cells were resuspended in ice-cold phosphate-buffered saline, spun down in microcentrifuge tubes, and immediately frozen as a pellet on dry ice. The cell pellets were thawed at a density of 107 cells/ml in a lysis buffer containing 62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 5 mM EDTA, 100 mM dithiothreitol, 2 M urea, and 0.02% bromphenol blue (23). Cells were homogenized by aspiration (15-20 strokes) using a 2-ml Hamilton syringe.
Samples containing the lysates of 2 × 105 cells (20 µl) were run on 7.5% alkaline Läemmli-type polyacrylamide gels and transferred onto nitrocellulose membranes. Saturation of nitrocellulose and immunostaining was performed in a buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 5% dry milk, and 0.1% Tween 20 as described previously (24). Luminescent signal was generated and detected using the Enhanced Chemiluminescence system (ECL) of Amersham. Luminograms were scanned and quantitated using an LKB Ultroscan XL Laser Densitometer. In control experiments we determined that the conditions used for immunostaining and detection gave signals proportional to the amount of SERCA protein loaded on the gels, and thus these conditions were suitable for the quantitative detection of SERCA protein in whole cell lysates. Immunofluorescent staining was performed on acetone-fixed cells as described previously (25).
SERCAPLIM430 and SERCA 2b mRNA levels were estimated using a semiquantitative reverse transcriptase-PCR method described previously (48). Briefly, total RNA from cells treated for 4 days with PMA plus ionomycin or vehicle control was reverse transcribed and amplified using the Perkin-Elmer GeneAmp RNA PCR kit and Taq DNA polymerase according to the manufacturers instructions by 30 cycles (each cycle consisting of 30 s at 94 °C, 2 min at 55 °C, and 2 min at 72 °C). The primers used to amplify SERCA 2b were TCATCTTCCAGATCACACCGCT and GTCAAGACCAGAACATATC, which cover the region from base pairs 2861 to 3132 of the human sequence (48). SERCAPLIM430 was amplified using the primers GAGTCACGCTTCCCCACCACC and TCAACTTCTGGCTCATTTCTT, which cover the region located between base pairs 2674 and 3000 (15). Data presented in this paper represent the results of at least four independent experiments.
JurE6-1 cells were treated for 4 days with 0.5 µM ionomycin, 10 nM PMA, or a combination of
the two drugs. Whole cell lysates were electrophoresed and
immunoblotted using the SERCA isoform-specific discriminating
antibodies IID8 and PLIM430. IL-2 secretion by the cells was
quantitated by ELISA. In accordance with previous studies (17, 18),
IID8 selectively recognized the SERCA 2b isoform at 100 kDa, whereas
PLIM430 stained the 97-kDa pump isoform, SERCAPLIM430. As
shown on Fig. 1, untreated cells expressed similar amounts of the two pump isoforms (Fig. 1A, lanes
1 and 5), whereas a combined treatment with the two
drugs resulted in an approximately 90% decrease of the expression of
SERCAPLIM430 (Fig. 1, A, lane 2, and
B) and an approximately 2-fold increase of the expression of
SERCA 2b (Fig. 1, A, lane 6, and B).
Ionomycin or PMA, when applied alone, did not modify significantly the
expression of either calcium pump isoform in this system (Fig. 1,
A, lanes 3, 4, 7, and
8, and B). The differential modulation of the
expression of the two pump species was also manifest on the mRNA
level. As shown on Fig. 1C, treatment of JurE6-1 cells by
PMA plus ionomycin resulted in an approximately 2-fold decrease of
SERCAPLIM430 mRNA (lane 3, untreated;
lane 4, treated), whereas in the same cells SERCA 2b
mRNA was increased approximately 2-fold (lanes 1 and 2).
Induction of Interleukin-2 Synthesis
Supernatants of JurE6-1 cells treated as above were collected, and their IL-2 content was quantitated by ELISA. In accordance with previous data (22, 26-30), a combined treatment by PMA together with ionomycin resulted in a marked induction of IL-2 synthesis by the cells (1.2 ng/105 cells at day 4). In accordance with data in the literature (22, 26, 29, 30), PMA or ionomycin, when applied alone, did not induce detectable IL-2 synthesis (i.e. less than 3 pg/105 cells).
Time Course of SERCAPLIM430 Down-modulationJurE6-1 cells were treated for various time
periods with PMA together with ionomycin, and SERCA expression was
determined by immunoblotting. As shown on Fig. 2
(A and B), SERCAPLIM430 expression
started to decline at hour 9 after treatment. The induction of SERCA 2b
expression followed a somewhat slower time course, being manifest
starting at day 2 (Fig. 2, A and C).
The state of activation of the cells was monitored in parallel by
immunostaining for the chain of the IL-2 receptor and by measuring
IL-2 secretion, two established markers of T lymphocyte activation.
SERCAPLIM430 down-regulation and the induction of IL-2
synthesis (Fig. 2E) followed a similar time course, whereas the expression of the
chain of the IL-2 receptor expression appeared somewhat delayed (Fig. 2, A and D). In
accordance with previous data (31), PMA plus ionomycin treatment
resulted in growth arrest of Jurkat cells with maintained
viability.
SERCAPLIM430
down-regulation by PMA together with ionomycin was also manifested at
the single cell level. In untreated cells a granular staining was seen
for both SERCA isoforms in the cytoplasmic space, corresponding to the
endoplasmic reticulum (Fig. 3, A and C). Although the fluorescence signal for SERCA 2b was not
detectably modified by a treatment with PMA plus ionomycin (Fig.
3B), a marked decrease of staining for
SERCAPLIM430 could be seen in PMA plus ionomycin-treated
cells (Fig. 3D).
Effect of the Immunosuppressant Cyclosporine-A
JurE6-1 cells
were preincubated with various concentrations of cyclosporine-A for
1 h and then treated for 4 days with PMA and ionomycin. As shown
on Fig. 4, cyclosporine-A in the submicromolar range
abolished in a concentration-dependent manner the
modulation of the expression of SERCAPLIM430 (Fig.
4A) and of SERCA 2b (Fig. 4B) as well as IL-2
synthesis (Fig. 4C) induced by PMA plus ionomycin. Cyclosporine-A applied alone had no effect on SERCA expression (not
shown).
Second messenger mediated calcium mobilization and protein kinase
C activation are key events in lymphocyte signaling. In this work these
signals were induced simultaneously by the combined treatment of cells
by ionomycin and PMA. This technique is widely employed in the
literature to obtain T cell activation and induces IL-2 synthesis and
the expression of the chain of the IL-2 receptor (22, 26-30, 32,
33).
As shown in the present work, such a treatment results in the isoform-specific modulation of the expression of the SERCA enzymes expressed in Jurkat T lymphoblastoid cells. The SERCAPLIM430 calcium pump isoform is almost completely down-modulated, whereas the expression of SERCA 2b is increased 2-fold in the same cells. This results in a more than 15-fold overall increase in the relative ratio of the expression of SERCA 2b versus SERCAPLIM430. Similar results were obtained with Molt-4 and CCRF-CEM T lymphocytes as well (not shown).
The modulation of SERCAPLIM430 expression in Jurkat cells
occurred in parallel with the induction of IL-2 secretion and preceded the induction of the expression of the chain of the IL-2 receptor. Similarly to the induction of IL-2 expression (22, 26), the effect on
SERCA expression was strictly dependent on the simultaneous presence of
both drugs, because PMA or ionomycin, when applied alone, was without
effect either on SERCA expression or on IL-2 expression.
Cyclosporine-A is a major, clinically used immunosuppressant that forms trimeric complexes with cyclophyllins and the calcium-calmodulin-dependent serin-threonin phosphatase, calcineurin. The formation of such a ternary complex leads to the inhibition of calcineurin enzyme activity and impaired signal transduction to the nucleus by NFAT (33-37). In this work the PMA plus ionomycin-induced IL-2 secretion and the modulation of SERCA 2b and SERCAPLIM430 expression were abolished by cyclosporine-A in the submicromolar range. This suggests that calcineurin-dependent signaling, a key component in T cell activation, is involved in the control of the expression of SERCA enzymes in lymphocytes.
It has been shown earlier that lymphocyte activation produces modifications of the calcium storage and release characteristics of the cell (38) and that SERCA enzyme activity is involved in the control of cell proliferation and of lymphocyte activation (39-42). However, the role and the modulation of the expression of the various SERCA isoforms, coexpressed in the cell, have not been previously investigated. The data presented in this work indicate that striking differences exist in the regulation of expression of the SERCA isoforms during lymphocyte activation. This phenomenon probably reflects that various SERCA isoforms play functionally distinct roles and are associated with functionally distinct subcompartments of the endoplasmic reticulum.
SERCAPLIM430 is believed to be a variant of SERCA 3, the expression of which is restricted to cells of hemopoietic origin (15-18). This enzyme, at least in platelets, is specifically associated with the IP3 mobilizable calcium pool (20, 21, 43). Lymphocyte activation as well as apoptosis are strictly dependent on the mobilization of the IP3-sensitive calcium pool (1, 8, 44-46). The down-modulation of SERCAPLIM430 during activation may lead to decreased filling of this pool. Because the depletion of the IP3-sensitive calcium pool via IP3 induced calcium release (2, 6, 7) or by direct inhibition of SERCA activity by drugs such as thapsigargin (39, 40) is known to result in the generation of activatory signals, SERCAPLIM430 down-modulation may contribute to the maintenance of the activated state or may alter the apoptotic potential of the cell.
The observation, shown in this paper, that SERCAPLIM430 and
SERCA 2b mRNA levels are differentially modulated by PMA plus ionomycin suggests that transcriptional regulation may be involved in
the control of SERCA expression during lymphocyte activation. Indeed,
simultaneous calcium mobilization and protein kinase C activation sets
in motion a complex intracellular signaling network including
calmodulin-dependent protein kinases and calcineurin (49),
as well as the Jun-N-terminal kinase pathway (47). This leads to the
modulation of the activity of several transcription factors such as
NFB, NFAT or AP-1 (9), resulting in a complex set of modifications
of gene expression, including, as shown in this work, intracellular
calcium pump isoenzymes. The use of specific inhibitors of the
different key factors of this complex signaling system and experiments
addressing SERCA gene transcription and mRNA stability will permit
a more detailed analysis of the mechanisms involved in the regulation
of SERCA expression.
The plasticity of expression of the various SERCA isoforms upon cell activation represents a previously unrecognized level of complexity of lymphocyte calcium homeostasis and shows that different SERCA isoforms and therefore presumably different calcium pools may play distinct roles in cell activation. This finding points at the dinamic nature of the structure and function of calcium homeostatic systems of differentiating cells and may open new perspectives in the understanding of intracellular calcium homeostasis and in the pharmacological control of lymphocyte function.
This work is dedicated to A. Tarkovsky.
We are grateful to Dr. Agnes Enyedi, Dr. Balàzs Sarkadi, Dr. Anna Berardi, and Dr. Ali Saïb for helpful discussions. The help of Dr. Jacques Maclouf with the IL-2 ELISA experiments is acknowledged. We express special thanks to Prof. Neville Crawford for giving us the PL/IM430 hybridoma.