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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4395-4405
By
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.
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
ACCUMULATION 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 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 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 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.
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-(4-chlorophenylthio)-adenosine 3':5'-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 Induction of cell differentiation.
Before the experiments, exponentially growing cells were harvested and
resuspended in the above-described 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.
Fluorescence-activated 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
transcriptase-polymerase chain reaction (RT-PCR) amplification of the
PML-RAR 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 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, 5' TCA TCT
TCC AGA TCA CAC CGC T 3' located at nt 2861-2882, and reverse
primer, 5' TCA AGA CCA GAA CAT ATC GC 3', corresponding to
the inverse complementary sequence of nt 3110-3129 of the human SERCA
2b sequence19; and SERCA 3b: forward primer, 5' GAG
TCA CGC TTC CCC ACC ACC 3' located at nt 2674-2694, and reverse
primer, 5' GGC TCA TTT CTT CCG GTG TGG TC 3', 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 3'-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 Bowman-Birk
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 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 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).
Modulation of calcium transport function by ATRA-induced
differentiation.
To gain insight into the functional consequences on cellular calcium
homeostasis of the modulation of SERCA expression during differentiation, we investigated ATP-driven active calcium transport into microsomal membrane preparations obtained from untreated and
retinoic acid-differentiated 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, PLIM430-inhibitable calcium transport accounted
for 32% of total SERCA-dependent calcium accumulation. Although total
SERCA-dependent 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 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
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 differentiation-resistant 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 selective 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 starting at day 1, as compared
with less than 5% in untreated cells), and a strong induction of CD11c
expression (Fig 7C).
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 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 The authors are indebted to Dr Robert Gallagher for the ATRA-resistant
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.
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.
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.
Address reprint requests to Béla Papp, PhD, U. 348 INSERM, Hôpital Lariboisière, 8, rue Guy Patin, 75475 Paris
Cedex 10, France; e-mail: bela.papp{at}inserm.lrb.ap-hop-paris.fr.
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FEBS Let |
TOP
ABSTRACT
INTRODUCTION
-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.
, RAR
, and RAR
; and the retinoid X receptors
(RXRs), which include RXR
6 mol/L) circumvents the differentiation blockage
and induces the maturation of the cells along the granulocytic
lineage.
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.
) ATRA-treated cells; (
) untreated cells. (C) NADPH
oxidase activity of the cells measured by NBT reduction. Data presented
are the mean ± 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 µmol/L ATRA, and SERCA mRNA
was amplified by RT-PCR using isoform-specific oligonucleotide primers.
As an internal control, RT-PCR using G3PDH-specific primers was used.
(H) (
) SERCA 3b. (I) (
) SERCA 2b; (
)
SERCAPLIM; (
B, NF-AT, or Oct/OAP.