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PHAGOCYTES
Lineage-Specific Modulation of Calcium Pump Expression
During Myeloid Differentiation
By Sophie Launay, Maurizio Giannı̀, Tünde Kovàcs, Raymonde Bredoux, Arlette Bruel, Pascal Gélébart,
Fabien Zassadowski, Christine Chomienne, Jocelyne Enouf, and Béla Papp
Calcium is accumulated from the cytosol into the endoplasmic reticulum by sarco-endoplasmic reticulum calcium transport ATPase (SERCA) enzymes. Because calcium stored in
the endoplasmic reticulum is essential for cell growth,
differentiation, calcium signaling, and apoptosis and because different SERCA enzymes possess distinct functional
characteristics, in the present report we explored SERCA
expression during in vitro differentiation of the human
myeloid/promyelocytic cell lines HL-60 and NB4 and of
freshly isolated acute promyelocytic leukemia cells. Two
SERCA species have been found to be coexpressed in these
cells: SERCA 2b and another isoform, SERCAPLIM, which is
recognized by the PLIM430 monoclonal antibody. Induction
of differentiation along the neutrophil granulocytic lineage
by all-trans retinoic acid or cyclic AMP analogs led to an
increased expression of SERCAPLIM, whereas the expression
of the SERCA 2b isoform was decreased. The modulation of
SERCA expression was manifest also on the mRNA level.
Experiments with retinoic acid receptor isoform-specific
retinoids indicated that SERCA expression is modulated by
retinoic acid receptor ␣-dependent signaling. SERCA expression of retinoic acid-resistant cell variants was refractory to
treatment. Differentiation along the monocyte/macrophage
lineage by phorbol ester resulted in an increased expression
of both SERCA isoforms. In addition, when cells were treated
by phorbol ester in the presence of the glucocorticoid
dexamethasone, a known inhibitor of monocyte differentiation, a selective blockage of the induction of SERCAPLIM was
observed. Altered SERCA expression modified the functional
characteristics of calcium transport into the endoplasmic
reticulum. These observations show for the first time that
the modulation of calcium pump expression is an integral
component of the differentiation program of myeloid precursors and indicate that a lineage-specific remodelling of the
endoplasmic reticulum occurs during cell maturation. In
addition, these data show that SERCA isoforms may serve as
useful markers for the study of myeloid differentiation.
r 1999 by The American Society of Hematology.
A
expression levels of the two coexpressed SERCA isoenzymes
vary depending on blood cell type,21 and the two enzymes are
associated with functionally distinct subcompartments of the
ER27 and possess distinct biochemical and pharmacological
characteristics such as calcium affinity24 or sensitivity to
inhibitors.21 Taken together, these observations suggest that the
two SERCA enzymes play functionally specialized, distinct
roles within the same cell.
Myeloid differentiation is accompanied by the acquisition of
new signaling, as well as effector functions, such as responsiveness to bacterial endotoxin, to chemotactic peptides, or to
growth factors and chemokines, phagocytosis, or respiratory
burst formation. Given that cellular calcium homeostasis and
calcium-dependent signaling are intimately involved in these
CCUMULATION OF CALCIUM ions from the cytosol
into the endoplasmic reticulum (ER) is accomplished by
various sarco-endoplasmic reticulum calcium transport ATPase
(SERCA) enzymes.1 Because calcium stored in the endoplasmic reticulum is required for second messenger-induced calcium mobilization2-4 as well as for the posttranslational processing of nascent proteins in the ER lumen,5,6 calcium pumping
into this organelle is essential for a large array of cell functions.
The direct inhibition of SERCA activity by cell permeable
drugs such as thapsigargin can induce cell activation leading to
differentiation,7,8 growth arrest,9 apoptosis,10-15 or enhanced
human immunodeficiency virus (HIV) production,16 depending
on cell type, indicating that SERCA activity represents an
important control mechanism of various types of cell activation.
Direct functional association of a SERCA enzyme with Bcl-2,
resulting in the modulation of the apoptotic potential of the cell,
has also been reported.17
The expression and alternative RNA splicing of the three
known human SERCA genes is tissue-dependent and developmentally regulated. The SERCA 1a and 1b isoenzymes are
expressed in adult and neonatal skeletal muscle, respectively.18
Whereas SERCA 2a is expressed in cardiac muscle, SERCA 2b
has been found ubiquitously in all nonmuscle cell types studied
so far.19,20 Cells of hematopoietic origin coexpress SERCA 2b,
recognized by the IID8 antibody and another SERCA-type
calcium pump, termed SERCAPLIM, recognized by the PLIM430
monoclonal antibody.21,22 Recent data on the tissue distribution
of SERCA 3 mRNA23,24 and analysis of the recognition pattern
by PLIM430 of recombinant SERCA 3 proteins25 suggest that
SERCAPLIM corresponds to an alternatively spliced SERCA 3
variant, the SERCA 3b isoform. Interestingly, and in accordance
with the presence of a GATA motif in the SERCA 3 promoter,26
the expression of the SERCAPLIM enzyme appears to be
restricted to the hematopoietic lineage.21 In addition, the
Blood, Vol 93, No 12 (June 15), 1999: pp 4395-4405
From U. 348 INSERM, IFR Circulation Lariboisière, Hôpital Lariboisière, Paris, France; CNRS UPR 9051, INSERM U. 301, and CNRS EP
107, Hôpital Saint Louis, Paris, France; and the National Institute of
Haematology, Budapest, Hungary.
Submitted August 18, 1998; accepted February 17, 1999.
This work is dedicated to the memory of Jacques Maclouf.
Supported by the Institut National de la Santé, et de la Recherche
Médicale Reseau Est-Ouest No. 4E004B, the Association pour la
Recherche sur le Cancer, and the Agence Nationale pour la Recherche
sur le SIDA, France. M.G. was supported by a fellowship from the Ligue
Nationale contre le Cancer and the Fondazione Italiana per la Ricerca
sul Cancro.
Address reprint requests to Béla Papp, PhD, U. 348 INSERM,
Hôpital Lariboisière, 8, rue Guy Patin, 75475 Paris Cedex 10, France;
e-mail: [email protected].
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1999 by The American Society of Hematology.
0006-4971/99/9312-0006$3.00/0
4395
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4396
LAUNAY ET AL
processes,28-30 intracellular calcium transport may be significantly remodelled during differentiation.
The NB4 promyelocytic31 and HL-60 myeloblastic32 leukemia cell lines offer a very useful model to study in vitro myeloid
differentiation. Upon treatment with all-trans retinoic acid
(ATRA), dimethylsulfoxide (DMSO), or cAMP analogs, these
cells readily undergo terminal neutrophil granulocytic differentiation.31-35 ATRA exerts a wide range of effects on cell
proliferation and differentiation.36 These activities are mediated
by at least two distinct classes of nuclear receptors37-39: the
retinoic acid receptors (RARs), which include RAR␣, RAR␤,
and RAR␥; and the retinoid X receptors (RXRs), which include
RXR␣, RXR␤, and RXR␥. RARs display high affinity towards
ATRA as well as 9-cis-retinoic acid,38 whereas RXRs have
9-cis-retinoic acid as a natural ligand.40 After the binding of
retinoids, RAR and RXR homodimers or heterodimers regulate
the expression of specific genes, coding for proteins involved in
differentiation and/or growth arrest.41 In basal conditions, NB4
and HL-60 cells express RAR␣ and RXRs, whereas expression
of RAR␤ and RAR␥ is not observed.38,39,42 ATRA-induced
differentiation of HL-60 is essentially mediated by RAR␣,35
whereas in NB4 cells, which have been derived from an acute
promyelocytic leukemia (APL) patient,31 differentiation may
also be mediated by the PML-RAR␣ oncogenic protein.43 In
NB4 or fresh APL cells, ATRA administered at pharmacological
concentrations (10⫺6 mol/L) circumvents the differentiation
blockage and induces the maturation of the cells along the
granulocytic lineage.44 The maturation of NB4 cells can be
induced also by other cyto-differentiating agents, such as cell
permeable cAMP analogs.34 This cell line represents an in vitro
model generally predictive for the behavior of freshly isolated
APL blasts upon culturing with ATRA, retinoic acid derivatives,
or other compounds. This finding is of clinical importance,
because differentiation therapy using retinoids is a very successful clinical modality in the treatment of APL in vivo.44,45
However, ATRA monotherapy may be complicated by the
emergence of a differentiation-resistant malignant cell population.46,47
In addition to their granulocytic differentiation potential,
phorbol ester treatment induces a monocyte/macrophage-like
phenotype in HL-60 cells12,29,48,49 that can be further modulated
by glucocorticoids.50-52 This bilineage differentiation potential
and the availability of various ATRA-resistant clonal derivatives53-56 of HL-60 and NB4 represent an interesting in vitro
system for the study of phenotypic changes occuring upon
drug-induced myeloid differentiation.
To gain insight into the involvement of SERCA enzymes in
myeloid differentiation, in differentiation-induction therapy of
APL, and in ATRA resistance, in the present report we
investigated the expression of the two SERCA isoforms during
in vitro differentiation of HL-60 and NB4 cells and their
differentiation-defective variants and of primary APL cells.
MATERIALS AND METHODS
Cells. HL-60 cells57 were obtained from the ATCC (Rockville,
MD). The ATRA-resistant HL-60 variant53 was a generous gift of Dr
Robert Gallagher (Montefiore Medical Center, Bronx, NY). NB4 cells,
as well as the NB4-R1 and R2 variants, were described previously.31,54-56 All cells were grown in RPMI-1640 medium (GIBCO-BRL
Paisley, UK) supplemented with glutamax-I, 2 mmol/L glutamine, and
10% heat-inactivated fetal calf serum at 37°C in a humidified atmosphere containing 5% carbon dioxide.
Chemicals. ATRA, phorbol 12-myristate 13-acetate (PMA), 8-(4chlorophenylthio)-adenosine 38:58-cyclic monophosphate (CTP-cAMP),
dexamethasone, and nitroblue tetrazolium (NBT) were purchased from
Sigma-Aldrich (St Louis, MO). TTNPB, Ro 41-5253, and Ro 61-8432
were kindly provided by Dr M. Klaus (Hoffman-la Roche, Basel, Switzerland). AM580 and CD2019 were synthetized by CIRD-Galderma (Sophia
Antipolis, Valbonne, France). SR 11237 was kindly provided by Dr H.
Gronemeyer (IGBMC, Strasbourg, Marseille, France). The Bear-1 and
the BU15 monoclonal antibodies directed against CD11b (integrin ␣M
subunit) and CD11c (integrin ␣X-chain), respectively, as well as
isotype-matched control antibody and fluorescein-conjugated antimouse IgG were obtained from Coulter/Immunotech (Marseille, France)
and were used for flow cytometry according to the instructions of the
manufacturer. The IID8 anti-SERCA 2 antibody was purchased from
BioMol (Plymouth Meeting, PA) and the PLIM430 antibody was
purified from hybridoma supernatant by protein-A chromatography.
Induction of cell differentiation. Before the experiments, exponentially growing cells were harvested and resuspended in the abovedescribed medium at a density of 2 ⫻ 105 cells/mL. Retinoids, PMA, or
dexamethasone were added to the cells from concentrated stock
solutions in DMSO. The amount of DMSO vehicle added to the cells
did not exceed 0.1%, was included in control experiments, and did not
interfere with the assays. In experiments in which retinoic acid was used
in combination with antagonists, these were preincubated with the cells
for 1 hour before the addition of retinoic acid. Differentiation of cells
was assessed by measuring NADPH-oxydase activity using NBT in the
presence of PMA over a period of 30 minutes at 37°C (as described
earlier58), by staining for naphtyl-acetate esterase using a commercially
available kit obtained from Sigma-Aldrich, and by light microscopy of
May-Grünwald-Giemsa–stained cytospin preparations. Fluorescenceactivated cell sorting (FACS) analysis of CD11b and CD11c antigen
expression of differentiating cells was performed on a FACS-Calibur
flow cytometer (Becton Dickinson, Mountain View, CA) by standard
protocols.
Primary APL cells. After we received informed consent, primary
APL cells were obtained by Ficoll gradient centrifugation from bone
marrow aspirates of 5 newly diagnosed APL patients presenting an
initial percentage of blasts of more than 80%. Cells were resuspended in
complete medium at a density of 106 cells/mL and treated for 1 week
with 0.1 µmol/L ATRA. Differentiation of the cells was evaluated by
NBT reduction and by microscopical examination. The presence of the
t(15;17) translocation in the cells was confirmed by reverse transcriptasepolymerase chain reaction (RT-PCR) amplification of the PML-RAR␣
fusion transcript.59
Immunoblotting. After treatment cell counts and viabilities were
determined, cells were washed once with PBS, resuspended in cold 5%
trichloroacetic acid, and kept at 4°C for 1 hour. The precipitate was then
centrifuged for 15 minutes at 12,000g at 4°C, the pellet was dissolved in
electrophoresis sample buffer as described,60 and the protein concentration of the lysate was determined. Five microliter to 25 µL samples
containing 20 µg cellular protein per well were run on sodium dodecyl
sulfate (SDS)-polyacrylamide gels, electroblotted onto nitrocellulose,
and immunostained as described previously.60 Luminograms were
quantitated using an LKB laser densitometer.60
RNA isolation and RT-PCR. Total RNA was isolated from cells
using the RNAPlus solution according to the manufacturers’ instructions (Quantum Bioprobe, Montreuil sous Bois, France). Reverse
transcription of 500 ng total RNA was performed essentially as
described,23 with the modifications as follows: RT reaction was used as
template for PCR reaction in a 50 µL reaction mixture including PCR
buffer, 2 or 1.5 mmol/L MgCl2 (for SERCA 2b or SERCA 3b,
respectively), 0.15 µmol/L primers, and 1.5 U of AmpliTaq DNA
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CALCIUM PUMPS AND MYELOID DIFFERENTIATION
polymerase. The reaction was heated to 94°C for 3 minutes and 10
cycles of touchdown PCR were performed as described previously61 to
increase the specificity of priming during the initial cycles of amplification. PCR was thereafter performed as described23 for 18 and 20
amplification cycles for SERCA 2b and SERCA 3b, respectively. One
amplification cycle consisted of 1 minute at 94°C; 1 minute at 55°C or
58°C for SERCA 2b and SERCA 3b, respectively; and 1 minute at
72°C. The last extension step at 72°C was performed for 7 minutes. As
an internal control, RT-PCR amplification of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was performed. The following primers
were used: SERCA 2b: forward primer, 58 TCA TCT TCC AGA TCA
CAC CGC T 38 located at nt 2861-2882, and reverse primer, 58 TCA
AGA CCA GAA CAT ATC GC 38, corresponding to the inverse
complementary sequence of nt 3110-3129 of the human SERCA 2b
sequence19; and SERCA 3b: forward primer, 58 GAG TCA CGC TTC
CCC ACC ACC 38 located at nt 2674-2694, and reverse primer, 58 GGC
TCA TTT CTT CCG GTG TGG TC 38, corresponding to the nucleotide
stretch located at nt 3058-3080 of the human SERCA 3b sequence.26
Amplification products were separated on 1.5% agarose gels, blotted
onto Hybond Z⫹ nylon membranes (Quantum Bioprobe), and visualized by Southern blotting using the Amersham ECL 38-oligolabeling
and detection system according to the instructions of the manufacturer
(Amersham, Little Chalfont, UK). The following oligonucleotide
probes were used for Southern detection: SERCA 2b: nt 2956-2992 of
the human SERCA 2 cDNA sequence19; SERCA 3b: nt 3017-3057 of
the human SERCA 3 cDNA sequence26; and G3PDH: nt 328-357 of the
human cDNA sequence.62 Prehybridization (30 minutes) and hybridization (2 hours) were performed at 42°C with 5 ng/mL labeled oligonucleotide probes. Membranes were then washed twice at 50°C in 0.5⫻ SSC,
0.1% SDS for 15 minutes and chemiluminescent signal was detected
with Hyperfilm ECL (Amersham). The molecular mass of the obtained
amplification products corresponded to that calculated based on the
published sequences. Moreover, the identity of the SERCA amplification products was also confirmed by direct sequencing (performed by
Eurogentec, Seraing, Belgium).
Membrane preparation and calcium transport. HL-60 cells grown
for 4 days in the presence or absence of 1 µmol/L ATRA were harvested
by centrifugation, washed once with 160 mmol/L KCl, 17 mmol/L
HEPES-K (pH 7.0), and lysed by 100 strokes in a teflon-glass
homogenizer on ice in a lysis buffer containing 10 mmol/L KCl,
10 mmol/L HEPES-K (pH 7.0), 50 µmol/L EDTA, 50 µmol/L EGTA,
100 µmol/L dithiothreitol, 0.1 mg/mL aprotinin, 0.1 mg/mL BowmanBirk trypsin-chymotrypsin inhibitor, 0.2 mg/mL soybean trypsin inhibitor, 0.125 mg/mL leupeptin, and 0.05 mg/mL pepstatin-A. The cell
lysate was centrifuged at 1,600g for 10 minutes at 4°C. Supernatant was
then centrifuged at 100,000g for 1 hour at 4°C. The obtained pellet was
resuspended in a buffer containing 30 mmol/L KCl, 17 mmol/L
HEPES-K (pH 7.0), and 0.2 mmol/L dithiothreitol, aliquoted, frozen
immediately in liquid nitrogen, and kept at ⫺80°C. Calcium influx into
membrane vesicles prepared from control and ATRA-treated HL-60
cells was measured at 37°C for 3 minutes by rapid filtration, as
described earlier.27 The transport medium contained 200 µg membrane
protein per milliliter, 119 mmol/L KCl, 43 mmol/L HEPES-K (pH 7.2),
2 mmol/L MgCl2, 1 mmol/L dithiothreitol, 0.01 mg/mL aprotinin, 0.01
mg/mL leupeptin, 100 µmol/L CaCl2 (labeled with 45Ca2⫹), and 110
µmol/L EGTA (to obtain 1.3 µmol/L free Ca2⫹ concentration). The free
calcium concentration was superior to the calcium affinities of both
SERCA 2 and SERCA 3 enzymes (0.2 and 1.1 µmol/L, respectively),63
permitting the simultaneous measurment of calcium transport activity
of both enzymes. Under these conditions, Ca2⫹ uptake by HL-60
membrane vesicles was linear for 6 to 8 minutes. To measure Ca2⫹
uptake by SERCAPLIM, the membrane vesicles were preincubated with
30 µg/mL purified PLIM430 antibody (which inhibits the transport
activity of SERCAPLIM selectively27,64) for 30 minutes before initiating
Ca2⫹ uptake by the addition of 0.5 mmol/L ATP, as described.27 Ca2⫹
4397
uptake by contaminating plasma membrane-type calcium pumps was
determined by preincubating the vesicles at 37°C for 10 minutes with 1
µmol/L thapsigargin (an inhibitor of total SERCA transport activity that
is inactive on plasma membrane-type calcium pumps) before the
addition of ATP. To calculate the total SERCA-dependent Ca2⫹ uptake,
thapsigargin-resistant Ca2⫹ uptake was substracted from total Ca2⫹
uptake values.
Experiments shown here were performed three or more times
(specified in the figure legends) and are presented as the means ⫾
standard error of the mean (SEM).
RESULTS
Time course of the modulation of SERCA expression in
ATRA-treated HL-60 cells. As shown in Fig 1A, E, and F,
treatment of HL-60 cells with 1 µmol/L ATRA for 5 days
resulted in an approximately fourfold (3.72- ⫾ 0.46-fold; n ⫽ 7)
overexpression of SERCAPLIM and a concomitant downregulation (to 41.6% ⫾ 8.7%; n ⫽ 7) of the expression of the SERCA
2b isoform, as detected by the PLIM430 and the IID8 antibodies, respectively. During this treatment, and in accordance with
data in the literature,35 the cells underwent terminal granulocytic differentiation as reflected by the aquisition of NADPH
oxidase activity measured by NBT reduction (Fig 1C), induction of CD11b expression (Fig 1D), appearence of U-shaped
cell nuclei and accumulation of granules in the cytosol (not
shown), and growth arrest (Fig 1B). Similar results were
obtained on SERCAPLIM expression also in NB4 cells (see
later).
Estimation of SERCA mRNA in ATRA-treated HL-60 cells.
Recent data in the literature indicate that SERCAPLIM recognizes the SERCA 3b splice variant, which is expressed preferentially in cells of hematopoietic origin. To estimate the modulation of SERCA mRNA levels during myeloid differentiation, we
designed RT-PCR systems for the amplification of SERCA 2b
and SERCA 3b and compared the relative mRNA levels in
control and ATRA-treated HL-60 cells. As shown Fig 1G, H,
and I, whereas SERCA 2b mRNA levels progressively decreased to 50% of the control value during ATRA treatment,
SERCA 3b mRNA was induced approximately 2.5-fold. These
data indicate that the modulation of SERCA expression during
ATRA-induced differentiation of HL-60 cells is controlled, at
least in part, on the transcriptional level. As an internal control,
G3PDH was also amplified. mRNA levels of this constitutively
expressed housekeeping enzyme did not change significantly
during the treatments. Whereas SERCA 2b levels decreased
steadily during the treatment, SERCA 3b mRNA peaked at day
1 posttreatment and remained elevated thereafter. The differences between the time course of the induction of SERCA
3b mRNA and protein are probably due to the combined effect
of time required for mRNA translation and posttranslational
processing of nascent SERCA 3b, to differences of half lives of
mRNA and protein, and to the fact that, during treatment, cells
assume growth arrest, permitting accumulation of newly formed
SERCA protein within the cell.
Concentration-dependent modulation of SERCA expression
by ATRA and cAMP. HL-60 cells were exposed for 4 days to
various concentrations of ATRA and SERCA expression was
determined by immunoblotting. In agreement with in vitro as
well as in vivo data in the literature and as determined by NBT
reduction here (Fig 2B), cell differentiation was induced at
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4398
LAUNAY ET AL
Fig 1. Time course of the
modulation of calcium pump expression in ATRA-treated HL-60
cells. HL-60 cells were treated
with 1 ␮mol/L ATRA during 5
days and calcium pump expression was determined by discriminating monoclonal antibodies. In
parallel, cell differentiation was
detected by NBT reduction,
CD11b expression, and growth
arrest. (A) Immunostaining for
SERCA 2b and SERCAPLIM with
the IID8 and PLIM430 antibodies,
respectively. (B) Inhibition of cell
proliferation by ATRA. (䉫) ATRAtreated cells; (䉬) untreated cells.
(C) NADPH oxidase activity of
the cells measured by NBT reduction. Data presented are the
mean ⴞ SE of 7 experiments.
(D) Induction of CD11b expression by ATRA-treated cells. (E
and F) Densitometric analysis of
SERCAPLIM and SERCA 2b expression, respectively. (G, H, and I)
Estimation of the relative abondance of SERCA mRNA species
in HL-60 cells during ATRA-induced differentiation. RNA was
isolated from HL-60 cells treated
with 1 ␮mol/L ATRA, and SERCA
mRNA was amplified by RT-PCR
using isoform-specific oligonucleotide primers. As an internal control, RT-PCR using G3PDHspecific primers was used. (H)
(䊉) SERCA 3b. (I) (䊐) SERCA 2b;
(䉬) G3PDH. Data presented are
the mean ⴞ SEM of 3 experiments.
submicromolar to low micromolar concentrations of ATRA.
This result was accompanied by the induction of the expression
of SERCAPLIM (Fig 2A and C) and the downmodulation of the
expression of SERCA 2b (Fig 2A and D) in the same concentration range. Retinoic acid treatment did not modify expression
levels of either SERCA isoform in the ATRA-resistant65 K-562
myelogenous leukemia cells (not shown).
The modulation of calcium pump expression could be
obtained by using other established inducers of granulocytic
differentiation such as 9-cis-retinoic acid and DMSO (not
shown) or by cAMP analogs (Fig 3A and B).
Modulation of calcium transport function by ATRA-induced
differentiation. To gain insight into the functional consequences on cellular calcium homeostasis of the modulation of
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CALCIUM PUMPS AND MYELOID DIFFERENTIATION
4399
Fig 2. Concentration dependence of ATRA-induced modulation of calcium pump expression. HL-60 cells were treated for
4 days with various concentrations of ATRA and calcium pump
expression; NBT reduction by the
cells was also determined. (A)
Immunostaining for SERCA 2b
and SERCAPLIM with the IID8 and
PLIM430 antibodies, respectively.
(B) NADPH oxidase activity of
the cells as measured by NBT
reduction. (C and D) Densitometric analysis of SERCAPLIM and
SERCA 2b expression. Data represent the mean ⴞ SEM of 5
experiments.
SERCA expression during differentiation, we investigated ATPdriven active calcium transport into microsomal membrane
preparations obtained from untreated and retinoic aciddifferentiated HL-60 cells. Calcium uptake was determined in
the absence or presence of thapsigargin (an inhibitor of total
SERCA activity, ie, SERCA 2b plus SERCAPLIM) or in the
presence of purified PLIM430 antibody. This antibody inhibits
calcium transport by its cognate antigen, SERCAPLIM selectively, and therefore can be used as a functional probe in the
analysis of calcium transport.27,64 As shown in Table 1, in
membranes prepared from undifferentiated cells, PLIM430inhibitable calcium transport accounted for 32% of total SERCAdependent calcium accumulation. Although total SERCAdependent calcium transport did not change significantly after
differentiation (0.55 v 0.57 nmol Ca2⫹/mg membrane protein),
in membranes prepared from retinoic acid-differentiated cells,
PLIM430-inhibitable transport was increased to 60%. Increased
SERCAPLIM expression combined with the concomitant decrease of SERCA 2b expression thus resulted in an approximately twofold shift towards calcium uptake into the
SERCAPLIM-associated calcium pool, indicating that the modification of SERCA protein levels upon differentiation results in
the modification of calcium transport function as well.
Effect of synthetic retinoids on SERCA expression. To
define the retinoic acid receptor isoforms involved in the
modulation of calcium pump expression, we treated HL-60 and
NB4 cells with activators and antagonists of different specificities towards the various retinoic acid receptor subtypes for 4
days. As shown in Fig 4, TTNPB (a pan-RAR agonist66),
similarly to ATRA, induced both SERCAPLIM expression
(Fig 4A) and granulocytic differentiation (assessed by NBT
reduction; not shown). As expected, Ro-618431 (a pan-RAR
antagonist67) inhibited the effect (Fig 4A). On the other hand,
SR 11237 (a pan-RXR agonist68) was without effect on NB4
Fig 3. Modulation of calcium pump expression by
cAMP. HL-60 cells were treated with various concentrations of the cell-permeable cAMP analog CTPcAMP for 4 days and SERCA expression was determined. (A and B) Concentration dependence of the
modulation of SERCAPLIM and SERCA 2b expression
by cAMP, respectively. Data represent the mean ⴞ
SEM of 3 experiments.
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LAUNAY ET AL
Table 1. Calcium Uptake Into Microsomal Membrane Vesicles
Prepared From Control and ATRA Differentiated HL-60 Cells
Calcium Uptake (pmol Ca2⫹/mg
membrane protein ⫾ SEM)
Total SERCA-dependent Ca2⫹
uptake
SERCA 2b-dependent Ca2⫹
uptake
SERCAPLIM-dependent Ca2⫹
uptake
Untreated Cells
ATRA-Treated Cells
554 ⫾ 13
572 ⫾ 43
375 ⫾ 13
222 ⫾ 46
181 ⫾ 6
346 ⫾ 21
tive induction of SERCAPLIM expression is seen (Fig 1), PMA
treatment of HL-60 cells resulted in the induction of the
expression of both SERCA isoforms. This was accompanied by
immediate growth arrest, assumption of an adherent phenotype,
and the induction of the expression of nonspecific esterase (all
cells becoming positive upon microscopical examination start-
Calcium transport by SERCA 2b and SERCAPLIM into membrane
vesicles prepared from untreated and ATRA-treated HL-60 cells was
determined. Although total SERCA-dependent calcium uptake did not
change significantly during differentiation, calcium transport into the
SERCAPLIM-associated calcium pool increased from 32% of total SERCAdependent calcium accumulation to 60% after ATRA treatment.
cells (not shown). These results suggest that the RAR family
rather than the RXR family is involved in the modulation of
SERCA expression. The RAR␣ selective agonist AM58042
induced SERCA expression (as well as differentiation) in the
submicromolar range (Fig 4B), whereas the RAR␤ specific
agonist CD201942 was without effect on these cells (not shown).
Ro 41-5253 (an RAR␣-selective antagonist69) could inhibit the
effect of ATRA (Fig 4C). Taken together, these data indicate that
the modulation of SERCA expression by ATRA proceeds via
RAR␣-dependent signaling.
Modulation of calcium pump expression by ATRA in fresh
APL cells. APL blasts isolated from 5 newly diagnosed
patients were treated with 0.1 µmol/L ATRA, and calcium pump
expression and cell differentiation was determined. As shown in
Fig 5, ATRA treatment specifically induced an approximately
threefold overexpression of SERCAPLIM in all 5 cases, whereas
the expression of SERCA 2b decreased or did not change
significantly. During treatment, the cells underwent terminal
granulocytic differentiation, as detected by NBT reduction and
morphological examination.
Lack of modulation of SERCA expression in ATRA-resistant
cells. Continuous ATRA therapy in APL patients after complete remission may be complicated by the emergence of a
malignant cell population that fails to differentiate upon ATRA
treatment. The expression of SERCA enzymes in differentiationresistant variants of HL-60 and NB4 cells was not modified by
ATRA. Whereas in wild-type cells ATRA induced a significant
overexpression of SERCAPLIM, in ATRA-resistant HL-60RES
and NB4-R2 cells (Fig 6), SERCA expression levels as well as
NADPH oxidase activity (not shown) remained essentially
unchanged upon ATRA treatment. In NB4-R1 cells, ATRA
treatment resulted in a very modest induction of SERCAPLIM
(Fig 6). This is in agreement with previous data indicating that
NB4-R1 cells undergo a very limited differentiation compared
with wild-type NB4 cells55 under the experimental conditions
used.
Modulation of SERCA expression during differentiation of
HL-60 cells towards macrophage-like cells. Upon PMA treatment, HL-60 cells display a mature macrophage-like phenotype.48 As shown in Fig 7A and B and in contrast with
ATRA-induced granulocytic differentiation, in which a selec-
Fig 4. Treatment of HL-60 and NB4 cells by retinoids in the
presence of retinoic acid receptor antagonists. (䊏) SERCAPLIM; (䊐)
SERCA 2b. (A) Treatment of NB4 cells by 0.1 ␮mol/L TTNPB (a
pan-RAR agonist) in the presence or absence of 10 ␮mol/L Ro61-8431
(a pan-RAR antagonist). (B) Treatment of NB4 cells with various
concentrations of AM580 (an RAR␣ selective agonist). (C) Treatment
of HL-60 cells with 0.1 ␮mol/L ATRA in the presence or absence of 10
␮mol/L Ro41-5253 (an RAR␣ specific antagonist). Data represent the
mean ⴞ SEM of 3 experiments.
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CALCIUM PUMPS AND MYELOID DIFFERENTIATION
Fig 5. Effect of ATRA treatment on the SERCA expression of fresh
APL cells. Freshly isolated APL blasts were treated by 0.1 ␮mol/L
ATRA over 7 days and SERCA expression; cell differentiation was also
determined. (A) Immunoblot analysis of SERCA 2b and SERCAPLIM
expression. (B) Modulation of SERCA expression upon ATRA treatment. One hundred percent refers to SERCA expression levels in
untreated cells. (䊏) SERCAPLIM; (䊐) SERCA 2b. Treatment resulted in a
marked induction of SERCAPLIM expression. (Inset) The percentage of
NBT-positive cells after ATRA treatment.
4401
mRNA level. Observations made with agonists and antagonists
of distinct selectivity towards different retinoic acid receptors
indicated that the modulation of calcium pump expression is
linked to RAR␣-dependent signaling. The modulation of SERCA
expression was a differentiation-associated phenomenon, because in differentiation-defective HL-60 and NB4 derivatives,
the same treatment failed to modulate SERCA expression
significantly. The modulation of calcium pump expression
during differentiation could also be demonstrated in primary
APL cells. Changes in SERCA protein expression resulted in
the modification of calcium transport function, as reflected by a
significant shift in calcium accumulation into SERCAPLIMversus SERCA 2b-associated calcium pool in differentiated
cells, as compared with untreated control.
In contrast to granulocytic differentiation, phorbol esterinduced differentiation towards the monocyte/macrophage lineage resulted in the simultaneous induction of the expression of
both SERCA isoforms. In addition, when the cells were treated
with phorbol ester in combination with the glucocorticoid
dexamethasone, a marked and selective blockage of the induction of SERCAPLIM was observed. This is in agreement with
data in the literature indicating the presence of a putative
glucocorticoid-responsive element in the SERCA 3 promoter,26
with the observation that PMA induces the expression of the
glucocorticoid receptor in HL-60 cells,70 and with the observation that dexamethasone inhibits the expression of markers of
monocytic differentiation and impairs macrophage function.50-52
The endoplasmic reticulum consists of a dynamically interconnected membrane network71,72 that is involved in several
distinct functions, such as protein posttranscriptional modification, sorting, secretion,5,6 and calcium-dependent signal transduction.3,4 In structural terms, ER multifunctionality is reflected
by the uneven distribution of ER-associated proteins within the
organelle,73-75 forming functionally distinct regions. In particular, regions possessing distinct densities of SERCA proteins and
D-myo-inositol 1,4,5-trisphosphate (IP3) receptors have been
ing at day 1, as compared with less than 5% in untreated cells),
and a strong induction of CD11c expression (Fig 7C).
Because dexamethasone has been described to impair the
differentiation of monocytes,50-52 we investigated the effect of
this glucocorticoid on PMA-induced SERCA expression of
HL-60 cells. When PMA treatment was performed in the
presence of dexamethasone, the induction of SERCAPLIM was
selectively and efficiently inhibited by submicromolar concentrations of the glucocorticoid (Fig 7D and E). Dexamethasone,
when applied alone, did not have an appreciable effect on
SERCA expression.
DISCUSSION
The data presented in this report show for the first time that
intracellular calcium pump expression is modulated during
myeloid differentiation in a lineage-specific manner. Granulocytic differentiation resulted in increased expression of
SERCAPLIM, whereas SERCA 2b expression was decreased.
This phenomenon was manifest on the protein as well as on the
Fig 6. SERCA expression in ATRA-resistant HL-60 and NB4 cells.
Wild-type and ATRA-resistant HL-60 and NB4 cells were treated with
1 ␮mol/L of ATRA for 4 days and SERCA expression was determined.
(䊏) SERCAPLIM; (䊐) SERCA 2b. Data are the mean ⴞ SEM of 3
experiments.
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4402
LAUNAY ET AL
Fig 7. Differentiation of HL-60
cells towards macrophage-like
cells by PMA. HL-60 cells were
treated with 10ⴚ8 mol/L PMA for
6 days and SERCA expression
was determined. (A) Immunoblot staining for SERCA 2b and
SERCAPLIM using the IID8 and the
PLIM430 antibodies, respectively.
(B) Densitometric analysis of
SERCA expression in PMAtreated cells. (䊐) SERCA 2b; (䊉)
SERCAPLIM. (C) Induction of CD11c
expression by PMA-treated HL-60
cells. (D and E) Selective inhibition of the PMA-induced overexpression of SERCAPLIM by dexamethasone. HL-60 cells were
treated for 5 days with 10ⴚ8
mol/L PMA in the presence of
various concentrations of the glucocorticoid dexamethasone. (D)
Immunostaining for SERCA 2b
and SERCAPLIM using the IID8 and
PLIM430 monoclonal antibodies,
respectively. (E) Densitometric
analysis of the expression levels
of (䊐) SERCA 2b and (䊏)
SERCAPLIM in cells treated with
PMA and dexamethasone (100%
refers to SERCA expression of
cells treated with PMA only).
Data represent the mean ⴞ SEM
of 6 experiments.
described in nonmuscle cells,73 and the association of distinct
SERCA isoenzymes with IP3 mobilizable and IP3-resistant
calcium pools has been observed.27,76,77 Taken together, these
observations strongly suggest that the ER comprises connected
but functionally specialized subcompartments.
The different homeostatic, synthetic, and signaling systems
of the ER undergo a profound reorganization in structural as
well as functional terms when a proliferating, undifferentiated
cell undergoes differentiation and assumes a mature, quiescent
phenotype. Moreover, differentiation is associated with the
acquisition of new cellular structures involved in functions
characteristic of the differentiated cell. Several functions of the
differentiated granulocyte and monocyte-macrophage, such as
respiratory burst, phagocytosis, degranulation, responsiveness
to chemotactic peptides, cytokines, or chemokines, as well as
apoptosis, are calcium dependent.11,15,28-30,78 The acquisition of
these functions during cell differentiation implies a complex
and cell-type–specific remodelling and de novo synthesis of
calcium-dependent signaling and effector systems and structures involved. This is demonstrated in the present report by the
complex and specific modifications of the expression levels of
endomembrane calcium transport ATPases during differentiation.
The cell-type–dependent and isoform-specific modulation of
SERCA expression during granulocytic or monocyte/macrophage differentiation of promyelocytic cells may have an
important modulatory effect on cell activation. The two SERCA
isoenzymes possess distinct biochemical characteristics such as
calcium affinity79 or sensitivity towards biochemical regulation80 or pharmacological manipulation21 and are located in
distinct ER subcompartments.27,75-77 In particular, SERCAPLIM
has been shown to be specifically associated with the IP3sensitive calcium pool.27 Taken together, these data strongly
suggest that changes in SERCA isoform expression levels can
exert significant effects on the calcium homeostasis and signaling of the cell, even if total endomembrane calcium pump mass
or activity is unaltered. In light of data in the literature81 and our
own observations (not shown) on ATRA-treated HL-60 cells,
indicating that IP3-receptor levels are increased upon granulocytic differentiation, it is tempting therefore to speculate that
increased SERCAPLIM expression may reflect the enhanced
synthesis by the cell of an IP3-sensitive intracellular calcium
storage organelle required for the augmentation of the intensity
of extracellular stimulus-dependent calcium mobilization and
influx observed in differentiated cells as compared with undifferentiated control,82 whereas increased SERCA 2b expression
may be involved in ER functions linked to effector functions
such as phagocytosis.
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CALCIUM PUMPS AND MYELOID DIFFERENTIATION
The modulation of calcium pump isoform levels may modify
gene expression as well. It has been shown recently that the
modification of the frequency and geometry of cytosolic
calcium oscillations has a dramatic effect on the activation of
calcium-dependent enzymes and transcription factors regulating gene expression.83-87 Cytosolic calcium oscillations are
maintained by a dynamic interplay of calcium release and
elimination mechanisms.88-91 Because the calcium affinity of
SERCA 3 is inferior to that of SERCA 2b, overexpression of
SERCA 3 may lead to slower calcium elimination, higher
resting calcium levels, and an overall modification of the
activity of proinflammatory transcription factors such as NF-␬B,
NF-AT, or Oct/OAP.83-87
Although the elucidation of the intricate interplay of mechanisms regulating ER organellogenesis during differentiation as
well as the interaction of different intracellular calciumdependent signaling systems and calcium pools during cell
activation requires further study, the present work indicates that
SERCA enzymes are intimately involved in hematopoietic
differentiation and that the endoplasmic reticulum undergoes a
significant remodelling upon this process. The modulation of
the calcium homeostasis of the endoplasmic reticulum may
therefore offer new approaches in the pharmacological modulation of cell growth, differentiation, and apoptosis.
ACKNOWLEDGMENT
The authors are indebted to Dr Robert Gallagher for the ATRAresistant HL-60 cells and to Dr Neville Crawford for the PLIM430
hybridoma. We thank Dr Anabelle Le Grand and Laurent Barbe for their
help with flow cytometry. The discussions and support of Dr Sylviane
Lévy-Tolédano, Dr Michel Lanotte, Prof Hugues de Thé, and Dr Balàzs
Sarkadi are gratefully acknowledged.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1999 93: 4395-4405
Lineage-Specific Modulation of Calcium Pump Expression During Myeloid
Differentiation
Sophie Launay, Maurizio Gianni?, Tünde Kovàcs, Raymonde Bredoux, Arlette Bruel, Pascal Gélébart,
Fabien Zassadowski, Christine Chomienne, Jocelyne Enouf and Béla Papp
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