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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2742-2747
PLENARY PAPER
Specific and rapid induction of the proapoptotic protein Hrk
after growth factor withdrawal in hematopoietic progenitor cells
Cristina Sanz,
Adalberto Benito,
Naohiro Inohara,
Daryoush Ekhterae,
Gabriel Nunez, and
Jose Luis Fernandez-Luna
From the Servicio de Inmunologia, Hospital Universitario Marques de
Valdecilla, INSALUD, Santander, Spain, and Department of Pathology,
University of Michigan Medical School, Ann Arbor, MI.
 |
Abstract |
Hrk is a newly described proapoptotic member of the Bcl-2 family
that is mainly expressed in hematopoietic tissues and cultured neurons.
In this study we have examined the expression and activity of Hrk in
hematopoietic progenitors. To address these issues, we used 3 growth
factor-dependent murine hematopoietic cell lines, HCD-57, FDCP-Mix, and
FL5.12. The expression of Hrk was undetectable in cells cultured with
growth factors, but it was rapidly up-regulated on growth factor
withdrawal. In contrast, the expression of Bcl-xL decreased
and that of proapoptotic Bax, Bad, and Bak was unchanged or
down-regulated after removal of growth factors. This pattern of
expression correlated with the induction of apoptosis. Hrk was also up-regulated in human cell lines and in bone marrow-derived CD34+ cells cultured in the absence of growth factors. In
addition, the levels of Hrk were up-regulated after treatment with the
chemotherapeutic drug etoposide. Expression of prosurvival
Bcl-xL or Bcl-2 proteins blocked the induction of Hrk. Hrk
was induced in FDCP-Mix cells treated with ionomicin in the presence of
IL-3, suggesting that cytosolic calcium may regulate the expression of
this proapoptotic protein. Furthermore, ectopic expression of Hrk
induced cell death of hematopoietic progenitors in the presence of
IL-3. Thus, Hrk is specifically and rapidly induced in hematopoietic
progenitors after growth factor deprivation or treatment with
chemotherapeutic drugs, and this may be sufficient
to induce apoptosis in these cells.
(Blood. 2000;95:2742-2747)
© 2000 by The American Society of Hematology.
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Introduction |
There is increasing evidence that most cells in
multicellular organisms require constant stimulation by extracellular
signals to survive. Committed hematopoietic progenitor cells require
defined growth factors for survival, differentiation, and
proliferation.1 Withdrawal of these growth factors leads to
apoptosis, and this cell death mechanism has been proposed to play a
critical role in the control of cell numbers in both hematopoietic
precursors and their fully differentiated cell
populations.2-4 Apoptosis is implemented by a death
machinery that is evolutionarily conserved and activated in the dying
cell. In mammals, the executory arm of apoptosis involves
a family of death proteases, called caspases, that are activated in a
proteolytic cascade to execute the cell death program.5 The
activation of upstream caspases represents a critical checkpoint in the
decision to survive or to die.6,7 Upstream caspases are
controlled by several molecules, including proteins of the Bcl-2
family. Members of this family possess at least 1 of 4 conserved motifs
known as Bcl-2 homology domains (BH1 to BH4), and can exert both
prosurvival and proapoptotic activity.8
In hematopoietic progenitor cells, several mechanisms have been
proposed to explain how growth factors promote survival through Bcl-2
family members. Several hematopoietins, including interleukin-3 (IL-3)
and erythropoietin, have been shown to maintain the expression of
prosurvival Bcl-2 family members, including Bcl-2, Bcl-xL, A1, and Mcl-1, at the transcriptional level.9,10 By
contrast, RNA levels of proapoptotic Bcl-2 members, including Bax, Bad, and Bak, appear to be regulated independently of IL-3 in hematopoietic progenitor cells. Additionally, IL-3 and perhaps other growth factors
can promote survival through phosphorylation and inactivation of
proapoptotic BAD via activation of the Akt kinase.11
Some proapoptotic Bcl-2 family members, including Bik/Nbk, Blk, Bad,
and Bid, contain only the BH3 motif that acts as a dimerization domain
for prosurvival proteins, including Bcl-2 and
Bcl-xL.12-15 The proapoptotic activity of these
BH3-only proteins is mediated by the BH3 region, as deletion or
mutation of this region abrogates their binding to Bcl-2 and
Bcl-xL and their killing activity.16,17 Thus,
these BH3-only Bcl-2 family proteins have been suggested to represent
the physiologic antagonists of prosurvival Bcl-2 family
members.8 Little is known about the regulation of the newly
described BH3 subfamily of proapoptotic Bcl-2 family members. These BH3
proteins are evolutionarily conserved, in that they are structurally
related to EGL-1, a nematode BH3-containing protein that interacts with
and inhibits CED-9.18 Human Hrk and its mouse homologue
(DP5) are members of this BH3 subfamily of proapoptotic proteins. Human
Hrk was isolated as a Bcl-xL and Bcl-2-interacting protein
that was found to be preferentially expressed in spleen and bone
marrow.19 Mouse Hrk was identified during a screening for
genes up-regulated after nerve growth factor withdrawal in primary
sympathetic neurons.20 We report here that the expression of Hrk, but not that of related proapoptotic Bcl-2 family members, including Bad, Bax, and Bak, is rapidly induced by growth factor deprivation at the messenger RNA (mRNA) and protein levels in hematopoietic progenitors. This up-regulation of Hrk was inhibited by
prosurvival Bcl-2 and Bcl-xL proteins. We also report that enforced expression of Hrk in the presence of growth factor induces cell death in hematopoietic progenitor cells.
| |
Materials and methods |
Cell culture
The erythropoietin-dependent HCD-57 cell line and its derivative
HCD-57-Bcl-xL were maintained in Iscove's modified
Dulbecco's medium (IMDM) (GIBCO-BRL, Grand Island, NY), supplemented
with 0.1 U/mL of recombinant erythropoietin (Boehringer Mannheim,
Indianapolis, IN) as previously described.9 FL5.12,
FL5.12-Bcl-2,21 FDCP-Mix, and UT7 cell lines were grown in
RPMI 1640 medium (Seromed Biochrom KG, Berlin, Germany), supplemented
with 10% fetal calf serum (FCS) (Flow Laboratories, Irvine, CA), and
10% of Wehi 3B culture supernatant as an IL-3 source for the murine
cells, or 5 ng/mL granulocyte-macrophage colony-stimulating factor
(GM-CSF) (Sigma, St Louis, MO) for UT7 cells. Mo7e cell line was
cultured in IMDM supplemented with 20% FCS and 5 ng/mL IL-3 (Sigma).
When indicated, cell lines were treated with 10 µg/mL etoposide or 1 µmol/L ionomycin (both from Sigma) for different time intervals and
then analyzed for expression of Hrk. Bone marrow was obtained from
healthy donors after appropriate informed consent. The CD34 positive
bone marrow population was selected using an immunomagnetic system as
previously described22 and then cultured in IMDM containing
20% FCS and recombinant stem cell factor, IL-3, and IL-6 (Immunex,
Seattle, WA) at a final concentration of 100 ng/mL.
Transfection and cell death analysis
FL5.12 and FDCP-Mix cells (5 × 106) were
transfected by electroporation (200 V, 950 µF) with a construct
containing the human Hrk cDNA in the sense or inverted orientation,
cloned into the unique EcoRI site of the pIRES-EGFP vector (Clontech
Lab, Palo Alto, CA) or with a control pIRES-Neo plasmid. After 24 hours of transfection, cells were analyzed for expression of the green fluoresce protein and the uptake of propidium iodide by flow cytometry using a FACScan analyzer (Becton Dickinson, San Jose, CA). For propidium iodide uptake, cells were incubated in a solution containing 0.1% sodium citrate and 0.1 mg/mL propidium iodide, for 10 minutes and
then analyzed.
In some experiments, 5 × 106 FL5.12 cells were
transfected with 1 µg of pcDNA3-Hrk19 or control pcDNA3
plasmid. After 48 hours, cells were seeded into 96-well plates at 2000 cells per well in the presence of 1 mg/mL G418 and the number of wells
containing viable cells were scored on day 12 after transfection.
Antibodies against Hrk and Western blot analysis
To examine the expression of Hrk, we synthesized a 35-amino acid
peptide corresponding to residues that encompass the BH3 region of
human Hrk.19 A rabbit was immunized and, after antigen boosting, the immune serum that recognized a protein of the expected molecular mass for human and mouse Hrk was used for Western blot analysis. The expression of Bcl-xL, Bax, Bad, Bak, and Hrk
proteins was determined by Western blotting as previously
described.9 Blots were incubated with rabbit antibodies
against Bcl-x (Transduction Lab, Lexington, KY), Bax (Santa Cruz
Biotechnology, Santa Cruz, CA), Bak (Upstate Biotechnology, Lake
Placid, NY), and Hrk, or mouse anti-Bad antibodies (Transduction Lab),
and then incubated with goat antirabbit or goat antimouse antibodies
conjugated to alkaline phosphatase (Tropix, Bedford, MA). Bound
antibody was detected by a chemiluminescence system (Tropix).
Messenger RNA expression analysis
Total RNA was prepared using TRIZOL reagent (GIBCO-BRL). To assess
mRNA expression, a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used as previously
described.9 The generated cDNA was amplified by
using primers for murine Bcl-x,  -actin,9 and Hrk
(5'TAGGCGACGAGCTGCA3' and
5'CTCCAAGGACACAGGGTT3'). The amplification profile was as
follows: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C
for 30 seconds. After 25 to 30 amplification cycles, the expected PCR
products were size fractionated onto a 2% agarose gel and stained with
ethidium bromide.
Assays for apoptotic cells
For DNA fragmentation analysis, cells (1 to
3 × 106) were washed with
phosphate-buffered saline and pelleted by centrifugation. Genomic
DNA was isolated from cell pellets as described
previously.23 DNA samples were electrophoresed on a 2%
agarose gel and stained with 0.1% ethidium bromide. The early
apoptotic cells were detected with annexin V, labeled with fluorescein
isothiocyanate (PharMingen, San Diego, CA) by flow cytometry.
| |
Results |
Hrk is up-regulated in hematopoietic progenitor cell lines cultured
in the absence of specific growth factors
To examine the regulation of Hrk, we used 3 hematopoietic progenitor
cell lines that required defined growth factors for survival and
proliferation. FDCP-Mix and FL5.12 are myeloid and lymphoid cell lines,
respectively, that are IL-3-dependent, whereas HCD-57 is an erythroid
cell line that requires erythropoietin for proliferation and survival.
When these 3 hematopoietic cell lines were cultured in the absence of
growth factor, the protein levels of Hrk were clearly up-regulated by
24 hours and greatly increased by 48 hours after growth factor
withdrawal (Figure 1A). In contrast,
expression of Bcl-xL gradually decreased within 48 hours of
culture in the absence of growth factor. In addition, the expression of
proapoptotic members of the Bcl-2 family, Bax, Bak, and Bad, either
remained unchanged or diminished, but none were up-regulated within 48 hours after growth factor withdrawal. To verify whether the increase in
Hrk protein expression correlated with the level of mRNA, we determined
the levels of Hrk by semiquantitative RT-PCR analysis (Figure 1B). In
agreement with the protein results, Hrk was up-regulated and
Bcl-xL mRNA levels were down-regulated within 48 hours of growth factor deprivation (Figure 1B). This expression pattern was
accompanied by a loss of cell viability caused by the activation of an
apoptotic process. The genomic DNA isolated from FDCP-Mix, HCD-57, and
FL5.12 cells cultured in the absence of growth factor was degraded into
oligonucleosomal fragments that are characteristic of apoptosis (Figure
1C). Similar results have been obtained also with human hematopoietic
progenitors. Induction of the Hrk protein but not of Bax was detected
in human megakaryoblastic UT-7 and Mo7e cell lines cultured in the
absence of GM-CSF and IL-3, respectively, for 24 and 48 hours, and in
bone marrow-derived CD34+ cell population cultured without
IL-3, IL-6, and stem cell factor for 36 hours (Figure
2).
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| Fig 1.
Analysis of Bcl-2 family members and apoptosis in murine
hematopoietic progenitor cell lines after growth factor withdrawal.
(A) Western blot analysis of Hrk, Bcl-xL, Bax, Bad, and Bak
in cells cultured in the presence (0 hours) or absence of IL-3
(FDCP-Mix, FL5.12) or erythropoietin (HCD-57) for 24 and 48 hours. (B)
Semiquantitative RT-PCR analysis of Hrk and Bcl-xL mRNA at
0, 24, and 48 hours after growth factor deprivation. PCR products were
electrophoresed onto a 2% agarose gel. -actin mRNA was used as an
amplification control. (C) DNA fragmentation analysis in cells cultured
with or without growth factor. Cells were incubated for the indicated
time points and genomic DNA fragmentation was monitored by
electrophoresis onto a 2% agarose gel and staining with ethidium
bromide.
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| Fig 2.
Expression of Hrk in human cell lines and
CD34+ progenitor cells.
Two megakaryoblastic cell lines were cultured in the absence of IL-3
(Mo7e) or GM-CSF (UT7). Bone marrow-derived CD34+ cells
were cultured with (+GFs) and without ( GFs) growth factors
(IL-3, IL-6, and stem cell factor) for 36 hours. At the indicated time
intervals, cells were analyzed for the expression of Hrk and Bax by
Western blot.
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Previous data showed that the treatment of rat cortical neurons with
inhibitors of calcium channels blocked the expression of Hrk mRNA and
prevented apoptosis induced by amyloid- protein.24 To
analyze whether calcium flux could also affect the expression of Hrk in
hematopoietic progenitors, we incubated FDCP-Mix cells with 1 µmol/L
ionomycin in the presence of IL-3. As shown in Figure 3A, the protein levels of Hrk but not of
Bax increased by 24 hours after addition of ionomycin, and the
expression was similar to that of cells cultured in the absence of
IL-3. Furthermore, this up-regulation of Hrk correlated with the
activation of an apoptotic process as assessed by genomic DNA
fragmentation analysis (Figure 3B).
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| Fig 3.
Analysis of Hrk and apoptosis in FDCP-Mix cells treated
with ionomycin.
Cells were cultured for 24 hours with ionomycin in the presence of
IL-3 and then analyzed. As controls, cells cultured in the
presence or absence of IL-3 were also analyzed. (A) Expression of Hrk
and Bax by Western blot. (B) DNA fragmentation analysis by
electrophoresis onto a 2% agarose gel and staining with ethidium
bromide.
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Induction of Hrk is rapid and unaffected by pretreatment
with cycloheximide
We focused next on the expression of Hrk during the first 2 hours
after IL-3 withdrawal, to study whether the induction of Hrk was an
early response to an apoptotic stimulus. The levels of Hrk mRNA in
FDCP-Mix cells began to increase within 15 minutes and continued to
accumulate by 2 hours after IL-3 deprivation (Figure
4A). The kinetics of Hrk induction
resembled that of immediate early genes such as
c-myc25 or oncostatin M.26 Indeed, as expected for an immediate early gene, when we culture growth
factor-starved cells with cycloheximide at a dose (20 µg/mL) that
inhibited more than 90% of protein synthesis (data not shown), the
expression of Hrk mRNA increased at a similar rate to that found in
cycloheximide-free cultures within 2 hours after IL-3 withdrawal
(Figure 4). In addition, the protein levels of Hrk reached after 24 hours of growth factor deprivation were clearly reduced by 6 hours
after re-addition of IL-3 and returned to baseline by 12 to 24 hours
(Figure 4B). By contrast, the steady-state protein levels of Bax were
not modified during the same interval (Figure 4B). This result was
consistent with the half-life of the Hrk protein, which was estimated
to be around 6 hours as detected by pulse-chase labeling analysis (data
not shown).
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| Fig 4.
Expression of Hrk in FDCP-Mix cells treated with a
protein synthesis inhibitor.
(A) IL-3-deprived cells were cultured with or without cycloheximide
(CHX), and at the indicated time intervals, the mRNA levels of Hrk were
analyzed by semiquantitative RT-PCR. As controls, cells
maintained with IL-3 in the absence (first lane) or in the presence
(second lane) of CHX were also analyzed and showed no induction of Hrk.
PCR products were electrophoresed onto a 2% agarose gel.
-actin mRNA was used as an amplification control. (B) Cells were
deprived of IL-3 for 24 hours and then stimulated for different time
intervals with IL-3. The expression of Hrk and Bax proteins was
determined by Western blotting with use of specific antibodies.
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Expression of Hrk is induced in hematopoietic progenitor cells after
treatment with etoposide
To determine whether the induction of Hrk was restricted to signals
associated with growth factor withdrawal or, alternatively, it might be
a response to an apoptotic signal, we analyzed the expression of Hrk in
FDCP-Mix cells treated with the topoisomerase II inhibitor etoposide,
an inducer of apoptosis.27 As shown in Figure
5, when cells were cultured with etoposide
in the presence of IL-3, a time-dependent increase in the number of
apoptotic cells was observed, as assessed by flow cytometry analysis
with annexin V (Figure 5A) and DNA fragmentation analysis (data not shown). By 12 hours of treatment, 53.3% of cells were annexin V
positive, and by 24 hours after etoposide, most of the cells (76.2%)
were apoptotic. Within 24 hours of treatment with etoposide, a clear
correlation was observed between the increase in the number of
apoptotic cells and the up-regulated expression of Hrk as assessed by
Western blot analysis (Figure 5B). By contrast, etoposide treatment did
not affect the expression of Bax. A similar pattern of expression was
observed in FL5.12 and HCD-57 cells treated with etoposide (data not
shown), indicating that in addition to growth factor withdrawal, the
expression of Hrk can be up-regulated by chemotherapeutic drugs.
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| Fig 5.
Analysis of Hrk and apoptosis in etoposide-treated
FDCP-Mix cells.
(A) Cells were treated for different times with etoposide in the
presence of IL-3 and then analyzed by flow cytometry with fluorescein
isothiocyanate-labeled annexin V. Numbers above the selected regions
indicate the percentage of apoptotic cells. (B) Western blot analysis
of Hrk and Bax expression in FDCP-Mix cells treated with etoposide for
12 and 24 hours in the presence of IL-3.
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Bcl-2 and Bcl-xL prevent the induction of Hrk
Overexpression of Bcl-2 and Bcl-xL has been shown to
inhibit or delay the apoptotic cell death in a number of cellular
systems,23,28-30 so that we studied whether ectopic
expression of Bcl-xL or Bcl-2 could impede the
up-regulation of Hrk in hematopoietic progenitor cell lines. HCD-57
cells transfected with Bcl-xL,9 and FL5.12 expressing constitutive levels of Bcl-221 were cultured in
the absence of erythropoietin and IL-3, respectively, and the levels of
Hrk were analyzed by Western blot. Figure 6
shows a representative experiment. Both parental FL5.12, and HCD-57
displayed an increase in the expression of Hrk after growth factor
withdrawal, in agreement with results shown in Figure 1. Furthermore,
by 24 hours of culture without IL-3 and 48 hours in the absence of
erythropoietin, most of the cells were apoptotic, as assessed by flow
cytometry with annexin V and DNA fragmentation analysis (data not
shown). Significantly, FL5.12-Bcl-2 cells and HCD-57-Bcl-xL
did not show any change in the expression of Hrk at the protein (Figure
6) and mRNA levels (data not shown) after 24 or 48 hours of
culture in the absence of IL-3 or erythropoietin, respectively.
Moreover, these cells did not show any evidence of apoptosis within the
same period after growth factor withdrawal (data not shown). These data
indicate that Bcl-2 and Bcl-xL can repress the induction of
Hrk and suggest that up-regulation of Hrk is triggered by an
intracellular apoptotic signal that is inhibited by these prosurvival
Bcl-2 family members.
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| Fig 6.
Analysis of the Hrk expression in cells transfected with
Bcl-2 or Bcl-xL.
FL5.12 cells transfected with human Bcl-2 cDNA and HCD-57 cells
transfected with human Bcl-xL cDNA were cultured in the
absence of IL-3 and Epo, respectively, for the indicated times and then
analyzed for the expression of Hrk and Bax proteins by Western blot. As
control, FL5.12 and HCD-57 cells subjected to the same culture
conditions were also analyzed (lane C).
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Enforced expression of Hrk induces cell death in the
presence of growth factor
We transfected a plasmid producing human Hrk into IL-3-dependent
FL5.12 cells but analysis of multiple clones selected after culture in
G418 showed no detectable Hrk expression (data not shown). To test
whether Hrk inhibits the expansion of these cells into colonies, we
compared colony formation in FL5.12 cells transfected with a plasmid
expressing Hrk or with a control plasmid. Transfection of the control
plasmid into FL5.12 cells generated 100 ± 13 colonies in the
presence of G418, whereas only 33 ± 18 were obtained by transfection of the Hrk plasmid (Figure 7),
indicating that expression of Hrk impedes the formation of colonies in
the presence of IL-3. To determine whether Hrk blocked the expansion of
these cells by inducing cell death, we constructed a plasmid to
coexpress Hrk and enhanced green fluorescence protein (EGFP). This
construct contains an internal ribosomal entry site, allowing
translation of Hrk and EGFP from a single bicistronic mRNA (Figure
8A), which permits identifying
Hrk-expressing cells on the basis of EGFP expression after transient
transfection. When we examined FL5.12 cells 24 hours after transfection
with Hrk in the sense orientation, 98% of the EGFP-positive cells were
dead as determined by their incorporation of propidium iodide (Figure
8B). In contrast, the great majority (more than 85%) of the cells
transfected with Hrk in the inverted orientation that coexpressed EGFP
remained alive as they excluded propidiun iodide (Figure 8B). Similar
results were obtained with FDCPMix cells (data not shown). These
results indicate that expression of Hrk causes the death of
hematopoietic progenitor cells in the presence of growth factor.
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| Fig 7.
Expansion of FL5.12 cells after transfection with Hrk.
Cells were transfected with pcDNA3 or pcDNA3-Hrk expression plasmids
and then cultured into 96-well plates at 2000 cells per well in the
presence of G418 for 12 days. Data are presented as the number of
colonies (wells containing viable cells) per
5 × 106 transfected cells (mean of triplicate
cultures ± SD).
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| Fig 8.
Cell death analysis of FL5.12 cells transiently
transfected with pIRES-EGFP-Hrk.
(A) Schematic structure of the bicistronic vector containing the
internal ribosomal entry site (IRES) and the Hrk and EGFP cDNAs. (B)
Flow cytometry analysis of cells after 24 hours of transfection with
pIRES-EGFP containing Hrk in the sense or inverted orientation (io).
Quadrants in the dot plots were set according to the green fluorescence
of cells transfected with a negative control vector (pIRES-Neo).
Histograms represent the percentage of cells stained with propidium
iodide in the green fluorescence positive population (R1) and the green
fluorescence negative population (R2). All histograms and dot plots are
from a representative experiment (n = 3).
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Discussion |
A number of cytokines and hematopoietic growth factors have been
shown to promote viability of subpopulations of primitive progenitor
cells, suggesting that the regulation of apoptosis plays a key role in
hematopoiesis.1 Apoptosis is tightly regulated by members
of the caspase and Bcl-2 families. Genetic and biochemical analyses
have demonstrated that the activation of caspases is influenced by the
Bcl-2 family members, which either suppress or induce
apoptosis.8 In this scenario, a reasonable expectation would be that the activity and/or expression of Bcl-2 family members is
regulated by signaling pathways that suppress or induce apoptosis.
Here we showed that Hrk, a newly described proapoptotic member of the
Bcl-2 family, is specifically and rapidly induced in hematopoietic
progenitor cell lines and bone marrow-derived CD34+ cells
after growth factor deprivation, which is accompanied by activation of
an apoptotic process. Consistent with this is the observation that the
mRNA of mouse Hrk (DP5) was induced in sympathetic neurons cultured in
the absence of nerve growth factor (NGF).20 However, the
expression of Hrk in hematopoietic progenitor cell lines can be induced
not only by growth factor withdrawal but also by treatment with
etoposide, a chemotherapeutic drug that induces apoptosis. On the basis
of these data, we hypothesize that the binding of growth factors (ie,
IL-3, erythropoietin, GM-CSF) to their cognate receptors in
hematopoietic progenitors activates a repressor pathway that silences
the expression of proapoptotic Hrk. Alternatively, this repressor
pathway may not be triggered by the growth factor but may be active
during the normal development of hematopoietic progenitor cells. On
growth factor withdrawal or treatment with certain chemotherapeutic
drugs, the repressor mechanism may be released (ie, by
posttranslational modification or binding to a specific factor),
allowing the expression of Hrk. A similar mechanism may operate in
neurons that up-regulate mouse Hrk and undergo apoptosis after NGF
withdrawal or treatment with amyloid- protein.20,24 The
induction of mouse Hrk in cultured neurons treated with amyloid-
protein is blocked by inhibitors of voltage-dependent calcium channels
and calcium release from the endoplasmic reticulum, suggesting that
calcium fluxes could be involved in the regulation of Hrk
expression.24 An increase in cytosolic calcium has been
implicated in signal transduction pathways that mediate
apoptosis.31,32 We have shown here that the expression of
Hrk is induced in FDCP-Mix cells after incubation with ionomycin, a
calcium ionophore that releases calcium from a variety of intracellular
stores, including the endoplasmic reticulum and
mitochondria.33 Thus, Hrk might be regulated in these cells by an intracellular calcium-sensitive pathway that is activated on
growth factor withdrawal or treatment with chemotherapeutic drugs.
Furthermore, it has been described that Bcl-2 either directly or
indirectly regulates the flux of calcium across the endoplasmic reticulum membrane, thereby abrogating calcium signaling of
apoptosis.34 Consistent with the model of calcium-regulated
expression of Hrk, we have shown that overexpression of Bcl-2 or
Bcl-xL inhibits the induction of Hrk and prevents apoptosis
after growth factor withdrawal.
Deletion of Bcl-x in mice leads to apoptosis of hematopoietic
progenitors and embryonic lethality.35 Thus, it appears
that Bcl-xL is an important mediator of hematopoietic cell
survival in vivo. Hematopoietic progenitor cells down-regulate the
expression of Bcl-xL on growth factor deprivation, in
addition to up-regulating Hrk. Furthermore, we demonstrate that
enforced expression of Hrk into hematopoietic progenitor cell lines is
sufficient to induce cell death in the presence of IL-3 within 24 hours. Consistent with this is the observation that transfection of Hrk
into embryonic kidney 293T cells resulted in a loss of cell viability
at 36 hours posttransfection.19 This suggests that
induction of Hrk, which is normally silenced or expressed at low
levels, might be a mechanism that participates in the activation of a
cell death program linked to growth factor withdrawal or treatment with
chemotherapeutic drugs, in hematopoietic progenitors. Because enforced
coexpression of Hrk and the antiapoptotic protein Bcl-2 or
Bcl-xL inhibits the death promoting activity of
Hrk,19 it appears that the balance between the steady-state
levels of Bcl-xL or Bcl-2 and those of Hrk likely
contribute to either suppress or induce apoptosis. Future studies will
need to address the molecular mechanisms that regulate the expression
of Hrk and, more importantly, the role for Hrk in the regulation of
normal hematopoiesis. To this end, mice deficient in Hrk will be very
useful to study whether this proapoptotic protein is needed for the
homeostasis of the hematopoietic compartment.
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Footnotes |
Submitted July 13, 1999; accepted January 3, 2000.
Supported by Comision Interministerial de Ciencia y Tecnologia
Grant No. SAF-96/0274 to J.L.F-L., and grant P01 CA75136 from the
National Institutes of Health to G.N. C.S. is a recipient of a
predoctoral fellowship from the "Fundacion Marques de
Valdecilla"; A.B. is a recipient of a NATO postdoctoral fellowship.
Reprints: Jose Luis Fernandez-Luna, Servicio de
Inmunologia, Hospital Universitario Marques de Valdecilla, INSALUD, 39008 Santander, Spain; e-mail: inmflj{at}humv.es.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction