Modulation of Endoplasmic Reticulum Calcium Pump Expression during T Lymphocyte Activation*

Calcium mobilization from intracellular storage or- ganelles 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 lympho- cyte cell lines a combined treatment by the two drugs resulted in an approximately 90% decrease of the ex- pression of the calcium pump isoform recognized by the PLIM430 isoform-specific antibody, whereas the expres- sion of the SERCA 2b isoform was increased approximately 2-fold. Phorbol ester or ionomycin applied sepa- rately was ineffective. In Jurkat T cells the down-modulation of expression of the SERCA isoform recognized by the PLIM430 antibody appeared concom-itantly with the induction of interleukin-2 expression and could be inhibited by the immunosuppressant drug cyclosporine-A. These data indicate that T cell activa- tion 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 activa- tion and may open new avenues in the understanding of the plasticity

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 downmodulation 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 (IP 3 ). 1 The for-mation of diacylglycerol results in the activation of various cellular protein kinase C isoenzymes, and the binding of IP 3 to its receptor induces the release of calcium into the cytosol from intracellular calcium storage organelles (1)(2)(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)(6)(7)(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)(13)(14)(15)(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 SERCA PLIM430 (17,18). The biochemical characteristics and the intracellular localization of these two pump species are different (17)(18)(19)(20)(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.

EXPERIMENTAL PROCEDURES
Materials-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. Peroxidaseconjugated as well as fluorescein-conjugated anti-mouse IgG antibody * This work was supported by the Agence Nationale de Recherches sur le SIDA, France, by the Ministère de l'Education Nationale, de l'Enseignement Supérieur, de la Recherche et de l'Insertion Professionnelle (ACC 9), and by the Association pour la Recherche sur le Cancer, France. 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.
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% CO 2 . 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 Treatments-Exponentially growing cells were harvested by centrifugation, resuspended in fresh complete medium at a density of 2 ϫ 10 5 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 Me 2 So. The concentration of Me 2 So 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 10 7 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 ϫ 10 5 cells (20 l) were run on 7.5% alkaline Lä emmli-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). SERCA PLIM430 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 TCATCTTCCAGATCACAC-CGCT and GTCAAGACCAGAACATATC, which cover the region from base pairs 2861 to 3132 of the human sequence (48). SERCA PLIM430 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.

Down-regulation of SERCA PLIM430 Expression by PMA Plus
Ionomycin-JurE6 -1 cells were treated for 4 days with 0.5 M 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, SER-CA PLIM430 . 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 SER-CA PLIM430 (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 differ-  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/10 5 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/10 5 cells).
Time Course of SERCA PLIM430 Down-modulation-JurE6 -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), SERCA PLIM430 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. SERCA PLIM430 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.
Immunofluorescent Staining-SERCA PLIM430 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 SERCA PLIM430 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 SERCA PLIM430 (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). DISCUSSION 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 SER-CA PLIM430 calcium pump isoform is almost completely downmodulated, 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 SERCA PLIM430 . Similar results were obtained with Molt-4 and CCRF-CEM T lymphocytes as well (not shown).
The modulation of SERCA PLIM430 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)(34)(35)(36)(37). In this work the PMA plus ionomycin-induced IL-2 secretion and the modulation of SERCA 2b and SERCA PLIM430 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.
SERCA PLIM430 is believed to be a variant of SERCA 3, the expression of which is restricted to cells of hemopoietic origin (15)(16)(17)(18). This enzyme, at least in platelets, is specifically associated with the IP 3 mobilizable calcium pool (20,21,43). Lymphocyte activation as well as apoptosis are strictly dependent on the mobilization of the IP 3 -sensitive calcium pool (1, 8, 44 -46). The down-modulation of SERCA PLIM430 during activation may lead to decreased filling of this pool. Because the depletion of the IP 3 -sensitive calcium pool via IP 3 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, SERCA PLIM430 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 SERCA PLIM430 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-Nterminal 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.