Potentiation of the Store-Operated Calcium Entry (SOCE ... · Potentiation of the Store-Operated Calcium Entry (SOCE) induces phytohemagglutinin-activated Jurkat T cell apoptosis

Accepted Manuscript

Title: Potentiation of the Store-Operated Calcium Entry(SOCE) induces phytohemagglutinin-activated Jurkat T cellapoptosis

Author: Alaeddine Djillani Isabelle Doignon Tomas LuytenBouchaib Lamkhioued Sophie C. Gangloff Jan B. ParysOliver Nu�e Christine Chomienne Olivier Dellis

PII: S0143-4160(15)00068-8DOI: http://dx.doi.org/doi:10.1016/j.ceca.2015.04.005Reference: YCECA 1673

To appear in: Cell Calcium

Received date: 9-12-2014Revised date: 14-4-2015Accepted date: 16-4-2015

Please cite this article as: A. Djillani, I. Doignon, T. Luyten, B. Lamkhioued, S.C.Gangloff, J.B. Parys, O. Nurmbetae, C. Chomienne, O. Dellis, Potentiation of theStore-Operated Calcium Entry (SOCE) induces phytohemagglutinin-activated Jurkat Tcell apoptosis, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.04.005

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

http://dx.doi.org/doi:10.1016/j.ceca.2015.04.005

http://dx.doi.org/10.1016/j.ceca.2015.04.005

Page 1 of 43

Accep

ted

Man

uscr

ipt

*Graphical Abstract

Page 2 of 43

Accep

ted

Man

uscr

ipt

A new store-operated Ca2+ entry potentiating agent

1

HIGHLIGHTS

MDEB potentiates store-operated Ca2+ entry in various cancer cell lines

The action of MDEB is more specific than that of the generally used inhibitor 2-APB

MDEB has a strong stimulatory effect of PHA-mediated Ca2+ increase in Jurkat cells

MDEB increases apoptosis in fully activated Jurkat cells

MDEB may represent the first member of a new family of immunomodulators

Page 3 of 43

Accep

ted

Man

uscr

ipt


2

Potentiation of the Store-Operated Calcium Entry (SOCE) induces

phytohemagglutinin-activated Jurkat T cell apoptosis

Alaeddine Djillani1,2, Isabelle Doignon2,1, Tomas Luyten3, Bouchaib Lamkhioued4, Sophie C.

Gangloff4, Jan B. Parys3, Oliver Nüβe1,2, Christine Chomienne5 and Olivier Dellis1,2

1 Univ Paris-Sud, Orsay, France2 UMR-S 757 INSERM, Orsay, France3 KU Leuven, Lab. Mol. Cell. Signaling, Leuven, Belgium4 EA 4691, Université de Reims Champagne Ardenne Reims, France5 UMR-S 1131 INSERM, Paris, France,

Corresponding author:

Dr Olivier DELLIS

University Paris Sud 11 / UMR-S 1174 INSERM

rue des Adèles

91405 Orsay

FRANCE

Phone: 33.1.69.15.49.59

Fax: 33.1.69.15.58.93

e-mail address: [email protected]

Keywords: Calcium influx, T-cell, Apoptosis, Store-operated calcium entry, Potentiation

Abbreviations: 2-APB, 2-aminoethyl diphenylborinate; 2-ABB, 2-aminoethyl

dibutylborinate; BC, Breast cancer; ER, endoplasmic reticulum; [Ca2+]cyt, cytosolic calcium

concentration; ∆[Ca2+]cyt, change in [Ca2+]cyt; CRAC, Calcium-Release Activated Calcium;

IL-2; interleukin-2; IP3; inositol 1,4,5-triphosphate; IP3R, inositol 1,4,5-triphosphate; MDEB,

Methyl diethylborinate; NCX, sodium-calcium exchanger; PMCA, plasma-membrane Ca2+

ATPase; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase; SOCE, Store-Operated

Calcium Entry; TG, thapsigargin; STIM, stromal interaction molecule; TRP, Transient

Receptor Potential.

Page 4 of 43

Accep

ted

Man

uscr

ipt


3

HIGHLIGHTS

MDEB potentiates store-operated Ca2+ entry in various cancer cell lines

The action of MDEB is more specific than that of the generally used inhibitor 2-APB

MDEB has a strong stimulatory effect of PHA-mediated Ca2+ increase in Jurkat cells

MDEB increases apoptosis in fully activated Jurkat cells

MDEB may represent the first member of a new family of immunomodulators

ABSTRACT

Store-operated Ca2+ entry (SOCE) is the main Ca2+ entry pathway of non-excitable

cells. In the past decade, the activation of this entry has been unveiled, with STIM1, a protein

of the endoplasmic reticulum able to sense the intraluminal Ca2+ content, and Orai1, the pore-

forming unit of the Ca2+ release activated Ca2+ (CRAC) channels. When Ca2+ ions are

released from the endoplasmic reticulum, STIM1 proteins oligomerize and directly interact

with Orai1 proteins, allowing the opening of the CRAC channels and a massive Ca2+ ion

influx known as SOCE. As Ca2+ is involved in various cellular processes, the discovery of

new drugs acting on the SOCE should be of interest to control the cell activity. By testing

analogues of 2-aminoethyl diphenylborinate (2-APB), a well known, though not so selective

effector of the SOCE, we identified methoxy diethylborinate (MDEB), a molecule able to

potentiate the SOCE in three leukocyte and two breast cancer cell lines by increasing the Ca2+

influx amplitude. Unlike 2-APB, MDEB does not affect the Ca2+ pumps or the Ca2+ release

from the endoplasmic reticulum. MDEB could therefore represent the first member of a new

group of molecules, specifically able to potentiate SOCE. Although not toxic for non-

activated Jurkat T cells, it could induce the apoptosis of phytohemagglutinin-stimulated cells.

Page 5 of 43

Accep

ted

Man

uscr

ipt


4

1. INTRODUCTION

Ca2+ signalling is involved in various cellular processes like activation, proliferation

and migration [1]. Due to this key role in cell physiology, the discovery of drugs acting on

Ca2+ signalling is of major interest. Thus, cyclosporine and FK-506 are usually used in

treatment after engraftment to inhibit calcineurin, a Ca2+-dependent phosphatase which is

absolutely required during T cell activation [2]. Upstream the Ca2+ signalling are the various

Ca2+ channels, and the control of channel activity by drugs is a common theme in

pharmacology.

When T cells are stimulated via the T cell receptor, inositol 1,4,5-trisphosphate (IP3)

is synthesised which induces, after the activation of the IP3 receptor (IP3R), the release of

Ca2+ ions from the endoplasmic reticulum (ER). This release causes in turn the opening of the

Ca2+ release activated Ca2+ channels (CRAC channels) [3] of the plasma membrane, allowing

a massive Ca2+ influx known as store-operated Ca2+ entry (SOCE), which is necessary for the

T cell activation [4]. Thus, a sustained increase of the resting cytosolic calcium concentration

([Ca2+]cyt) must persist for at least 2 hours to induce the full T cell commitment [5-7]. SOCE

channels have been first characterized on mast cells and T lymphocytes in the early 90s [8,

9]. Recently, the key proteins responsible for the SOCE have been identified with STIM1

(stromal interaction molecule 1) acting as a Ca2+ sensor in the ER, and Orai1 representing the

plasma membrane resident CRAC channel pore [10, 11]. It has been shown that after cell

stimulation and Ca2+ release by the ER, STIM1 proteins oligomerize and translocate in the

ER toward the plasma membrane where they physically interact with Orai1 channels to allow

their opening whereby the resulting Ca2+ influx is instrumental to sustain the increase of the

cytosolic Ca2+ concentration ([Ca2+]cyt) [12].

Mutations of STIM1 or Orai1 genes, responsible for the absence or deficiency of the

corresponding protein, induce a severe combined immunodeficiency (SCID) phenotype: T

cells are well developed, but cannot be activated [11]. There exist two Orai1 paralogues,

Orai2 and Orai3. The three Orai proteins can elicit SOCE when expressed in a heterologous

system [13-15]. However, only Orai1 expression can fully correct the SCID phenotype in

Orai1-deficient cells [13], highlighting the key role of Orai1 in lymphocyte activation,

whereas Orai2 and Orai3 functions are still to be elucidated. However, it has recently been

proposed that Orai2 and Orai3 channels are the channels responsible for the native SOCE in

dendritic cells and estrogen receptor positive breast cancer cells respectively [16, 17].

Furthermore, the arachidonate-regulated Ca2+ channel is made of a Orai1/Orai3 mixed

proteins [18].

Page 6 of 43

Accep

ted

Man

uscr

ipt


5

Due to its key role, it is of interest to develop effectors of the SOCE that could

represent new immunomodulators or a new way to cure cancers as e.g. leukemias. Econazole

and ketotifen, two compounds known to block the SOCE have already been described to

purge bone marrow of leukemia cells suggesting a higher sensitivity of these cells to SOCE

inhibitors [19].

The aim of this study therefore was to develop new effectors of SOCE. Interestingly,

2-aminoethyl diphenylborinate (2-APB, Fig. 1), commonly used to inhibit SOCE, seems to be

the most promising molecule, as it has a dual effect on SOCE: potentiating at 1-5 µM and

inhibiting at > 30 µM [20]. Unfortunately 2-APB lacks specificity as it can also affect the

function of various other Ca2+-transporting mechanisms. Depending on the concentration, 2-

APB can inhibit IP3Rs, sarco- and endoplasmic Ca2+ ATPase (SERCA) pumps, as well as

some transient receptor potential (TRP) channels of the TRPC family [21-25] while opening

some members of the TRPV family [26, 27]. A few teams have already documented the

synthesis of 2-APB analogues, but only with the purpose of developing more specific

inhibitors [28-32].

In an attempt to determine which part of the 2-APB molecule is needed for the SOCE

potentiating and/or inhibiting ability, we previously recorded SOCE in three leukemia cell

lines in the presence of 2-APB or of several analogues and reported that the boron-oxygen

core (BOC) of the molecule is necessary for the potentiating capacity [33]. However, when

the free rotation of the two phenyl groups is blocked or when they are replaced by larger

groups like benzothienyl groups, Ca2+ influx potentiation is impaired [34]. A drawback was

that all the new potentiating molecules we identified were still inhibitory at high

concentrations [33].

In this new study, we therefore focussed our interest on a new 2-APB analogue,

methyl diethylborinate (MDEB, Fig. 1), which almost only contains the BOC. MDEB is the

first molecule only able to potentiate the SOCE, without any inhibitory action, and therefore

could represent the first member of a promising family. Indeed, when used alone, MDEB

does not show any effect on Jurkat cell viability. However MDEB inhibits the synthesis of

interleukin-2 by partially activated cells and also induces the apoptosis of fully activated

Jurkat T cells. Thus, it appears that MDEB could specifically eliminate fully activated cells.

2. MATERIAL AND METHODS

2.1. Cells

Page 7 of 43

Accep

ted

Man

uscr

ipt


6

Jurkat (acute T cell leukemia), BL-41 (Burkitt B lymphoma), U937 (monocytic cell

line), MDA-MB231 and BT-20, two breast carcinoma (BC) cell lines, were from ATCC.

Leukocyte cell lines were basically maintained in RPMI-1640 medium (Lonza, Verviers,

Belgium) supplemented with 10 % heat-inactivated fetal calf serum and 2 mM L-glutamine.

BC cell lines were maintained in DMEM medium (Lonza, Verviers, Belgium) supplemented

with 10 % heat-inactivated fetal calf serum and 2 mM L-glutamine. L15 fibroblasts stably

overexpressing IP3R1 [35] were a generous gift of Pr Mikoshiba (RIKEN Brain Science

Institute, Japan). They were maintained in DMEM medium (Gibco®, Life Technologies,

Gent, Belgium) supplemented with 10 % heat-inactivated fetal calf serum, 3.8 mM L-

glutamine, 1% non-essential amino acids, 85 IU ml-1 penicillin, 85 g ml-1 streptomycin, 400

g ml-1 geneticin and 25 mM Na-Hepes (pH 7.4). Epstein-Barr Virus immortalized B cells

from Orai1-deficient and healthy patients were a kind gift of Dr Picard and Pr Fischer (Study

center of primary immunodeficiencies, AP-HP, Hôpital Necker, Paris, France). Written

informed consent was obtained from the parents of the patients. The experiments were

conducted after approval was given by the institutional review boards at Necker–Enfants

Malades Hospital (Paris, France). All cell lines were maintained at 37 °C in a 5% CO2

humidified atmosphere, except for L15 fibroblasts which were maintained at 10% CO2.

2.2. Measurement of cytosolic Ca2+ concentration ([Ca2+]cyt)

[Ca2+]cyt was recorded by a fluorimetric ratio technique [36, 37]. Leukocytes were

spun and resuspended at a density of 106 cells/ml in Phosphate-Buffered Saline (PBS)

supplemented with 1 mg/ml bovine serum albumin and incubated in the dark with 4 μM

Fura-2-AM for one hour at room temperature under slow agitation. Cells were then

centrifuged and resuspended in Ca2+-free Hepes Buffered Saline solution (HBS; 135 mM

NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 11.6 mM Hepes, 11.5 mM glucose adjusted to pH 7.3

with NaOH) prior to measurement. After centrifugation, 0.5 to 1 x 106 cells were suspended

in 2 ml HBS in a quartz cuvette and inserted into a spectrofluorophotometer (RF-1501

Shimadzu Corporation, Kyoto, Japan) connected to a PC computer (Dell Computer Corp.,

Montpellier, France). BC cells were grown on glass coverslips, and prior experiments, rinsed

with PBS. The coverslips were incubated for 30 minutes with 1 mM Ca2+-containing HBS

supplemented with 4 µM Fura-2-AM. Prior to experiments, glass coverslips were inserted in

a 2 ml Ca2+-free HBS-containing cuvette. A temperature of 37°C was maintained by

Page 8 of 43

Accep

ted

Man

uscr

ipt


7

circulating water from a bath. Ultraviolet light of 340 and 380 nm were used for excitation of

Fura-2, and emission at 510 nm was recorded. Background and autofluorescence of the cell

suspension were subtracted from the recordings. The maximum Fura-2 fluorescence (Rmax)

was obtained by adding 1 μM ionomycin to the cell suspension in the presence of 10 mM

CaCl2. Minimum fluorescence was determined following depletion of external Ca2+ with 5

mM EGTA. [Ca2+]cyt was calculated according to the equation [Ca2+]cyt = Kd (R-Rmin)/(Rmax-

R), where Kd is the apparent dissociation constant of Fura-2-calcium complex (250 nM), and

R is the ratio of fluorescence values recorded at 510 nm after excitation at 340 and 380 nm

[36]. In a large number of experiments, cells placed into a quartz cuvette, were treated with 1

μM thapsigargin (TG) during 10 min in Ca2+-free HBS to induce Ca2+ release from the ER

and the opening of SOCE channels [37]. Then, 1 mM CaCl2 was added to measure the

change in [Ca2+]cyt subsequently to Ca2+ calcium influx [37]. At the end of each experiment, 1

µM GdCl3 was added to verify that MDEB only acts on the SOCE [17].

2.3. Measurement of SOCE channel activity via Mn2+ quenching

To study directly the divalent ion influx through the SOCE channels, we performed

Mn2+ quenching experiments. Mn2+ ions bind to Fura-2 and quench the 510 nm emission

wavelength fluorescence after excitation at 360 nm. The decrease of fluorescence is directly

dependent on the Mn2+ entry through SOCE channels. For measurements, cells were treated

for 10 min with 1 µM TG, whereupon 100 µM MnCl2 was added instead of CaCl2. To test the

effects of MDEB on the Ca2+ release from the ER, the Jurkat cells were stimulated with 10

µg/ml PHA instead of TG, still in the absence of extracellular Ca2+. PHA cross-links the T

cell receptor (TCR), inducing the synthesis of IP3 and Ca2+ release through the IP3Rs [37].

Cells were pre-treated for 5 min with MDEB and then stimulated with 10 µg/ml PHA. Values

are representative of at least 3 independent experiments.

2.4. Down-regulation of Orai1

MDA-MB231 cells were seeded in a 6 well-plate on day 1 (2.105 cells/well). At day 2,

cells were transfected with the Lipofectamine® RNAiMax (Life Technologies, SAS, Saint

Aubin, France) according to the supplier recommandations together with 100 nM of Orai1

siRNA or of a scrambled siRNA (FlexiTube, Qiagen, SAS. Courtaboeuf, France). After 3

days, cells were used for [Ca2+]cyt measurement (see 2.2) or for extraction of proteins. For

cellular protein extraction, cells were lysed using RIPA buffer (50 mM Tris-HCl pH8, 150

Page 9 of 43

Accep

ted

Man

uscr

ipt


8

mM NaCl, 1% Triton X100, 0.5% deoxycholate, 0.2% SDS and 0.2 mM EDTA)

supplemented with an antiprotease pellet (cOmplete Mini, Roche Diagnostics, Meylan,

France). SDS-PAGE electrophoresis (10%) was subsequently performed (50 µg

proteins/lane). Proteins from the gels were electrotransferred onto Hybond ECL membrane

(Amersham-GE Healthcare, Velizy-Villacoublay, France). After blocking with 5% non-fat

dry milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (“Tris buffer”) for 1h at

room temperature, blots were washed three times with Tris buffer and probed 3h with

specific primary antibody in 5 % non-fat dry milk in Tris buffer. The primary antibodies used

against Orai1 (1:1000) and actin (1:500) were from Sigma-Aldrich, Saint Quentin Fallavier,

France). The anti-actin antibody was used to verify that equal amounts were loaded in each

lane. Detection was performed using the enhanced chemiluminescence reagent (ECL Western

blotting detection reagents, Amersham-GE Healthcare). Quantification of the bands was

performed with the ImageJ software.

2.5. Patch-clamp measurements

Cells for patch-clamp experiments were allowed to adhere to a poly-D-ornithine-

coated Petri dish for 10 min. Each dish was then rinsed twice with the bath solution

containing 160 mM NaCl, 4.5 mM KCl, 10 mM CaCl2 (or 1 mM when indicated), 1.2 mM

MgCl2, 5 mM Hepes, adjusted to pH 7.4 with NaOH, and kept in this solution during the

complete experiment. Pipettes were pulled from borosilicate glass capillaries (GC150F, Clark

Electromedical Instruments, England). Membrane currents were recorded using a Heka EPC9

amplifier (Heka Elektronik, Lambrecht, Germany) interfaced to a PC computer (Dell

Computer Corp.). Current were sampled at 5 kHz and filtered at 2.9 kHz. Voltage-clamp

protocols were implemented and data analyzed using Pulse/Pulsefit softwares (Heka

Elektronik, Lambrecht, Germany). Data were corrected for the liquid junction potential. ICRAC

was mainly recorded in the whole-cell configuration using the following pipette solution: 140

mM Cs-aspartate, 2 mM MgCl2, 10 mM BAPTA (or EGTA when indicated), 10 mM Hepes,

pH adjusted to 7.2 with CsOH [37]. Typical resistance of the pipette was 10-15 M. The

chelation of Ca2+ ions by BAPTA / EGTA induced the passive depletion of the stores, and the

opening of the CRAC channels [37]. Just after reaching the whole-cell configuration, the

holding potential (VH) was set to 0 mV to minimize the basal Ca2+ influx. The Jurkat cells

have a capacitance of 6.6 ± 0.3 pF (n > 50). ICRAC was evoked by applying a 100 ms step to -

100 mV every 2 s; the current amplitude was calculated and expressed as current over cell

Page 10 of 43

Accep

ted

Man

uscr

ipt


9

capacitance. ICRAC time course was monitored over ~ 15-20 min time range by measuring

ICRAC amplitude 5 ms after pulse clamping every 2 s. At the end of all the experiments, 1 µM

GdCl3 was added to inhibit ICRAC. The remaining current was considered the leak current.

2.6. Unidirectional 45Ca2+ flux experiments

L15 fibroblasts were seeded in gelatin-coated 12-well plates (Greiner, Frickenhausen,

Germany) at a density of 60 000 cells per well. Experiments were carried out on confluent

cell monolayers, 7 days after seeding. 45Ca2+ unidirectional flux experiments were performed

at 30 C on saponin-permeabilized cells, essentially as described [38]. Permeabilization was

for 10 min in a solution containing 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 2 mM

MgCl2, 1 mM ATP, 1 mM EGTA and 40 µg ml-1 saponin. The non-mitochondrial Ca2+ stores

were subsequently loaded for 45 min in 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM

MgCl2, 5 mM ATP, 0.44 mM EGTA, 10 mM NaN3 and 150 nM free 45Ca2+ (28 μCi ml-1).

Efflux was initiated by incubation in efflux medium (120 mM KCl, 1 mM EGTA, 30 mM

imidazole-HCl pH 6.8) containing TG (10 µM) and the indicated concentrations of MDEB.

Every two minutes the medium was replaced and the Ca2+ release from the stores assessed.

At the indicated time point IP3 (0.7 µM) was added to the efflux medium for 2 min to elicit

IP3-induced Ca2+ release. To assess its potential effect on the ATP-driven Ca2+ uptake in the

stores, MDEB was added in separate experiments during the 45Ca2+-loading phase.

Alternatively, TG (2 M) was added during this phase to completely block SERCA activity.

By subtraction of the TG-insensitive Ca2+ uptake from the total uptake for the various

conditions [39], the active Ca2+ uptake in the ER was calculated and compared.

2.7. Interleukin-2 (IL-2) assay

48-well plates were seeded with 106 Jurkat T cells per well. Cells were then incubated

for 24 h with 0, 1 or 10 µg/ml PHA and 0 to 1 mM MDEB in complete culture medium. After

24 h, supernatants were collected and IL-2 amounts were quantified using a Quantikine

Human IL-2 Immunoassay (R&D Systems Europe, Lille, France). Optical density (OD) was

measured at 450 nm using a Victor3 plate reader (Perkin Elmer, Courtaboeuf, France). The

concentration of supernatant IL-2 was determined from the OD curve obtained with the

standard. Results were the mean of four repeats. In parallel plates, cell viability was accessed

after treatment with trypan blue by counting with a Malassez hematimeter.

Page 11 of 43

Accep

ted

Man

uscr

ipt


10

2.8. Apoptosis detection by TUNEL method

We measured the apoptosis levels in Jurkat cells by the "in situ cell death detection"

kit (Roche Applied Science, Meylan, France). Briefly, 50 000 cells/well were seeded in a 96-

well plate and treated with or without 10 µg/ml PHA and 1 mM MDEB for 6, 12 or 24 h.

Then according to the manufacturer’s instructions, cells were spun, fixed, permeabilized and

the Terminal transferase dUTP Nick End Labelling (TUNEL) reaction was performed during

60 min at 37°C in darkness. For visualization of results, an inverted epifluorescence

microscope (Axioskop, Karl Zeiss, Le Pecq, France) was used at an excitation wavelength of

488 nm, and an emission wavelength of 545 nm. Cells were also loaded with 4', 6' –

diamidino-2-phenylindole (DAPI) to visualize the nuclei. Total cell number and TUNEL-

positive cells were counted in several fields and the ratio of apoptotic cells was calculated.

This experiment was repeated three times.

2.9. Chemicals

2-aminoethoxydiphenyl borate (2-APB), 2-aminoethoxydibutyl borate (2-ABB),

methyl-diethylborinate (MDEB) and trimethylborate (TMB) were from Sigma-Aldrich (Saint

Quentin Fallavier, France), TG was purchased from Calbiochem-Merck (Nottingham, UK).

The structures of 2-APB and its analogues are given in Figure 1.

2.10. Statistical analysis

Given values are representative of at least 3 independent experiments and are given as

mean ± SEM. When used, a t-test < 0.05 is considered as significant.

3. RESULTS

3.1. MDEB has only SOCE potentiating capacity

To visualize the SOCE, we treated the Jurkat cells with 1 µM thapsigargin (TG) in

Ca2+-free HBS during 10 min to allow the release of Ca2+ ions from the ER and subsequently

Page 12 of 43

Accep

ted

Man

uscr

ipt


11

the opening of the SOCE channel (Fig. 2A, arrow i). Addition of 1 mM CaCl2 induced an

increase of [Ca2+]cyt due to the entry of Ca2+ ions through the SOCE channels (Fig. 2A).

In these conditions, a progressive increase of the MDEB concentration added 30s

prior CaCl2 (arrow ii) was accompanied by an increase of the peak [Ca2+]cyt, stabilizing at

MDEB concentrations 1 mM (Fig. 2A and B). Noteworthy, high MDEB concentrations

were devoid of any inhibition ability, allowing the calculation of an apparent potentiation

constant of 44 ± 11 µM (n = 5, Figs. 2A and B). MDEB potentiation ability peaked at 243 ±

20 % (n = 5) at a concentration of 1 mM (peak ∆[Ca2+]cyt = 2171 ± 78 nM vs 895 ± 34 nM in

the absence of MDEB, p < 0.01), followed at higher concentrations by a non-significantly

different plateau at 216 ± 19 % (n = 5, Fig. 2 B). This MDEB-induced increase of [Ca2+]cyt

was totally abolished by 1 µM GdCl3, showing that MDEB acted on SOCE (Fig. 2A, dashed

line). In view of this unexpected result, we used 2-APB and 2-ABB (Fig. 1) as controls. As

shown in Figure 2B, 2-APB and 2-ABB had their usual dual effects on Jurkat cell SOCE:

potentiation at 3 µM ( 220 %) and inhibition at a concentration > 50 µM for 2-APB

(respectively at 20 and at > 100 µM for 2-ABB). The potentiation amplitude of MDEB, 2-

APB and 2-ABB was however similar (respectively 243 ± 20, 218 ± 21 and 203 ± 17 %).

Trimethylborate (TMB, Fig. 1), a molecule similar to MDEB (the two ethyl groups

are replaced by two methoxyl groups) was devoid of any potentiation or inhibition capacity

(Fig. 2B).

As the [Ca2+]cyt represents the equilibrium between Ca2+ influx and Ca2+ efflux, it is

impossible to know directly whether the increase of [Ca2+]cyt is the result of an increase in

Ca2+ entry amplitude or a decrease in Ca2+ exit amplitude. To answer this question, we next

performed Fura-2 fluorescence quenching experiments with Mn2+ ions. Mn2+ ions enter the

cell through SOCE channels, but are not pumped back to the extracellular medium as they

inhibit the Na+/Ca2+ exchanger (NCX) and are not transported by the plasma membrane Ca2+-

ATPases (PMCA) [40-42]. As Mn2+ ions quench the fluorescence of the Fura-2, an

acceleration of the divalent ion entry is associated with an increase of the quenching rate. As

shown in Fig. 2C, after a 10 min TG stimulation of Jurkat cells, addition of 100 µM MnCl2

induced a clear decrease of the Fura-2 fluorescence (-0.24 ± 0.01 % F0/s, n = 4). The

subsequent addition of 1 mM MDEB induced an about 3-fold acceleration of the Fura-2

quenching rate (-0.69 ± 0.03 % F0/s, p<0.05, n = 4). Thus, MDEB is able to enhance the

divalent ion influx.

3.2. MDEB has a potentiating ability on SOCE of various leukocytes

Page 13 of 43

Accep

ted

Man

uscr

ipt


12

We next tested MDEB on two other leukocyte cell lines (BL-41, Burkitt B lymphoma

and U937, monocytic cells).

In the same conditions as for Jurkat cells, MDEB was also able to potentiate the

SOCE of the two other leukocyte cell lines with a non significantly different potentiation

constant of 58 ± 7 µM (n = 4) and 66 ± 12 µM (n = 5) respectively for BL-41 and U937 cell

lines vs 44 ± 11 µM for Jurkat cell SOCE (Fig. 3A). MDEB was slightly more powerful on

BL-41 and U937 cells than on Jurkat cells, increasing the amplitude of the [Ca2+]cyt

respectively to 328 ± 23 % (n = 4) and 282 ± 42 % (n = 5) vs. 243 ± 20 %.

3.3. MDEB only potentiates the SOCE when Orai1 is expressed

Although 2-APB has a dual effect on Orai1 channels, it only potentiates Orai3

channels [17, 43]. However, even if T cells express the three Orai isoforms, only the presence

of Orai1 is requested for the potentiating/inhibiting effects of 2-APB on SOCE [33]. To

verify that Orai1, and not the other Orai isoforms, is also the target of MDEB in physiological

conditions, we compared wild type B cell that express all 3 Orai isoforms and a B cell line

which only express Orai2 and Orai3 but not Orai1 [44]. TG treatment in absence of

extracellular Ca2+ ions clearly induced the same kind of Ca2+ release from the ER of B cells

expressing Orai1 ("B-Orai1 wt") or from Orai1-deficient B cells ("B-Orai1 deficient", new

Fig. 3B). However, after readdition of extracellular Ca2+ ions, the [Ca2+]cyt increased by ~ 900

nM (896 ± 58 nM, n = 5) in cells expressing Orai1. In contrast, with the same protocol,

extracellular Ca2+ readdition only induced a rise of 75 ± 18 nM (n = 5, Fig 3B) in Orai1-

deficient cells, which is significantly less than in cells expressing Orai1 (-92 %, p < 0.01).

When 1 mM MDEB was added 200s after Ca2+ ion readdition on Orai1-expressing cells, it

induced a rebound due to its potentiation ability on the SOCE (maximal increase to 1568 ±

142 nM, n = 5). In contrast, in cells lacking Orai1, MDEB did not induce any rebound (Fig.

3B).

To confirm this result, Mn2+-quenching experiments were performed on these cells.

Similarly to 2-APB [33], MDEB was able to increase the Mn2+-induced quenching of Fura-2

only in B cells expressing Orai1: -0.52 ± 0.01 % F0/s vs -0.35 ± 0.01 % F0/s (n = 5, p < 0.01)

in absence of MDEB (Fig. 3C). Mn2+ ions were not able to induce the Fura-2 quenching in

Orai1-deficient cells, and MDEB was in these cells without effect (Fig. 3C). The recordings

Page 14 of 43

Accep

ted

Man

uscr

ipt


13

appeared noisier in B cells lacking Orai1 (Fig 3C), probably due to a weaker loading of Fura-

2.

3.4 MDEB has the same potentiating ability on breast cancer cell SOCE

On both BC cell lines, by using the same protocol as for the leukocyte cell lines,

MDEB also potentiated SOCE but with higher affinity: 25 ± 7 µM (n = 4) and 14 ± 2 µM (n

= 4) respectively for the MDA-MB231 and BT-20 cell lines vs. 44 ± 11 nM for Jurkat cells (p

< 0.05, Fig. 4A). On the contrary, the amplitude of the SOCE increase was significantly

smaller than in the leukocyte cell lines: 162 ± 4 % (MDA-MB231 cells: 575 ± 24 nM vs. 390

± 15 nM without MDEB, n = 4) and 166 ± 3 % (BT-20 cells: 581 ± 30 nM vs. 346 ± 16 nM,

n = 4) vs. 243 ± 20 % (Jurkat cells, Fig. 4B, p < 0.01).

To verify that Orai1 is also the main protein allowing the SOCE in the BC cell lines,

we used siRNA targeted against Orai1 in MDA-MB231 cells. As shown in figure 4B, 100

nM siRNA abolished the expression of Orai1 proteins by 78 ± 6 % (n = 3).

TG treatment in absence of extracellular Ca2+ ions clearly induced the same kind of

Ca2+ release from the ER of Orai1-expressing MDA-MB231 cells ("control siRNA") or from

cells in which the expression of Orai1 was largely impaired ("Orai1 siRNA", Fig. 4C),

showing that important downregulation of Orai1 expression had no effect on the ER Ca2+ ion

content. After readdition of Ca2+ ions, the [Ca2+]cyt increased by ~ 500 nM (516 ± 20 nM, n =

5) in cells expressing Orai1. In contrast, using the same protocol, extracellular Ca2+

readdition induced only a rise of 118 ± 21 nM (n = 5, Fig 4C) in the cells in which Orai1 was

downregulated, which was significantly less than in cells expressing normal Orai1 levels (-77

%, p < 0.01). This result clearly shows that Orai1 is the main responsible for the SOCE in

MDA-MB231 cells. When 1 mM MDEB was added 200s after Ca2+ readdition on Orai1-

expressing cells, it clearly induced a rebound due to its potentiation ability on the MDA-

MB231 cell SOCE (686 ± 30 nM, n = 5) but was devoid of any significant effect on cells

with a decreased expression of Orai1 (Fig 4C).

Taken together, the results indicate that in both leukocyte and BC cell lines, MDEB

had only a potentiating capacity on the SOCE when Orai1 is expressed.

3.4. Competition between 2-APB/MDEB and between 2-ABB/MDEB on Jurkat cell SOCE

Page 15 of 43

Accep

ted

Man

uscr

ipt


14

As 2-APB and MDEB have similar structures, we hypothesize that they bind to the

same site(s), and therefore performed competition experiments between the two molecules.

For this purpose we used a concentration of 100 µM for all compounds. At this concentration,

2-APB inhibits ~ 95% of the peak [Ca2+]cyt increase ("∆[Ca2+]cyt",,45 ± 9 nM vs. 1049 ± 21

nM, n = 5, p < 0.01, Fig. 5A, green line), while MDEB potentiates this increase (2075 ± 49

nM vs. 1049 ± 21 nM, n = 5, p < 0.01, Fig. 5B, cyan line).

As clearly shown in Figure 5A, MDEB was not able to potentiate the SOCE in

presence of 2-APB (both compounds were added at the same time, arrow ii, green line vs. red

line, (∆[Ca2+]cyt = 60 ± 9 nM , n = 5, not significantly different to the measured ∆[Ca2+]cyt

with 2-APB alone). Furthermore, when 2-APB had totally impaired the Ca2+ entry, MDEB

could not reverse the inhibition (orange line, MDEB was added at t = 630 s, arrow iii,

∆[Ca2+]cyt = 63 ± 10 nM , n = 5, not significantly different to the measured ∆[Ca2+]cyt with 2-

APB alone).

In the parallel experiments (Fig. 5B), we treated the cells with MDEB prior using 2-

APB. Clearly, either when both compounds were added together (Fig. 4B, red line) or even

when 2-APB was added (arrow iii) after potentiation with MDEB (∆[Ca2+]cyt = 76 ± 8 nM , n

= 5, not significantly different to the measured ∆[Ca2+]cyt with 2-APB alone), 2-APB totally

inhibited the Ca2+ entry (Fig. 5B, velvet line vs. cyan line). These results implied that at

equimolar concentrations, inhibition by 2-APB was stronger than potentiation by MDEB.

In a previous work, we showed that the removal of the two phenyl rings of the 2-APB

molecule, which leads to 2-ABB, did not impair its potentiation/inhibition capacity, but

decreased the sensitivity of the SOCE to 2-ABB [33].

As shown in Figure 5C, 100 µM 2-ABB almost totally inhibited the SOCE by ~ 90%

(∆[Ca2+]cyt = 107 ± 7 nM vs. 1049 ± 21 nM, n = 5, green line). However, when MDEB was

added simultaneously with 2-ABB, only an inhibition of 20 % was observed (∆[Ca2+]cyt =

225 ± 17 nM vs. 1049 ± 21 nM, n = 5, p < 0.01, red line). Thus MDEB restored 10 % of

the Δ[Ca2+]cyt. Similarly, when MDEB was added after 2-ABB had fully exerted its inhibitory

ability, it was able to partially counteract the inhibition (∆[Ca2+]cyt = 184 ± 15 nM vs. 1049 ±

21 nM, n = 5, p < 0.01, orange line).

Also, when 2-ABB was added after MDEB exerted its potentiating effect, the SOCE

was only partially inhibited (∆[Ca2+]cyt = 236 ± 24 nM, n =5, velvet line vs. cyan line, Fig.

5D). These results showed that MDEB could, although only partially, counteract the

inhibition of the SOCE by the less potent 2-ABB, but not by the more potent 2-APB.

Page 16 of 43

Accep

ted

Man

uscr

ipt


15

We next performed experiments where increasing concentrations of MDEB (1 µM to

30 mM) were applied 30s prior Ca2+ with various inhibiting concentrations of 2-APB (20, 30,

50 and 100 µM, Fig 5E). The potentiating effect of MDEB was totally abolished by 50 or 100

M 2-APB even at the highest concentration (30 mM). However at lower concentrations of 2-

APB (20 and 30 µM), MDEB could partly counteract the inhibition due to 2-APB. In the

presence of 30 mM MDEB + 20 µM 2-APB, 46 ± 4 % (n = 4) of the control ∆[Ca2+]cyt was

observed vs. 6 ± 1 % with 20 µM 2-APB alone (n = 4, p < 0.01). This value was still however

far from the potentiation capacity of 30 mM MDEB alone (212 ± 8%, n = 4, p < 0.01). These

results confirm that the inhibition by 2-APB was stronger than the potentiation by MDEB.

3.5. MDEB increases Jurkat cell ICRAC amplitude

ICRAC is the plasma membrane Ca2+ current activated by passive depletion of the

internal stores [37]. Upon break-in with the pipette solution containing 10 mM BAPTA,

hyperpolarization of the Jurkat cell membrane evoked a weak inward current in the absence

of external Ca2+ ions (Fig. 6A). Addition of 1 or 10 mM extracellular CaCl2 induced the

development of an inward current (Fig. 6A), which was inhibited by 1 µM GdCl3 (Fig. 6B

and C), 1 mM CdCl2 or 5 mM NiCl2 (data not shown) and showed a fast inhibition during the

100 ms pulse. The current/voltage relationships revealed an inwardly rectifying current with a

reversal potential at a positive voltage (Fig. 6D). The time course of the peak current showed

that after an increase of the current amplitude, there was a slow inactivation (first 6 min of

Fig. 6B): furthermore, the high affinity chelating agent BAPTA allowed the development of a

larger Ca2+ current than the low affinity chelating agent EGTA (Fig. 6B). These kinetic and

pharmacological characteristics are typical of ICRAC's [45, 46].

In the presence of 10 mM CaCl2 in the bath, 1 mM MDEB quickly increased the

amplitude of ICRAC (Fig. 6B), and this effect was inhibited by addition of 1 µM GdCl3 (Fig.

6B and C). As the trace recorded in presence of GdCl3 could be considered as the leak

through the clamped membrane, we observed a peak amplitude increase at the steady state

(95-100 ms) from -3.6 ± 0.1 pA/pF to -20.5 ± 0.1 pA/pF (~ 6 fold), followed by a slow decay

(Fig. 6B). Furthermore, when EGTA, a less efficient chelator of Ca2+ ions than BAPTA was

used in the pipette solution, the peak amplitude increase induced by 1 mM MDEB was

weaker: from -2.7 ± 0.2 pA/pF to -11.1 ± 0.3 pA/pF (~ 4 fold, p < 0.01, Fig. 6B). As shown

in Figure 6D, the effect of MDEB on ICRAC was not voltage-dependent as its amplitude was

Page 17 of 43

Accep

ted

Man

uscr

ipt


16

increased at all the voltage values. Thus, these results confirmed that MDEB increased the

SOCE of Jurkat cells by directly increasing ICRAC amplitude.

3.6. MDEB does not inhibit ER Ca2+ release and sequestration

One of the main limitations of the use of 2-APB as an immunomodulator is its lack of

specificity and especially the fact that it also inhibits both Ca2+ release from the ER and

SERCA activity [21]. Some 2-APB analogues have been developed with higher ability to

block the SOCE, but they still inhibit Ca2+ release [30, 33, 47].

To test the possibility that MDEB inhibits the Ca2+ release from the ER, we pre-

treated the Jurkat cells with 1 mM MDEB for 10 min. To induce the Ca2+ release from the

ER, Jurkat cells were then treated with 10 µg/ml PHA, allowing synthesis of IP3, activation

of the IP3R and Ca2+ release from the ER [37].

In Ca2+-free HBS, PHA induced a rapid and transient increase of [Ca2+]cyt: the

[Ca2+]cyt peaked at 39 ± 7 nM after 2 min (Fig. 7A). When 1 mM MDEB was added 5 min

prior to PHA, the [Ca2+]cyt increase was not significantly different as both curves

superimposed: [Ca2+]cyt peaked at 29 ± 4 nM after 2 min. The same kind of curves was

obtained with 10 mM MDEB (data not shown). These experiments therefore strongly suggest

that MDEB had no effect on the PHA-induced Ca2+ release from the ER.

As the response to PHA was the same in the absence or the presence of 1 mM MDEB,

it seems that the ER Ca2+ amount was not modified by MDEB. To confirm these results, we

performed the same kind of experiment in a HBS medium where all the Na+ ions were

replaced by Li+, and where 0.4 µM HgCl2 was added: this protocol allows the blockade of the

NCX and the PMCA respectively. Therefore, when TG is added, all the Ca2+ ions are

released by the ER but could neither be pumped back to the ER, nor exit from the cell by

NCX or PMCA, and are thus trapped in the cytosol [48].

As shown in Figure 7B, in absence of MDEB, TG induced a rapid increase of

[Ca2+]cyt, that peaked after 90 s, reaching a plateau of 100 nM. When the cells were pre-

treated with 1 mM MDEB for 10 min, TG induced the same kind of [Ca2+]cyt increase and

plateau level, indicating again that the ER Ca2+ amount was not modified by presence of

MDEB.

In order to take into account the possibility that in the above-mentioned experiments

MDEB did not reach the IP3Rs and SERCA pumps because it would be cell impermeant, we

Page 18 of 43

Accep

ted

Man

uscr

ipt


17

also performed experiments on permeabilized cells. We have previously demonstrated that

gentle permeabilization of the plasma membrane of cells grown in monolayers, followed by 45Ca2+ loading of the ER stores and measurement of the unidirectional Ca2+ efflux allows for

a quantitative analysis of IP3R activity in the presence of various activators or inhibitors [25,

38, 49-51]. As this type of experiment can however not be performed on cells in suspension

or loosely attached cell lines, we used as model system the L15 fibroblast cell line

overexpressing IP3R1 [51]. MDEB between 10 µM and 1 mM was present during the

complete release phase and IP3-induced Ca2+ release was induced by addition of 0.7 µM IP3

for 2 min (Fig. 7C). As can be observed (Fig. 7C and D) MDEB up to 1 mM had no effect on

IP3-induced Ca2+ release.

In the same model system we investigated whether MDEB could affect the ER

loading capacity by adding the compound during the loading phase. However, even high

MDEB concentrations only very slightly affected active Ca2+ uptake in the ER (at 100 M

MDEB 98 2 % and at 1 mM MDEB 94 2 % active Ca2+ uptake remaining, n=3)

indicating only a very weak inhibitory effect on SERCA pump activity.

3.7. MDEB differently modifies the [Ca2+]cyt rise induced by suboptimal (1 µg/ml) and

optimal (10 µg/ml) concentrations of PHA

Having shown that MDEB was able to increase the Ca2+ influx amplitude, we next

performed experiments to see whether MDEB had a functional effect on the IL-2 synthesis

induced by PHA. IL-2 is synthesized by activated cells, and is absolutely necessary for T cell

proliferation and immune function [52]. IL-2 synthesis requires an influx of extracellular

Ca2+ ions through SOCE channels during at least 2 hours, while a short-term Ca2+ rise is not

sufficient [5-7, 53].

Prior to performing the IL-2 synthesis experiment, we tested the effect of MDEB on

the [Ca2+]cyt of PHA-stimulated Jurkat cells during the 1st 10 min as well as, after 3 and 24h

of stimulation (Fig. 8). Jurkat cells had a resting [Ca2+]cyt of 121 14 nM (n > 50). After

stimulation with 10 µg/ml PHA, a concentration known to fully activate Jurkat cells

[54],[Ca2+]cyt quickly rised to 424 34 nM (n = 5, p < 0.01) for a time period that exceeded

the complete duration (10 min) of the experiment (Fig.8A). After 3h of 10 µg/ml PHA

stimulation, the [Ca2+]cyt of Jurkat cells had started to decrease (198 47 nM, n = 4, p < 0.01)

and was back to the level observed in control cells after 24 h (126 21 nM, n = 5, Fig. 8C).

Page 19 of 43

Accep

ted

Man

uscr

ipt


18

In contrast, 1 mM MDEB induced a very slight and transient increase of the resting

[Ca2+]cyt to 141 2 nM (Fig. 8A). After 10 min, the resting [Ca2+]cyt of MDEB-treated cells

was not different from the value measured in untreated cells (104 13 nM vs. 121 14 nM, n

> 50). Longer exposures to 1 mM MDEB (3 and 24h) did not modify the resting [Ca2+]cyt that

were 127 9 nM (n = 3) and 118 13 nM (n = 4) respectively (Fig. 8C). 30 µM 2-APB,

known to block the SOCE, significantly decreased the resting [Ca2+]cyt to 60 5 nM (n = 3, p

< 0.01), showing that under resting conditions, some channels responsible for the SOCE are

already open, and that they are sensitive to inhibition by 2-APB as well as to potentiation by

MDEB (Fig. 8A).

Finally, when the Jurkat cells were stimulated with 10 µg/ml PHA in the presence of 1

mM MDEB, the [Ca2+]cyt increase was significantly enhanced to 780 53 nM after 10 min (n

= 5, p < 0.01, Fig. 8A). After 3 h of 10 µg/ml PHA stimulation in the presence of MDEB, and

although the [Ca2+]cyt of the cells had started to decrease, it still remained significantly higher

(251 36 nM, n = 3, p < 0.01) than after PHA stimulation in the absence of MDEB.

Moreover, after 24h of the same treatment, the resting [Ca2+]cyt was also still higher (190 8

nM, n = 3, p < 0.01) than in untreated cells (Fig. 8C). These results show that MDEB

reinforced the [Ca2+]cyt increase in both amplitude and time.

When the Jurkat cells were stimulated with a suboptimal concentration of PHA (1

µg/ml, [54]), [Ca2+]cyt rised slowly to 190 2 nM ( n = 4, p < 0.01, Fig. 8B) after 10 min

value. Even if after 3h, [Ca2+]cyt started to decrease, it was still higher than in control cells:

166 4 nM (n = 3) vs.121 14 nM (p < 0.01) and was back to the control [Ca2+]cyt after 24h

(112 6 nM, n = 4, Fig. 8 D).

The stimulation of the cells with 1 µg/ml PHA in presence of 1 mM MDEB, induced

a faster rise of [Ca2+]cyt that peaked at 224 6 nM (n = 4, p < 0.01), followed by a slow

decrease to 197 6 nM after 10 min (n = 4, p < 0.01, Fig. 8B). Interestingly, after 10 min, the

[Ca2+]cyt rises of cells stimulated with 1 µg/ml PHA in absence and presence of 1 mM MDEB

were not significantly different (190 2 nM vs. 197 6 nM). Furthermore, the use of 1 µg/ml

PHA + 1 mM MDEB could not induce a rise similar to the one obtained with 10 µg/ml PHA

after 10 min (197 6 nM vs. 424 34 nM, p < 0.01). Surprisingly, after 3h, [Ca2+]cyt was

significantly lower than in controls cell (96 4 nM, (n = 4) vs. 121 14 nM, p < 0.05, Fig.

8D) or in cells only stimulated with 1 µg/ml PHA (166 4 nM, p < 0.01), and came back to

control values after 24 h (114 5 nM, n = 4). Thus, the presence of MDEB led after

treatment with 1 µg/ml PHA to a more transient [Ca2+]cyt rise that totally finished after 3h.

Page 20 of 43

Accep

ted

Man

uscr

ipt


19

3.8. MDEB inhibits IL-2 synthesis induced by PHA

We next measured the synthesis of IL-2 by the Jurkat cells stimulated for 24 h with 1

or 10 µg/ml PHA in the presence or absence of MDEB. To compensate for any effect of

MDEB on cell survival, we first controlled their viability by counting the living vs. dead cells

in the presence of Trypan blue. In the absence of any stimulation, the percentage of dead cells

in the Jurkat cell culture represented 4.4 1.8% (n > 20).

Even if MDEB did not change the proportion of dead cells when stimulated by 1

µg/ml PHA (from 2 to 7.2% of dead cells, Fig. 9A), increasing MDEB concentrations

induced in the presence of 10 µg/ml PHA, a progressive rise of the cell death to reach a value

of 28.2 2.8 % at 1 mM MDEB (n = 3, p < 0.01, Fig. 9A). This means that while almost all

the Jurkat cells stimulated by 1 µg/ml PHA were able to synthetize IL-2, only about ¾ of

them would be able to do so when stimulated by 10 µg/ml PHA stimulation.

In the absence of PHA stimulation, MDEB was not able to induce the synthesis of IL-

2 at any concentration (Fig. 9B), indicating that the small increase in resting [Ca2+]cyt

observed with 1 mM MDEB (Fig. 7A) was not enough to activate the Jurkat cells. However,

MDEB significantly decreased in a concentration-dependent way the IL-2 synthesis

(expressed as IL-2 synthesized per 100% living cells) elicited by 24 h of treatment with 1

(grey bars) or 10 µg/ml (black bars) PHA by respectively ~ 70% (24 1 vs. 86 2 pg/ml, p <

0.01) and ~ 30% (113 5 vs. 168 3 pg/ml, p < 0.01) at the maximal MDEB concentration

used (1 mM). Surprisingly, no MDEB potentiation of the IL-2 synthesis was observed at the

sub-optimal concentration of 1 µg/ml PHA.

These results indicate that MDEB is an inhibitor of IL-2 synthesis and of Jurkat T cell

activation.

3.9. MDEB induces apoptosis of Jurkat cells stimulated by 10 µg/ml PHA

Staining with Trypan blue showed that MDEB increased the number of dead cells

when stimulated with 10 µg/ml PHA. We next performed a TUNEL assay to assess the

capacity of MDEB to induce apoptosis of the Jurkat cells. In control conditions, less than 1%

of cells were apoptotic (Fig. 10 A and B). After stimulation with 10 µg/ml PHA, or in the

presence of 1 mM MDEB, no increase in apoptosis level was detectable (Fig. 10A).

Page 21 of 43

Accep

ted

Man

uscr

ipt


20

Remarkably, only PHA-stimulated cells treated with 1 mM MDEB demonstrated a significant

time-dependent level of apoptotic cells (27 ± 3%, n = 3, Fig. 10B).

Page 22 of 43

Accep

ted

Man

uscr

ipt


21

4. DISCUSSION

During the past decade, several effectors of the SOCE have been developed, and

among them, 2-APB is probably the one with the most interesting properties: at low

concentrations, it potentiates the SOCE, while at higher concentrations it is inhibitory.

However, despite these properties, 2-APB is not specifically targeting the SOCE, and is a

well-established modulator of other Ca2+ transporters: SERCA, IP3R and some TRP channels

[21, 23, 25, 55, 56].

In a recently published work [47], we showed that the use of 2-APB analogues can

help to understand which part of the molecule is requested for the potentiation or the

inhibition capacity. Thus, the two phenyl groups seem to play a key role in the sensitivity to

the inhibition process, the ethanolamine group plays a role only in the inhibition, and the

central BOC in the potentiation.

Even if the Orai1 proteins seem to form the pore of the channel allowing the SOCE in

the Jurkat cells, it has been shown that several other proteins could associate with Orai1:

TRPC1 [57], CRACR2A [58], SARAF [59] and junctate [60]. Thus, the SOCE channel

molecular nature is not restricted only to Orai1 proteins, and SOCE studies should be

performed with "complete" SOCE channels. Furthermore, the expression ratio of

Orai1/STIM1 is known to modify the effect of 2-APB on the SOCE. For all these reasons, we

have chosen to study the effect of MDEB in more physiological conditions, by using cell

lines.

In this work, we focussed our attention to MDEB, which is a derivative of the 2-APB

molecule restricted to its BOC. When used on leukocyte cell lines and two breast cancer cell

lines, MDEB has unique properties: this molecule can only potentiate the SOCE, and is

totally devoid of inhibition capacity. Furthermore, unlike 2-APB, MDEB is not able to

influence the Ca2+ release by the IP3R of the ER. Noteworthy, MDEB cannot modify the Ca2+

amount of the ER, probably due to a lack of effect on the SERCA. Thus, this molecule seems

to have a better selectivity for the SOCE than the other 2-APB analogues [30, 31].

Furthermore the cell lines we used showed different sensitivity to MDEB: with a potentiation

constant of ~ 20 µM, breast cancer MDA-MB231 and BT-20 cells SOCE are more sensitive

to MDEB than leukocyte cell lines SOCE (between 44 and 66 µM). This difference could

reflect differences in the SOCE channel composition and/or the expression ratio between

Orai1 and the activating protein STIM1. Indeed, it is known that the sensitivity to 2-APB

depends on the relative expression ratio between Orai1 and STIM1 proteins [61], and the

Page 23 of 43

Accep

ted

Man

uscr

ipt


22

same could be possible for the sensitivity to MDEB. Sub-unit composition of the SOCE is

still under debate, and each type of cells could have its own type of SOCE according to the

expressed proteins. Thus, Orai1 proteins could associate to different TRPC proteins to form a

SOCE, like TRPC1, TRPC3 or TRPC6 [57, 62]. Noteworthy, studies on Orai1-deficient

lymphocytes and fibroblasts clearly established a central role for Orai1 in Ca2+ entry in these

cells [44]. In our hands, we confirmed that lymphocytes lacking Orai1 expression were

devoid of a discernible SOCE, and in addition demonstrated that MDEB was useless to evoke

SOCE. Previous studies have also shown that Orai1 is important for the SOCE of MDA-

MB231 cells and that when expression of Orai1 is reduced in those cells by using siRNA

protocols, the SOCE is hugely decreased [17, 63]. In line with this, MDEB was in our hands

also devoid of any significant SOCE potentiation ability in MDA-MB231 cells with a

reduced Orai1 protein expression. Thus, it seems that the potentiating effect of MDEB on

SOCE is linked to the presence of Orai1 proteins.

Interesting is the comparison between MDEB and 2-ABB: 2-ABB resembles MDEB

with the main difference that an ethanolamine group replaces the methyl group (the two ethyl

groups are also replaced by two butyl groups, but this change probably does not play a main

role in the difference of the behaviour of the two molecules). 2-ABB has, like 2-APB, a dual

effect on SOCE [33]. Thus, we can conclude that the B-N internal coordinate of 2-ABB (and

2-APB) has an inhibitory ability. However, dimesityl borinic acid (DMBA), a 2-APB

analogue without the ethanolamine group, and not able to form the B-N internal coordinate, is

a strong inhibitor of the SOCE [33]. Thus, the structure function analysis of 2-APB leads to a

complex conclusion: the BOC is an absolute requirement for the molecule to be potentiating,

and the phenyl groups and/or the B-N internal coordinate are needed for the inhibitory

capacity of the molecule. Noteworthy, we can hypothesize that 2-APB inhibits the SOCE by

two ways: via the phenyl rings (as with DMBA) or by the B-N internal coordinate (as with 2-

ABB).

The great advantage of MDEB is that this molecule is devoid of any rings or internal

coordinate. Thus, it confirms that the BOC is responsible for the potentiating capacity of the

borinate esters and we can hypothesize that any molecule with a BOC and without any rings

or internal coordinate will have the ability to only potentiate the SOCE. It also confirms that

the presence of rings or B-N internal coordinate plays a key role in the sensitivity of the

SOCE to the modulation by the borinate esters: thus, if we count the number of closed

structures, we have the following sequence of efficacy: DPBA (4 phenyl groups = 4 rings) >

Page 24 of 43

Accep

ted

Man

uscr

ipt


23

2-APB (2 phenyl groups + 1 internal coordinate ~ 3 rings) > 2-ABB (1 internal coordinate ~

1 ring) > MDEB (0 ring) [33].

Another main difference between MDEB and the ring-containing borinate esters is

that this molecule is not able to inhibit the Ca2+ release by the ER. One of the limits of 2-APB

and its analogues is this lack of specificity between Ca2+ release and Ca2+ influx. Mikoshiba’s

group had designed analogues with more selectivity for the Ca2+ influx [30, 31]. However,

these molecules could still inhibit Ca2+ release by the ER. Furthermore, investigating both

intact and permeabilized cells we could demonstrate that MDEB only minimally affects the

SERCA activity. Although the present work therefore clearly shows that two of the main

targets of 2-APB, are nearly completely insensitive to this analogue, more work is still

needed to further document the specificity of MDEB, and especially to investigate whether

the compound can affect TRP channels.

In this work, we also demonstrated that the special properties of MDEB could be used

to selectively inhibit or eliminate target cells. MDEB is able to induce a very faint increase of

[Ca2+]cyt by itself. In contrast, addition of 30 µM 2-APB decreases the [Ca2+]cyt, showing that

some Orai1 channels are already open to regulate [Ca2+]cyt allowing the constant replenishing

of the ER with Ca2+ ions under the control of STIM2 [64]. However this tiny and transient

MDEB induced [Ca2+]cyt increase is not sufficient to activate the Jurkat T cells and the

synthesis of IL-2. We expected MDEB to potentiate the IL-2 synthesis of cells stimulated by

a suboptimal concentration of PHA. On the contrary after 24 h of treatment with 1 µg/ml

PHA, 1 mM MDEB was already able to inhibit the IL-2 synthesis. Surprisingly, even if

MDEB 1 mM potentiates the [Ca2+]cyt rise induced by 1 µg/ml PHA, [Ca2+]cyt rapidly returns

to the level of control cells. We can hypothesize that in the first minutes of stimulation, the

Jurkat cells increased their capacity to remove Ca2+ ions from the cytosol as usual, except that

the stimulation is not optimal, not allowing the full mobilisation of Ca2+ ions from the ER. In

the absence of MDEB, the increase of [Ca2+]cyt is weaker, the cells did not fully activate the

extrusion of Ca2+ ions: this rise can last for at least 3h, allowing the full synthesis of IL-2.

At higher concentration of PHA (10 µg/ml), MDEB induced the cell death of ~ 30%

of the Jurkat cells. Furthermore, the IL-2 synthesis of the remaining cells is also reduced by ~

30%. Thus, MDEB, by potentiating the Ca2+ influx, inhibits the synthesis of IL-2 induced by

full activation. Interestingly, SKF96365 inhibition of IL-2 synthesis in peripheral blood

lymphocytes did not affect their viability [53]. MDEB is by itself not toxic for the cells, but is

toxic for 10 µg/ml PHA-stimulated cells. Thus after 6h of 10 µg/ml PHA + 1 mM MDEB, a

significant part of the cells have shown apoptotic signs, like DNA fragmentation that reached

Page 25 of 43

Accep

ted

Man

uscr

ipt


24

almost one third after 24h. Since the discovery of the Orai1 protein as pore of the SOCE

channels, several mutations have been created which revealed some crucial parts of the

protein. The glycine in position 98 forms a hinge in the first transmembrane domain. Even if

the G98A mutation abolished the SOCE and has a dominant-negative action on the wild type

Orai1 subunit, G98P and G98D mutations create constitutively active Orai1 channels [65].

This constitutive SOCE induces an increase of the resting [Ca2+]cyt compared to the wild type

SOCE, leading to cell death 2 days after transfection [65]. As shown in the present study,

MDEB extends the [Ca2+]cyt rise induced by PHA. The inability of the cells to regain in the

presence of MDEB a normal [Ca2+]cyt after PHA stimulation seems to induce their cell death

by apoptosis.

In contrast to SOCE inhibitors like SKF96365, MDEB decreases the Jurkat cell

activation by two ways according to the intensity of the cell stimulation:

(i) by shortening the [Ca2+]cyt rise at suboptimal PHA concentration, MDEB impairs

the full activation of the machinery leading to IL-2 synthesis,

(ii) by inducing the cell apoptosis of an increasing number of cells at optimal PHA

concentration. This latter property could be of interest as MDEB targets only activated cells,

and does not seem to be toxic for untreated cells. Thus, we can hypothesize that cells where

the [Ca2+]cyt is already increased could be sensitive to MDEB. In a previous work we showed

that the [Ca2+]cyt of B cells expressing the Epstein-Barr Virus oncogenic protein LMP-1 rises

to almost 200 nM due to an increase of the SOCE amplitude [48, 66]. The next step of our

work will be to test the ability of MDEB to induce the apoptosis of these LMP-1 – expressing

B cells. For memory, LMP-1 is expressed by infected cells of some Hodgkin diseases,

polyclonal lymphomas or the nasopharyngeal carcinoma for example [67].

In summary, we have characterized the first molecule having only a potentiating

ability on SOCE from different cell lines. It has kept the potentiating ability of the 2-APB

molecule, but lost the inhibitory capacity. Thus, it could represent the first member of a

promising new compound family, able for example to regulated the activity of immune cells

or for inducing the apoptosis of target cells.

Page 26 of 43

Accep

ted

Man

uscr

ipt


25

5. REFERENCES

1. Berridge MJ, Lipp P, Bootman MD. (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol, 1, 11-21.

2. Liu JO. (2009) Calmodulin-dependent phosphatase, kinases, and transcriptional corepressors involved in T-cell activation. Immunol Rev, 228, 184-98.

3. Varga-Szabo D, Authi KS, Braun A, Bender M, Ambily A, Hassock SR, Gudermann T, Dietrich A, Nieswandt B. (2008) Store-operated Ca(2+) entry in platelets occurs independently of transient receptor potential (TRP) C1. Pflugers Arch, 457, 377-87.

4. Zweifach A, Lewis RS. (1993) Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci U S A, 90, 6295-9.

5. Goldsmith MA, Weiss A. (1988) Early signal transduction by the antigen receptor without commitment to T cell activation. Science, 240, 1029-31.

6. Weiss A, Shields R, Newton M, Manger B, Imboden J. (1987) Ligand-receptor interactions required for commitment to the activation of the interleukin 2 gene. J Immunol, 138, 2169-76.

7. Crabtree GR. (1989) Contingent genetic regulatory events in T lymphocyte activation. Science, 243, 355-61.

8. Hoth M, Penner R. (1992) Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature, 355, 353-6.

9. Lewis RS, Cahalan MD. (1989) Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells. Cell Regul, 1, 99-112.

10. Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A. (2006) A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature, 441, 179-85.

11. Feske S. (2010) CRAC channelopathies. Pflugers Arch, 460, 417-435.12. Putney JW, Jr. (2007) New molecular players in capacitative Ca2+ entry. J Cell Sci,

120, 1959-65.13. Gwack Y, Srikanth S, Feske S, Cruz-Guilloty F, Oh-hora M, Neems DS, Hogan PG,

Rao A. (2007) Biochemical and functional characterization of Orai proteins. J Biol Chem, 282, 16232-43.

14. Soboloff J, Spassova MA, Tang XD, Hewavitharana T, Xu W, Gill DL. (2006) Orai1 and STIM reconstitute store-operated calcium channel function. J Biol Chem, 281, 20661-5.

15. Lis A, Peinelt C, Beck A, Parvez S, Monteilh-Zoller M, Fleig A, Penner R. (2007) CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional properties. Curr Biol, 17, 794-800.

16. Bandyopadhyay BC, Pingle SC, Ahern GP. (2010) Store-operated Ca2+ signaling in dendritic cells occurs independently of STIM1. J Leukoc Biol, 89, 57-62.

17. Motiani RK, Abdullaev IF, Trebak M. (2010) A novel native store-operated calcium channel encoded by Orai3:Selective requirement of Orai3 versus Orai1 in estrogen receptor positive versus estrogen receptor negative breast cancer cells. J Biol Chem, 285, 19173-19183.

18. Mignen O, Thompson JL, Shuttleworth TJ. (2008) Both Orai1 and Orai3 are essential components of the arachidonate-regulated Ca2+-selective (ARC) channels. J Physiol, 586, 185-95.

19. Soboloff J, Zhang Y, Minden M, Berger SA. (2002) Sensitivity of myeloid leukemia cells to calcium influx blockade: application to bone marrow purging. Exp Hematol, 30, 1219-26.

Page 27 of 43

Accep

ted

Man

uscr

ipt


26

20. Prakriya M, Lewis RS. (2001) Potentiation and inhibition of Ca(2+) release-activated Ca(2+) channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP(3) receptors. J Physiol, 536, 3-19.

21. Maruyama T, Kanaji T, Nakade S, Kanno T, Mikoshiba K. (1997) 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem, 122, 498-505.

22. Lievremont JP, Bird GS, Putney JW, Jr. (2005) Mechanism of inhibition of TRPC cation channels by 2-aminoethoxydiphenylborane. Mol Pharmacol, 68, 758-62.

23. Bilmen JG, Wootton LL, Godfrey RE, Smart OS, Michelangeli F. (2002) Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites. Eur J Biochem, 269, 3678-87.

24. Ma HT, Venkatachalam K, Parys JB, Gill DL. (2002) Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes. J Biol Chem, 277, 6915-22.

25. Missiaen L, Callewaert G, De Smedt H, Parys JB. (2001) 2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2+ pump and the non-specific Ca2+ leak from the non-mitochondrial Ca2+ stores in permeabilized A7r5 cells. Cell Calcium, 29, 111-6.

26. Chung MK, Lee H, Mizuno A, Suzuki M, Caterina MJ. (2004) 2-aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J Neurosci, 24, 5177-82.

27. Hu HZ, Gu Q, Wang C, Colton CK, Tang J, Kinoshita-Kawada M, Lee LY, Wood JD, Zhu MX. (2004) 2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3. J Biol Chem, 279, 35741-8.

28. Dobrydneva Y, Blackmore P. (2001) 2-Aminoethoxydiphenyl borate directly inhibits store-operated calcium entry channels in human platelets. Mol Pharmacol, 60, 541-52.

29. Dobrydneva Y, Abelt CJ, Dovel B, Thadigiri CM, Williams RL, Blackmore PF. (2006) 2-aminoethoxydiphenyl borate as a prototype drug for a group of structurally related calcium channel blockers in human platelets. Mol Pharmacol, 69, 247-56.

30. Zhou H, Iwasaki H, Nakamura T, Nakamura K, Maruyama T, Hamano S, Ozaki S, Mizutani A, Mikoshiba K. (2007) 2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry. Biochem Biophys Res Commun, 352, 277-82.

31. Suzuki AZ, Ozaki S, Goto JI, Mikoshiba K. (2010) Synthesis of bisboron compoundsand their strong inhibitory activity on store-operated calcium entry. Bioorg Med Chem Lett, 20, 1395-1398.

32. Ozaki S, Suzuki AZ, Bauer PO, Ebisui E, Mikoshiba K. (2013) 2-Aminoethyl diphenylborinate (2-APB) analogues: regulation of Ca2+ signaling. Biochem Biophys Res Commun, 441, 286-90.

33. Dellis O, Mercier P, Chomienne C. (2011) The boron-oxygen core of borinate esters is responsible for the store-operated calcium entry potentiation ability. BMC Pharmacol, 11, 1.

34. Djillani A, Nusse O, Dellis O. (2014) Characterization of novel store-operated calcium entry effectors. Biochim Biophys Acta, 1843, 2341-7.

35. Miyawaki A, Furuichi T, Maeda N, Mikoshiba K. (1990) Expressed cerebellar-type inositol 1,4,5-trisphosphate receptor, P400, has calcium release activity in a fibroblast L cell line. Neuron, 5, 11-8.

36. Grynkiewicz G, Poenie M, Tsien RY. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem, 260, 3440-50.

Page 28 of 43

Accep

ted

Man

uscr

ipt


27

37. Dellis O, Gangloff SC, Paulais M, Tondelier D, Rona JP, Brouillard F, Bouteau F, Guenounou M, Teulon J. (2002) Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160. J Biol Chem, 277, 6044-50.

38. Luyten T, Bultynck G, Parys JB, De Smedt H, Missiaen L. (2014) Measurement of intracellular Ca2+ release in permeabilized cells using 45Ca2+. Cold Spring Harb Protoc, 2014, 289-94.

39. Missiaen L, Van Acker K, Parys JB, De Smedt H, Van Baelen K, Weidema AF, Vanoevelen J, Raeymaekers L, Renders J, Callewaert G, Rizzuto R, Wuytack F. (2001) Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store. J Biol Chem, 276, 39161-70.

40. Chatamra KR, Chapman RA. (1996) The effects of sodium-calcium exchange inhibitors on protein loss associated with the calcium paradox of the isolated Langendorff perfused guinea-pig heart. Exp Physiol, 81, 203-10.

41. Graf E, Verma AK, Gorski JP, Lopaschuk G, Niggli V, Zurini M, Carafoli E, Penniston JT. (1982) Molecular properties of calcium-pumping ATPase from human erythrocytes. Biochemistry, 21, 4511-6.

42. Owen CS. (1993) Simultaneous measurement of two cations with the fluorescent dye indo-1. Anal Biochem, 215, 90-5.

43. Mercer JC, Dehaven WI, Smyth JT, Wedel B, Boyles RR, Bird GS, Putney JW, Jr. (2006) Large store-operated calcium selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1. J Biol Chem, 281, 24979-90.

44. McCarl CA, Picard C, Khalil S, Kawasaki T, Rother J, Papolos A, Kutok J, Hivroz C, Ledeist F, Plogmann K, Ehl S, Notheis G, Albert MH, Belohradsky BH, Kirschner J, Rao A, Fischer A, Feske S. (2009) ORAI1 deficiency and lack of store-operated Ca2+ entry cause immunodeficiency, myopathy, and ectodermal dysplasia. J Allergy Clin Immunol, 124, 1311-1318.

45. Zweifach A, Lewis RS. (1995) Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback. J Gen Physiol, 105, 209-26.

46. Zweifach A, Lewis RS. (1995) Slow calcium-dependent inactivation of depletion-activated calcium current. Store-dependent and -independent mechanisms. J Biol Chem, 270, 14445-51.

47. Djillani A, Nusse O, Dellis O. (2014) Characterization of novel store-operated calcium entry effectors. Biochim Biophys Acta.

48. Dellis O, Arbabian A, Brouland JP, Kovacs T, Rowe M, Chomienne C, Joab I, Papp B. (2009) Modulation of B-cell endoplasmic reticulum calcium homeostasis by Epstein-Barr virus Latent Membrane Protein-1. Mol Cancer, 8, 59.

49. Missiaen L, De Smedt H, Droogmans G, Casteels R. (1992) Ca2+ release induced by inositol 1,4,5-trisphosphate is a steady-state phenomenon controlled by luminal Ca2+ in permeabilized cells. Nature, 357, 599-602.

50. Parys JB, Missiaen L, De Smedt H, Droogmans G, Casteels R. (1993) Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells. Pflugers Arch, 424, 516-22.

51. Mills SJ, Luyten T, Erneux C, Parys J, Potter BV. (2012) Multivalent benzene polyphosphate derivatives are non-Ca2+-mobilizing Ins(1,4,5)P3 receptor antagonists. Messenger, 1, 167-181.

52. Emmel EA, Verweij CL, Durand DB, Higgins KM, Lacy E, Crabtree GR. (1989) Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science, 246, 1617-20.

Page 29 of 43

Accep

ted

Man

uscr

ipt


28

53. Chung SC, McDonald TV, Gardner P. (1994) Inhibition by SK&F 96365 of Ca2+ current, IL-2 production and activation in T lymphocytes. Br J Pharmacol, 113, 861-8.

54. Mills GB, Lee JW, Cheung RK, Gelfand EW. (1985) Characterization of the requirements for human T cell mitogenesis by using suboptimal concentrations of phytohemagglutinin. J Immunol, 135, 3087-93.

55. Peppiatt CM, Collins TJ, Mackenzie L, Conway SJ, Holmes AB, Bootman MD, Berridge MJ, Seo JT, Roderick HL. (2003) 2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels. Cell Calcium, 34, 97-108.

56. Kukkonen JP, Lund PE, Akerman KE. (2001) 2-aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca(2+) discharge and store-operated Ca(2+) influx. Cell Calcium, 30, 117-29.

57. Ambudkar IS, Ong HL, Liu X, Bandyopadhyay BC, Cheng KT. (2007) TRPC1: the link between functionally distinct store-operated calcium channels. Cell Calcium, 42, 213-23.

58. Srikanth S, Jung HJ, Kim KD, Souda P, Whitelegge J, Gwack Y. (2010) A novel EF-hand protein, CRACR2A, is a cytosolic Ca2+ sensor that stabilizes CRAC channels in T cells. Nat Cell Biol, 12, 436-46.

59. Palty R, Raveh A, Kaminsky I, Meller R, Reuveny E. (2012) SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling. Cell, 149, 425-38.

60. Srikanth S, Jew M, Kim KD, Yee MK, Abramson J, Gwack Y. (2012) Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1). Proc Natl Acad Sci U S A, 109, 8682-7.

61. Scrimgeour N, Litjens T, Ma L, Barritt GJ, Rychkov GY. (2009) Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins. J Physiol, 587, 2903-2918.

62. Liao Y, Erxleben C, Abramowitz J, Flockerzi V, Zhu MX, Armstrong DL, Birnbaumer L. (2008) Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels. Proc Natl Acad Sci U S A, 105, 2895-900.

63. McAndrew D, Grice DM, Peters AA, Davis FM, Stewart T, Rice M, Smart CE, Brown MA, Kenny PA, Roberts-Thomson SJ, Monteith GR. (2011) ORAI1-mediated calcium influx in lactation and in breast cancer. Mol Cancer Ther.

64. Brandman O, Liou J, Park WS, Meyer T. (2007) STIM2 Is a Feedback Regulator that Stabilizes Basal Cytosolic and Endoplasmic Reticulum Ca(2+) Levels. Cell, 131, 1327-39.

65. Zhang SL, Yeromin AV, Hu J, Amcheslavsky A, Zheng H, Cahalan MD. (2011) Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91. Proc Natl Acad Sci U S A, 108, 17838-43.

66. Dellis O, Arbabian A, Papp B, Rowe M, Joab I, Chomienne C. (2011) Epstein-Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells. J Biol Chem, 286, 18583-92.

67. Khanna R, Burrows SR, Moss DJ. (1995) Immune regulation in Epstein-Barr virus-associated diseases. Microbiol Rev, 59, 387-405.

Page 30 of 43

Accep

ted

Man

uscr

ipt


29

6. FOOTNOTESThis work was supported by the Association pour la Recherche contre le Cancer (ARC), the Association de Biologie du Cancer et Dynamiques complexes (ABCD) and a starting grant from the University Paris-Sud (to OD). We thank Sylvie Prigent for her help.

7. FIGURE LEGENDS

Figure 1: Structure of 2-APB and analogues.Structures of 2-aminoethoxydiphenyl borate (2-APB, 1), 2-aminoethoxydibutyl borate (2-ABB, 2), methyl-diethylborinate (MDEB, 3) and trimethylborate (TMB, 4).

Figure 2: MDEB potentiates the divalent ion influx in Jurkat cells.A. Cytosolic Ca2+ concentration ([Ca2+]cyt) measurement in Jurkat cells using Fura-2 fluorescence. Cells were treated for 10 min with thapsigargin (1 µM, arrow i) to allow Ca2+

release from ER and opening of the SOCE channels. After 10 min, 1 mM CaCl2 was added, allowing Ca2+ entry through the SOCE channels. MDEB at the indicated concentrations wasapplied 30 s prior to CaCl2 (arrow ii). The SOCE inhibitor GdCl3 (1 µM) was added at the same time as MDEB. Results are representative of 5 experiments.B. 2-APB, 2-ABB and MDEB regulate in a different way SOCE in Jurkat cells, while TMB is ineffective. Experiments were performed as in Figure 2A, and the peak [Ca2+]cyt was expressed as % of the peak [Ca2+]cyt recorded in the absence of any compounds. Compounds were added 30 s prior to CaCl2. Results are expressed as mean ± SEM (n ≥ 3).C. Mn2+ quenching of Fura-2 in Jurkat cells is enhanced by 1 mM MDEB. Cells were pre-treated for 10 min with thapsigargin (TG, 1 µM) to allow Ca2+ release from the ER and opening of the SOCE channels. 60 s after beginning of the recordings, 100 µM MnCl2 was added. At 120 s, 1 mM MDEB was added. Fluorescence of Fura-2 was recorded at 510 nm after excitation at 360 nm. Results are representative for 3 experiments and express thefluorescence relative to that prior to MnCl2 addition. The dotted line represents the slope of Fura-2 quenching by Mn2+ ions in absence of MDEB. Results are representative of 4experiments.

Figure 3: MDEB potentiates SOCE in other leukocyte cell lines.The same protocol as in Figure 2 was used on Burkitt B lymphoma (BL-41) and monocytic(U937) cells.A. MDEB dose-response curves could be fitted with a simple sigmoidal curve, giving potentiation constant of 44 ± 11 µM (Jurkat cells, n = 5), 58 ± 7 nM (BL-41 cells, n = 4) and 66 ± 12 nM (U937 cells, n = 5).B. MDEB has no effect on the SOCE of Orai1-deficient B cells. The same protocol as in Figure 2A was used on a lymphoblastoid cell line expressing wild type Orai1 (B-Orai1 wt)and an Orai1-deficient lymphoblastoid cell line (B-Orai1 deficient) established from a patient presenting a SCID phenotype due to a double mutation in the Orai1 gene allele, resulting in the absence of Orai1 protein in the plasma membrane of B cells [44]. At t = 800s, 1mM MDEB was added.C. MDEB only potentiates SOCE when Orai1 is expressed. Mn2+ experiments were performed as in Figure 2C. A comparison was performed between a lymphoblastoid cell lineexpressing wild type Orai1 (B-Orai1 wt) and an Orai1-deficient lymphoblastoid cell line (B-Orai1 deficient). Results are representative of 5 experiments. Recordings done on Orai1 deficient cells are noiser due to a weaker Fura-2 loading.

Page 31 of 43

Accep

ted

Man

uscr

ipt


30

Figure 4: MDEB potentiates SOCE in two breast cancer cell lines.The same protocol as in Figure 2 was used on MDA-MB231 and BT20 cells.A. MDEB dose-response curves could be fitted with a simple sigmoidal curve, giving potentiation constant of 44 ± 11 µM (Jurkat cells, n =5), : 25 ± 7 nM (MDA-MB231, n = 4) and 14 ± 2 µM (BT20, n =4).B. MDA-MB231 were transfected with 100 nM siRNA against Orai1 or a control siRNA. Proteins were extracted after 72h. Western-blotting was then conducted to assess the Orai1 protein knockdown. Actin was used as a control and showed no difference in the quantity of protein loaded per lane (not shown). The use of 100 nM siRNA against Orai1 mRNA decreased expression of Orai1 protein by 78 ± 6 % (n=3).C. Decreasing Orai1 expression impaired the SOCE of MDA-MB231. The same protocol used in figure 2A was used on control siRNA-transfected MDA-MB231 (“control siRNA”) cells and Orai1 siRNA-transfected MDA-MB231 (“Orai1 siRNA”). At t = 800s, 1mM MDEB was added and was only efficient on cells still expressing normal Orai1 protein level.Results are representative of 5 experiments.

Figure 5: MDEB partly counteracts the inhibition of the SOCE by 2-ABB, but not that by 2-APB.[Ca2+]i measurement was performed using Fura-2 in Jurkat cells in the presence of either MDEB and 2-APB (A and B) or MDEB and 2-ABB (C and D). MDEB, 2-APB and 2-ABB were used at a concentration of 100 µM. A and B. Cells were treated for 600 s with thapsigargin (1 µM, arrow i) in Ca2+-free medium to allow Ca2+ release from the ER and opening of the SOCE channels. Then, 1 mM CaCl2 was added, allowing Ca2+ entry through the SOCE channels. 2-APB, MDEB or 2-APB + MDEB were applied 30 s prior to CaCl2 (A and B, arrow ii). In competition experiments, MDEB was applied 30 s after CaCl2 addition tocells treated with 2-APB (“2-APB prior MDEB”, A, arrow iii) or 2-APB was applied 30 s after CaCl2 addition to cells treated with MDEB (“MDEB prior 2-APB”, B, arrow iii). C and D: similar experiments were performed as in A and B, except that 2-APB was replaced in all cases by 2-ABB. Results are representative for 5 experiments. E. MDEB dose-response curves were obtained as in figure 2B. The various concentrations of MDEB were added 30s prior CaCl2 with different concentrations of 2-APB. The curves could be fitted with a mono-exponential curve.

Figure 6: MDEB directly enhances ICRAC.Jurkat cells were dialyzed with a pipette solution containing 10 mM BAPTA to induce store depletion, and the whole-cell current was measured in the presence of extracellular Ca2+.A. ICRAC was evoked by a 100 ms pulse at -100 mV from a holding potential of 0 mV. Bath medium contained 0, 1 or 10 mM CaCl2. Results are representative for 3 experiments.B. Time courses of ICRAC recorded with a pipette solution containing 10 mM BAPTA or 10 mM EGTA, in the presence of 10 mM external CaCl2. Patch disruption allowed appearance of an inward current that peaked quickly with EGTA or after ~1 min with BAPTA in the pipette. Then, the current decayed. At t = 6 min, the 1 mM MDEB was directly added to the bath and ICRAC amplitude immediately increased. At t = 6 min, 1 µM GdCl3 was added. The remaining Gd3+ ions - resistant current was considered as the leak current and subtracted from the current amplitude recordings. These recordings are typical of 3 experiments.C. Same protocol as in Fig. 6A. ICRAC was recorded in the presence of 10 mM extracellular CaCl2 before MDEB addition (black line), at the peak of 1 mM MDEB potentiation (red line), and after 1 µM GdCl3 addition (blue line).

Page 32 of 43

Accep

ted

Man

uscr

ipt


31

D. Current-voltage relationships of the currents recorded in the presence of 10 mM CaCl2

before (black trace) and after (red trace) 1 mM MDEB addition. Results are representative for5 experiments

Figure 7: MDEB does not inhibit the Ca2+ release from the ER.A. Jurkat [Ca2+]cyt was measured as in Figure 2. To induce Ca2+ release by the ER, cells were treated at t = 30 s with 10 µg/ml PHA, which is known to induce the synthesis of IP3 and subsequent Ca2+ release through the ER IP3Rs. When indicated, 1 mM MDEB was added 5 min prior to PHA stimulation. Results are express as mean ± SEM (n = 3).B. To estimate the Ca2+ amount of the ER, Jurkat cells were placed in Li+-HBS, in order to impair the activity of the Na+/Ca2+ exchanger and in the presence of 0.4 µM HgCl2 to block the activity of the plasma membrane Ca2+ ATPases. When indicated, 1 mM MDEB was added 5 min prior thapsigargin (TG) addition used to induce the Ca2+ release by the ER.Results are express as mean ± SEM (n = 3). C. and D. The effect of MDEB on the IP3–induced Ca2+ release was investigated in permeabilized L15 fibroblasts. C. The ER Ca2+ stores of saponin-permeabilized cells were loaded until steady-state with 45Ca2+ and release was induced by incubation in a Ca2+-free efflux medium in the presence of thapsigargin (10 M) and the indicated concentration of MDEB. Samples were taken every 2 minutes. IP3 (0.7 M) was added for 2 min (white bar) and IP3-induced Ca2+ release measured. 45Ca2+ release is expressed as fractional loss, i.e. the amount of Ca2+ released during 2 min relative to the total amount of Ca2+ present at that moment in the stores. Representative traces are shown for 6 independent experiments, each performed in two-fold. Standard deviation is indicated when larger than the symbol. D. Bar graph showing the IP3-induced Ca2+ release in the presence of the indicated MDEB concentrations. The fractional loss induced by IP3 in the absence of MDEB was taken as 100% and all values are shown relative to this value. Average S.E.M is shown for 6 independent experiments, each performed in two-fold.

Figure 8: MDEB modifies the [Ca2+]cyt rise of PHA-stimulated Jurkat cells.Jurkat cell [Ca2+]cyt was measured using Fura-2 fluorescence in the presence of 1 mM extracellular CaCl2. Cells were stimulated with 1 mM MDEB, 30 µM 2-APB, 1 (B and D) or 10 µg/ml PHA (A and C) alone or with MDEB. Statistically significant differences from control are indicated by: *: p < 0.01, #: p< 0.05.A. At t = 30 s, cells were kept untreated (“control”, black line), treated by 1 mM MDEB(green line), or stimulated with 10 µg/ml PHA alone (blue line) or in the presence of 1 mM MDEB (red line). 30 mM 2-APB was used as a control (grey line). Results are representative for 3 experiments.B. At t = 30 s, cells were kept untreated (“control”, black line), treated by 1 µg/ml PHA alone (red line), or with 1 mM MDEB (green line). A recordings made with 10 µg/ml PHA was added for comparison. Results are representative for 3 experiments.C. Several batches of Jurkat cells were stimulated in cell culture conditions for 3 and 24 h with 10 µg/ml PHA alone or in the presence of 1 mM MDEB, or treated with 1 mM MDEB alone. After Fura-2 loading, the cells were placed in a 1 mM CaCl2-containing HBS medium, and their mean [Ca2+]cyt was calculated based on the Fura-2 fluorescence. Values at 10 min were measured at the end of the experiments shown in Figure 8A. Results are mean ± SEM from 3 experiments. The red line represents the mean resting [Ca2+]cyt of untreated cells ( n = 10).D. Same protocol as in Figure 8C except that cells were stimulated with 1 µg/ml PHA alone or in the presence of 1 mM MDEB, or treated with 1 mM MDEB alone. The red line represents the mean resting [Ca2+]cyt of untreated cells ( n = 10).

Page 33 of 43

Accep

ted

Man

uscr

ipt


32

Figure 9: MDEB inhibits IL-2 synthesis of PHA-stimulated Jurkat cells by decreasing cell viability and/or IL-2 synthesis capacity.Jurkat cells were stimulated by 1 or 10 µg/ml PHA in the presence of MDEB at the indicated concentrations. After 24h dead cells were counted using a Malassez hematimeter (A) or the supernatants were collected, and IL-2 concentrations were measured by Elisa. The values of produced IL-2 were corrected by the number of living cells (B). Statistically significant differences from control are indicated by: *: p < 0.01, #: p< 0.05.

Figure 10: MDEB induces apoptosis of 10 µg/ml PHA-stimulated Jurkat cells.Cells were stained with DAPI (blue, top) to visualize the nuclei and with the TUNEL method (green, bottom) to visualize apoptotic cells. Each staining was merged with the phase contrast image to visualize whole cells. In control conditions or after stimulation by 10 µg/ml PHA or 1 mM MDEB for 6, 12 or 24 h, no apoptotic cells were detectable. Only 10 µg/ml PHA-stimulated cells treated with 1 mM MDEB demonstrated a significant level of apoptotic cells.A. Representative pictures of cells after 24h of treatment with 10 µg/ml PHA alone or with 1 mM MDEB, or 1 mM MDEB alone. Scale bar = 10µm.B. The number of apoptotic cells was counted at 6, 12 and 24 h of treatment. Only 10 µg/ml PHA + 1 mM MDEB induced a significant increase of apoptosis. Statistically significant differences from control are indicated by: *: p < 0.01.

Page 34 of 43

Accep

ted

Man

uscr

ipt

Figure 1

2-ABB (2)

B

O

NH 2

CH 3 CH 3

2-APB (1)

B

O

NH 2

MDEB (3)

B

O

CH 3 CH 3

CH 3

TMB (4)

B

O

O O

CH 3 CH 3

CH 3

Figure 1

Page 35 of 43

Accep

ted

Man

uscr

ipt

Figure 2

-9 -8 -7 -6 -5 -4 -3 -2 -10

100

200

300 2-APBMDEB

TMB2-ABB

log ([X]) (M)

Peak

[Ca2+

] cyt

. (%

con

trol)

B

C

0 30 60 90 120 150 1802030405060708090

100110

Time (s)

% F

0

A + Ca2+ 0 Ca2+

0 200 400 600 800 10000

1000

2000

3000control

1 mM10 mM

100 µM10 µM1 µM

10 mM + GdCl3

Time (s)

[Ca2+

] cyt

. (nM

)

1 mM MDEB

1 µM TG

100 µM MnCl2

i ii

Figure 2

Page 36 of 43

Accep

ted

Man

uscr

ipt

Figure 3

C

A

0 30 60 90 120 150 18040

50

60

70

80

90

100

110

B-Orai1 wtB-Orai1 deficient

Time (s)

% F

0

1 mM MDEB

1 µM TG

100 µM MnCl2

B

-8 -7 -6 -5 -4 -3 -2 -10

100

200

300

400

500

JurkatBL-41U937

log ([MDEB]) (M)

Peak

[Ca2+

] cyt

. (%

con

trol)

0 200 400 600 800 10000

500

1000

1500

2000

B-Orai1 wtB-Orai1 deficient

Time (s)

[Ca2+

] cyt

(nM

)

+ Ca2+ 0 Ca2+

MDEB

Figure 3

Page 37 of 43

Accep

ted

Man

uscr

ipt

Figure 4

A

-8 -7 -6 -5 -4 -3 -2 -10

100

200

300 JurkatMDA-MB231BT-20

log ([MDEB]) (M)

Peak

[Ca2+

] cyt

. (%

con

trol)

0 200 400 600 800 10000

200

400

600

800

1000

control siRNAOrai1 siRNA

Time (s)

[Ca2+

] cyt

(nM

)

+ Ca2+ 0 Ca2+

MDEB

B

37 25

C

Figure 4

Page 38 of 43

Accep

ted

Man

uscr

ipt

Figure 5

2-APB vs. MDEB

2-ABB vs. MDEB

+ Ca2+ 0 Ca2+ A B

0 200 400 600 800 10000

500

1000

1500control2-APB2-APB + MDEB2-APB prior MDEB

Time (s)

[Ca2+

] cyt

. (nM

)

0 200 400 600 800 10000

500

1000

1500

2000

2500controlMDEB2-APB + MDEBMDEB prior 2-APB

Time (s)[C

a2+] c

yt. (

nM)

+ Ca2+ 0 Ca2+

0 200 400 600 800 10000

500

1000

1500

2000controlMDEB2-ABB + MDEBMDEB prior 2-ABB

Time (s)

[Ca2+

] cyt

. (nM

)

+ Ca2+ 0 Ca2+ C D + Ca2+ 0 Ca2+

0 200 400 600 800 10000

200

400

600

800

1000

1200

1400control2-ABB2-ABB + MDEB2-ABB prior MDEB

Time (s)

[Ca2+

] cyt

. (nM

)

i ii

iii

i ii

i ii

iii

i ii

iii

iii

E

-8 -7 -6 -5 -4 -3 -2 -10

100

200

300

no 2-APB

50 µM 2-APB100 µM 2-APB

20 µM 2-APB30 µM 2-APB

log ([MDEB]) (M)

Peak

[Ca2+

] cyt

. (%

con

trol)

Figure 5

Page 39 of 43

Accep

ted

Man

uscr

ipt

Figure 6

A

0 25 50 75 100-15

-10

-5

0

no CaCl21 mM CaCl210 mM CaCl2

Time (ms)

I(pA

/pF)

0 25 50 75 100-40

-30

-20

-10

0

10 mM CaCl2+ 1 mM MDEB+ 1 µM GdCl3

Time (ms)

I (pA

/pF)

C D -100 -80 -60 -40 -20 20

-15

-10

-5

5

MDEBno MDEB

B 1 mM MDEB

GdCl3

0 2 4 6 8 10 12 14-40

-30

-20

-10

0

10 mM EGTA10 mM BAPTA

Time (min)

I (pA

/pF)

pA/p

F

V (mV)

10 mM CaCl2

Figure 6

Page 40 of 43

Accep

ted

Man

uscr

ipt

Figure 7

0 100 200 300 400 500 60050

75

100

125

150control+ 1 mM MDEB

Time (s)

[Ca2+

] cyt

(nM

)

10 µg/ml PHA

0 100 200 300 400 500 6000

50

100

150

control+ 1 mM MDEB

Time (s)[C

a2+] c

yt (n

M)

1 µM TG A B

C

0 2 4 6 8 10 12 14 16 18 200

20

40

60

80MDEB 10 µMMDEB 30 µMMDEB 100 µMMDEB 300 µMMDEB 1000 µM

vehicle

Time (s)

Frac

tiona

l loss

(% /

2 m

in)

0

50

100

IP3

- in

duce

dC

a2+ re

leas

e (%

)

10 30 100 300 1000 MDEB (µM)

D

Figure 7

Page 41 of 43

Accep

ted

Man

uscr

ipt

Figure 8

A B

10 min 3 h 24 h0

200

400

600

800

1000

10 µg/ml PHA1 mM MDEB10 µg/ml PHA + 1 mM MDEB

control

Time

Rest

ing

[Ca2+

] cyt

(nM

)

0 200 400 6000

200

400

600

800

1000

control+ 1 mM MDEB+ 10 µg/ml PHA+ 10 µg/ml PHA + 1 mM MDEB+ 30 µM 2-APB

Time (s)

[Ca2+

] cyt

(nM

)

0 200 400 6000

200

400

600

800

1000control+ 10 µg/ml PHA

+ 1 µg/ml PHA + 1 mM MDEB+ 1 µg/ml PHA

Time (s)

[Ca2+

] cyt

(nM

)

C D

10 min 3 h 24 h0

100

200

300

4001 µg/ml PHA1 mM MDEB1 µg/ml PHA + 1 mM MDEB

control

Time

Rest

ing

[Ca2+

] cyt

(nM

)

*

*

* * * * #

#

#

Figure 8

Page 42 of 43

Accep

ted

Man

uscr

ipt

Figure 9

0 1 10 100 10000

10

20

30

40

50 PHA 1 µg/mlPHA 10 µg/ml

[MDEB] (µM)

Dea

d ce

lls (%

)

0 1 10 100 10000

50

100

150

200

PHA 0PHA 1 µg/mlPHA 10 µg/ml

[MDEB] (µM)

IL-2

(pg/

ml)

per l

iving

cel

ls

A

B

* * * *

*

* * * *

# * *

Figure 9

Page 43 of 43

Accep

ted

Man

uscr

ipt

Figure 10

Control + MDEB + PHA + PHA + MDEB

DAPI

Tunel

0 6 12 240

10

20

30

40PHA 10 µg/ml + MDEB 1 mM

Time (h)

% a

popt

otic

cel

ls

A

B

* * *

Figure 10

Documents

Potentiation of the Store-Operated Calcium Entry (SOCE ... · Potentiation of the Store-Operated Calcium Entry (SOCE) induces phytohemagglutinin-activated Jurkat T cell apoptosis