From the Institute of Signalisation, Developmental Biology and Cancer, INSERM 470, Centre de Biochimie, Université de Nice, Faculté des Sciences, 06108 Nice, France
Received for publication, September 10, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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Depriving primary bone marrow-derived macrophages
of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the
expression of anti-apoptotic Bcl-xL protein and the
Ets2 and PU.1 proteins of the Ets transcription factor family.
Macrophages require both priming and triggering signals independent of
CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression of
bcl-xL, ets2, and
PU.1. Furthermore, we show that only Ets2 and PU.1,
but not Ets1, function in a synergistic manner to transactivate the
bcl-x promoter. The synergy observed between PU.1 and Ets2
is dependent on the transactivation domains of both proteins. Although
other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are
implicated in the activation of macrophage competence or in CSF-1
signaling, no synergy was observed between Ets2 and these transcription
factors on the bcl-x promoter. We demonstrate that the
exogenous expression of both Ets2 and PU.1 in macrophages increases the
number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can
functionally replace Bcl-xL in inhibiting Bax-induced
apoptosis. Together, these results demonstrate that PU.1 and Ets2
dramatically increase bcl-x activation, which is necessary
for the cytocidal function and survival of macrophages.
The Ets family of transcription factors consists of ~30 members
conserved from sea urchins to man. Ets members contain a conserved DNA-binding domain of ~85 amino acids known as the Ets domain (reviewed in Ref. 1). Ets1, the progenitor of v-Ets found in the E26
retrovirus, and Ets2 are 97% conserved in the Ets domain, whereas
PU.1/Spi.1 is highly divergent in this domain (37% identity) (2).
These differences in sequence identity of the Ets domain allow Ets1,
Ets2, and PU.1 to bind to common as well as distinct optimal DNA target
sequences. Ets proteins bind to DNA as monomers to activate
transcription alone or in conjunction with other transcription factors
binding to their respective sites (reviewed in Ref. 1).
PU.1 is expressed in early progenitor cells as well as in fully
differentiated B cells, neutrophils, and macrophages. The importance of
PU.1 in hematopoietic development has been confirmed by PU.1
gene disruption studies showing that PU.1-deficient mice lack mature B cells, neutrophils, and macrophages (3, 4). ets2 null mice die early during embryonic development (5). However, transgenic studies using a dominant-negative form of Ets2
under the control of a monocyte/macrophage-specific promoter have
provided insight into the role of Ets2 in macrophages (6). Abnormal
macrophage development occurs in these transgenic mice during the first
40 days following birth, and peritoneal macrophages obtained from these
transgenic animals do not have the characteristic macrophage morphology
when cultivated in vitro with
CSF-1.1 These results imply
the importance of Ets2 in the development of macrophages, yet the
molecular mechanisms by which Ets2 functions had not been elucidated in
these studies.
The level of PU.1 is highly abundant in immature myeloid progenitor
cells, and this level of expression remains high throughout macrophage
differentiation (7, 8). In contrast, Ets2 is not expressed in early
myeloid progenitors, but later in more mature myeloid cells (9-11),
and Ets2 become rapidly up-regulated upon induction of macrophage
differentiation or activation of primary macrophages (10).
Bcl-xL is believed to be the key anti-apoptotic protein
expressed in myeloid precursors and macrophages (12-17). In contrast, Bcl-2 is down-regulated in these systems. Recently, we showed that the
induction of bcl-xL results from an increase in
bcl-x promoter activity and that de novo protein
synthesis is required for this activation of bcl-x
transcription (17). The human bcl-x promoter contains nine
potential EBS. The capacities of PU.1 and Ets2 to individually
transactivate the bcl-x promoter led us to ask whether PU.1
and Ets2 could compete or act in synergy to transactivate this
promoter. In parallel, we wanted to determine the biological relevance
of the coexpression of PU.1, ets2 and bcl-xL in primary macrophages upon induction of
proliferation and differentiation and activation of macrophage
competence and during programmed cell death by apoptosis.
Cell Culture--
293 cells, NIH3T3 cells, and NIH3T3 cells
exogenously expressing the CSF-1R (NIH3T3-cfms) (18) were maintained in
Dulbecco's modified Eagle's medium and 10% fetal calf serum. Primary
bone marrow-derived cells were isolated from femurs of 2-3-month-old male C57BL/6 mice. Femurs were flushed with PBS to recover cells. After
several washes in PBS, cells were cultivated either in Dulbecco's modified Eagle's medium, 20% fetal calf serum, and 30% conditioned medium from L cells as a source of CSF-1 (19) or in Dulbecco's modified Eagle's medium and different concentrations of human recombinant CSF-1. After 4-5 days, fully differentiated macrophages were obtained. When recombinant CSF-1 was used, the concentrations are
indicated below. IFN-
The BAC1.2F5 and BACets2.1D macrophage cell lines have been previously
described (17). For transfection studies, BAC1.2F5 macrophages were
electroporated either with 5 µg of pRK-ets2 and 5 µg of
pRK-PU.1 or with 5 µg of pRK- Transactivation Studies--
The cloning of the 5'-regulatory
sequences of the bcl-x gene upstream of the luciferase gene
has been described previously (17). Ets2, a dominant-negative mutant of
Ets2 (
AP-1 activity was also measured using the full-length
pXP-Bcl-xPr reporter construct. pXP-Bcl-xPr was
cotransfected in 24-well dishes with 200 ng of pRK5,
pRK-fos, or pRK-jun (21) and 20 ng of pCMV-
Several independent experiments using the different cell types and
different DNA preparations were performed in triplicate or
quadruplicate. Cell lysates were prepared as previously described (22).
Briefly, 48-72 h after transfections, cells lysates were prepared in
25 mM Tris (pH 7.5), 10% glycerol, 1% Triton X-100, and 2 mM dithiothreitol and analyzed for luciferase (Promega) and
Northern Hybridization Analysis--
Cells were washed twice in
1× PBS. Cells were then lysed in RNA Insta-Pure (Eurogentec) as
described by the manufacturer. 5 µg of total RNA was loaded and
electrophoresed on a 2.2 M formaldehyde-containing 1%
agarose gel and then transferred to a nylon membrane (Amersham Pharmacia Biotech) as described by the manufacturer. Purified ets2, PU.1/spi.1,
bcl-xL, lysozyme M, IP10 (gift of
T. A. Hamilton), and S26 cDNA fragments were used as probes.
Probes with equal high specific activities were generated using the
Stratagene Prime-It kit as described by the manufacturer.
Prehybridization and hybridization were carried out at 42 °C in a
solution of 6× SSC, 5× Denhardt's solution, 0.5% SDS, and 50%
formamide containing 20 µg/ml denatured salmon sperm DNA. Normal
stringency washes were performed at 50 °C using 0.1× SSC and 0.1%
SDS. All mRNA transcripts were visualized after exposure to Biomax
film (Eastman Kodak Co.) at Western Analyses--
Cells were lysed in Laemmli buffer, and
equal amounts of total protein from each lysate were electrophoresed on
10-15% polyacrylamide/bisacrylamide gels. Migrated proteins
were transferred to a Polyscreen polyvinylidene difluoride transfer
membrane (PerkinElmer Life Sciences) as described by the
manufacturer; immunoblotted using anti-HA tag (12CA5), anti-Bcl-xL/S (S-18, Santa Cruz Biotechnology),
anti-PU.1 (T-21, Santa Cruz Biotechnology), anti-Ets2 (C-20, Santa Cruz
Biotechnology), or anti-p42 MAPK (gift of Jacques Pouyssegur)
antibodies; and revealed by ECL (Amersham Pharmacia Biotech) as
described by the manufacturer.
Detection of Apoptotic Cells--
Apoptotic macrophage cells
were detected with fluorescein isothiocyanate-conjugated annexin V
(Roche Molecular Biochemicals). Interaction of annexin V with
phosphatidylserines on the outer surface of cells was performed as
described by the manufacturer with the following modifications. After
incubating cells with annexin V and washing in binding buffer, they
were fixed in PBS containing 3% paraformaldehyde for 15 min at
20 °C. Cells were washed in 1× PBS and incubated with a 1:5000
dilution of 4,6-diamidino-2-phenylindole for 5 min at 37 °C. Cells
were then washed three times in 1× PBS and twice in water, and
Moviol was added to the slide and mounted. At least 350 cells
(in the absence of CSF-1) or 700 cells (in the presence of CSF-1)
visualized by 4,6-diamidino-2-phenylindole staining were counted to
determine the number of viable cells. The number of annexin V-positive
cells was calculated with respect to the number of viable cells.
FACS Analysis--
293T cells were seeded in 100-mm culture
dishes and transfected the following day using the calcium phosphate
procedure with 10 µg of various combinations of plasmid DNA as
indicated in the figure legend. 3 µg of a green fluorescent
protein reporter plasmid was included in all transfections. 20 h
post-transfection, cells were scraped and labeled with Alexa-conjugated
annexin V (Roche Molecular Biochemicals) following the manufacturer's
instructions. The cells were then analyzed on a Beckman FACSCalibur
using the FL1-H window to detect transfected cells (>50% of total
cells), and the annexin V-positive cells found among these were
quantitated using the FL3-H window.
Expression of ets2, PU.1, and bcl-xL upon CSF-1
Induction of Macrophage Differentiation--
CSF-1 induces primary
bone marrow-derived precursor cells to proliferate and then to
differentiate into macrophages (BMM). We (23) and others (24) have
shown that cotreatment of hematopoietic progenitors or myeloblastic
cells with leukemia inhibitory factor enhances the hematopoiesis
process. We obtained murine BMM using different concentrations of CSF-1
(12 and 120 ng/ml) in the presence of 0.1 ng/ml leukemia inhibitory
factor. Based on morphology (data not shown) and the expression of a
macrophage-specific marker with bacteriolytic functions (lysozyme M
(lysM)), BMM were obtained with both concentrations of CSF-1
used (Fig. 1B). However, fewer macrophages were obtained with low doses of CSF-1 (Fig. 1A).
Northern analysis revealed that PU.1 was abundantly
expressed under both culture conditions, paralleling the expression of
lysozyme M. However, ets2 expression was detected when a
high concentration of CSF-1 was used. It is worth noting that even
small increases in Ets2 expression are sufficient to induce profound
biological changes since a <2-fold induction of ets2
mRNA expression has been shown to greatly affect bone and cartilage
development in ets2 transgenic mice (25). Interestingly,
bcl-xL expression was clearly activated when
ets2 was expressed (Fig. 1B). These results show
that there is a correlation of ets2 and
bcl-xL expression upon treatment of primary
precursor cells with concentrations of CSF-1 necessary to induce
maximal proliferation and subsequent differentiation.
Depriving Primary Macrophages of CSF-1 Results in the
Down-regulation of ets2 and bcl-xL Expression and Induces
Apoptosis of These Cells--
In addition to inducing proliferation
and differentiation, CSF-1 is required for the survival of BMM. To
determine the expression patterns of PU.1, ets2,
and bcl-xL upon death or survival signals, we
first treated bone marrow-derived cells with conditioned medium
containing CSF-1 for 5 days to obtain BMM (control BMM, 0 h). BMM
were then either starved of CSF-1 (0 ng) or maintained with decreasing
amounts of CSF-1 (120, 12, or 6 ng/ml) for 36 h. Macrophages were
photographed at 24 and 36 h. Visualized in Fig.
2A is the morphology of
primary macrophages upon the different treatments. At 24 and 36 h,
a higher number of floating, round, refractile cells corresponding to
dying cells was observed at 0, 6, or 12 ng of CSF-1. The number of
adherent macrophages also decreased in the absence or presence of low
doses of CSF-1 compared with the number of cells maintained at 120 ng/ml CSF-1. Total numbers of viable cells were confirmed by trypan
blue exclusion for each test condition at 36 h (Fig.
2B). To demonstrate that the cell death observed in the
absence of CSF-1 was indeed due to programmed cell death by apoptosis,
macrophages maintained with or starved of CSF-1 were incubated with
annexin V, an early marker of apoptosis. As shown in Fig.
3, in the absence of CSF-1, 15% of the
remaining cells were labeled with annexin V. The lower number of
4,6-diamidino-2-phenylindole-stained CSF-1-depleted cells (52% fewer
compared with CSF-1-treated cells) and the typical compacted aspect of
these nuclei reflect that the apoptotic process was underway.
To confirm that the cell death observed in CSF-1-starved BMM is
accompanied by decreases in Bcl-xL protein expression,
Western blot analysis was performed using lysates obtained from BMM
starved of CSF-1 for 24 h and then restimulated with decreasing
amounts of CSF-1 (120, 60, 12, or 0 ng/ml). After migration and
transfer, the blot was incubated with an anti-Bcl-xL/S
antibody recognizing both anti-apoptotic Bcl-xL and
pro-apoptotic Bcl-xS proteins. As shown in Fig.
4A, Bcl-xL levels
decreased in the absence of CSF-1, correlating with increases in cell
death. The pro-apoptotic Bcl-xS product migrating at ~25
kDa was not detected in these experiments. The amount of Ets2 and PU.1
proteins also decreased with decreasing concentrations of CSF-1. The
conclusion from these results is that there is a tight correlation
between the levels of Ets2, PU.1, and Bcl-xL protein
expression and CSF-1. p42 MAPK was used as a control for the amount of
protein loaded on the gel.
IFN- Synergistic Effects of Ets2 and PU.1 on the bcl-x
Promoter--
Our Northern and Western results show that PU.1, Ets2,
and Bcl-xL are expressed upon CSF-1 survival,
proliferation, or differentiation signals in primary macrophages. In
addition, treatment of macrophages with IFN-
By Western analysis, we verified that exogenously added HA-tagged Ets1,
To investigate in greater detail this apparent cooperative effect,
bcl-x promoter activity was monitored in 96-well plates to
allow for the analysis of a wide range of Ets2 versus PU.1 concentrations. As the concentrations of either PU.1 or Ets2 alone increased, an increase in transcriptional activation was observed (Fig.
6). As the concentrations of both PU.1
and Ets2 increased, so did their capacities to transactivate the
bcl-x promoter. The inductions observed with Ets2 and PU.1
together result from a synergistic (and not an additive) effect between
these factors.
Synergy Is Specific for Ets2 and PU.1 Transcription
Factors--
To address whether other Ets proteins could synergize
with PU.1 or Ets2, the following experiments were performed. 293 cells were transiently transfected with Ets2 and Ets1 (Fig.
7A) or Ets1 and PU.1 (Fig.
7B). In the 96-well assay at the concentrations of DNA used,
Ets1 alone transactivated the bcl-x promoter
approximately two times better than Ets2 (Fig. 7A). As
the concentrations of Ets1 or Ets2 increased, so did the luciferase
activities. The relative bcl-x promoter activity was not at
all affected by cotransfections with increasing concentrations of both
Ets1 and Ets2. Similar results were obtained using Ets1 and PU.1 (Fig.
7B). Cotransfection with Ets1 and PU.1 did not result in a
synergistic response on the bcl-x promoter. These results
indicate that the capacity to transactivate the bcl-x
promoter in a synergistic manner is specific to the Ets proteins PU.1
and Ets2.
The full-length bcl-x promoter contains binding sites for
other transcription factors, including an AP-1 site for Fos and Jun, an
E-box for Myc and USF-1, and GAS sites for STAT proteins. Activation of macrophage competence is accompanied by increases in Fos
and Jun expression (for review, see Ref. 31). Since CSF-1 stimulation
of NIH3T3 fibroblasts exogenously expressing the CSF-1R also results in
the up-regulation of Fos, Jun, and Myc (32), and CSF-1 activates STAT
phosphorylation in BMM and in the BAC1.2F5 cell line (33), we asked
whether these transcription factors could activate transcription on the
bcl-x promoter in cooperation with Ets2. Cotransfection of
Fos and c-Jun led to a 2-3-fold increase in luciferase activity
compared with a 4-5-fold increase for Ets2. An additive effect was
observed following cotransfections of the bcl-x promoter
reporter construct with Fos, c-Jun, and Ets2 (~6-7-fold induction of
luciferase activity) (Fig.
8A). Although USF-1,
FIP, and Myc all weakly transactivated the bcl-x
promoter (2-3-fold inductions) (data not shown), no synergy was
observed with any of these E-box-binding proteins and Ets2 (data not
shown). CSF-1 stimulation induces STAT proteins to migrate to the
nucleus and become functional transcription factors (33). STAT5a, but
not STAT3 (data not shown), weakly transactivates the bcl-x
promoter (~2-fold) after activation of NIH3T3 cells exogenously
expressing the CSF-1R (32) with CSF-1. Similar inductions (~3-fold)
have been obtained upon interleukin-3 or erythropoietin stimulation of
pro-B or erythroid cells, respectively (reviewed in Ref. 34). But
when STAT5a and Ets2 were coexpressed, the transactivation capacities
of Ets2 appeared to be inhibited by STAT5a (Fig. 8B). Together, these results suggest that CSF-1 activation of these transcription factors is not necessary for the regulation of
transcription of the bcl-x gene, but more likely for the
regulation of other downstream target genes.
Transactivation Domains of Both Ets2 and PU.1 Are Necessary for
Their Synergistic Effects on the bcl-x Promoter--
To determine
whether the transactivation domain or the DNA-binding domain of the
Ets2 protein is necessary for this synergistic response with PU.1, we
cotransfected PU.1 with
Similarly, the transactivation domain of PU.1 was deleted to generate
Synergistic Expression of PU.1 and Ets2 Permits Survival of
Macrophages--
The above experiments demonstrate that the expression
of Ets2, PU.1, and Bcl-xL decreases in primary BMM starved
of CSF-1 and that this decrease in expression correlates with the onset of apoptosis. We also showed that Ets2 and PU.1 function in synergy to
transactivate the bcl-x promoter, yet these experiments do not directly demonstrate that the correlation between expression of
Ets2, PU.1, and Bcl-xL and inhibition of apoptosis is
biologically relevant. To address this question, the following
experiments were performed using three different cell lines.
The BAC1.2F5 macrophage cell line is dependent on CSF-1 for its growth
and survival, although CSF-1 depletion of these cells results in
slightly slower kinetics of cell death than observed with primary
macrophages. We previously described a BAC1.2F5 clone that
constitutively expresses Ets2 even in the absence of CSF-1 (BACets2.1D) (17). To determine whether PU.1 and Ets2 work
together in macrophages to inhibit apoptosis induced by CSF-1
depletion, we electroporated BAC1.2F5 cells with PU.1 and Ets2
expression plasmids. In parallel experiments, BAC1.2F5 cells were
electroporated with transcriptionally inactive
The first striking observation
is that cell survival of BAC1.2F5 cells, as well as of the
Ets2-expressing cell line BACets2.1D, depends on the transfected
plasmids. Indeed, electroporation of constructs encoding dominant
negative forms of PU.1 and Ets2 results in a dramatic increase in cell
death compared to electroporation of a control vector or expression
vectors encoding full-length Ets2 and PU.1 proteins (54-69% fewer
cells) (Fig. 10A). This massive increase in cell death is
independent of CSF-1, reinforcing the notion that Ets2 and PU.1 may be
involved in other cell survival processes in macrophages independent of
CSF-1, as shown above with LPS and IFN treatment. It is worth noting
that in parallel experiments, 293 cells were transfected with a control
expression plasmid or
The second observation is that, among the remaining cells, sensitivity
to CSF-1 depletion is doubled in the Ets2-expressing BACets2.1D line
when the dominant negative form of PU.1 is expressed (Fig.
10B). Similarly, CSF-1 depletion of parental BAC1.2F5 cells resulted in 4 times more cell death when both PU.1 and Ets2 dominant negative forms are expressed (Fig. 10B). Taken together,
these results show that Ets2 and PU.1 participate in macrophage
survival, in both a CSF-1-dependent and -independent manner.
Previous studies showed that the expression of the pro-apoptotic
Bax protein in 293 cells induces apoptosis, whereas apoptosis is
inhibited when Bax heterodimerizes with Bcl-xL in these
cells (35). Since Ets2 and PU.1 activate the transcription of the bcl-x gene and, as a consequence, the expression of the
Bcl-xL protein, we asked whether Ets2 and PU.1 could
functionally replace the exogenous expression of Bcl-xL to
inhibit Bax-induced apoptosis. 293 cells were transfected in the
presence of an enhanced green fluorescent protein expression
plasmid and with an empty expression plasmid as a control; with Bax;
with Bcl-xL and Bax together; or with Ets2, PU.1, and Bax
together. 20 h after transfection, the cells were labeled with
annexin V, fixed, and analyzed by FACS. Visualized in Fig.
11 are the enhanced green fluorescent protein-positive cells labeled with annexin V. In agreement with previous studies (35), Bax induced apoptosis as visualized by the
detection of annexin V-positive cells within the green fluorescent protein-positive ones. In contrast, the proportion of annexin V-positive cells detected in cotransfections with Bax and
Bcl-xL was comparable to control cells. The profile
obtained with Ets2, PU.1, and Bax was identical to that obtained with
Bax and Bcl-xL. In other words, Ets2 and PU.1 functionally
replaced Bcl-xL in these studies. Taken together, these
results clearly demonstrate that both PU.1 and Ets2 function together
to inhibit apoptosis and do so by transcriptionally activating the
expression of the bcl-x gene, which results in the induction
of Bcl-xL protective functions.
CSF-1 is necessary for the survival, proliferation, and
differentiation of myeloid cells. In this report, we show that there is
a tight correlation of expression of ets2 and
bcl-xL in macrophages differentiated from primary
bone marrow-derived progenitor cells. When ets2 is
up-regulated, so is bcl-xL, but only when doses of
CSF-1 are sufficient to induce maximal differentiation. Depriving fully
differentiated primary BMM of CSF-1 induces these cells to die by
apoptosis. This cell death is accompanied by decreased levels of Ets2,
PU.1 and Bcl-xL, and when primary BMM are CSF-1-starved and
then restimulated with CSF-1, the expression of both Ets2, PU.1 and
Bcl-xL is up-regulated, and survival/proliferation pathways
are restored in a CSF-1 dose-dependent manner.
The induction of macrophage tumoricidal and microbicidal activities by
IFN- We previously showed that the bcl-xL transcript has
a short half-life (17). In this report, we show that programmed cell
death of primary BMM induced by CSF-1 withdrawal correlates with
decreasing Bcl-xL protein expression and is not due to the generation of the pro-apoptotic Bcl-xS form. Instead,
growth factor withdrawal-induced apoptosis of primary BMM results from
silencing of the bcl-x gene as evidenced by decreased
bcl-xL transcript expression. Our results using
terminally differentiated macrophages are in agreement with those of
Packham et al. (15), who showed that apoptosis of primary
myeloid stem cells induced upon interleukin-3 withdrawal results in
decreased bcl-xL mRNA levels and not in caspase
cleavage of the Bcl-xL protein. Together, these studies
demonstrate that Bcl-xL is more ubiquitous than previously thought, but is tightly regulated in myeloid cells independent of their
maturation state or their specific cell survival factors.
Although the expression of PU.1 is necessary to induce macrophage
differentiation from immature precursor cells, the expression of PU.1
alone is not sufficient to keep the BAC1.2F5 macrophage cell line alive
in the absence of CSF-1. In contrast, constitutive Ets2
expression protects CSF-1-depleted BAC1.2F5 macrophages from cell
death by apoptosis while PU.1 levels remain elevated (17). The question
that we addressed in this study is what are the functions of these two
members of the Ets family in terminally differentiated macrophages.
To answer this question, we investigated the transcriptional activity
of both factors individually and together on the bcl-x promoter. We showed that although both Ets2 and PU.1 were able to
individually transactivate the bcl-x promoter, a strong
synergistic activation was observed when both were present. It thus
appears that although a constitutively elevated level of PU.1 would
ensure some basal bcl-x expression, the concomitant
expression of the highly regulated Ets2 protein results in a dramatic
increase in bcl-x activation. Abundant levels of
Bcl-xL expression would then enable cell survival and
ensure a long life span of macrophages when both Ets proteins are
simultaneously expressed.
It is striking to note that the synergy between Ets family members PU.1
and Ets2 on the bcl-x promoter is specific since
cotransfections of Ets2 and Ets1 or of PU.1 and Ets1 do not result in
increased transactivation. Interestingly, although Ets1 is not
expressed in macrophages, Ets1 is highly expressed during certain
stages of developing thymocytes (37, 38) when Bcl-xL is not
expressed (39, 40). Together, these observations suggest that the
expression patterns of Ets family members parallel functions on the
bcl-x promoter. Finally, we showed that both PU.1 and Ets2
transactivation domains are required for this synergy. The integrity of
the bcl-x promoter is necessary for maximal synergy between
Ets2 and PU.1 since deletion of two or more sites greatly reduces the
capacity of these two transcription factors to synergize (data not shown).
We observed that other transcription factors like Fos and Jun or Myc
(data not shown), which are up-regulated after CSF-1 stimulation,
surprisingly only weakly transactivate the bcl-x promoter
through their AP-1 or E-box sites, respectively. Cotransfections with
Ets2 result in only an additive effect in increasing transcriptional activation. STAT3 and STAT5a transcription factors become activated and
translocate into the nucleus after CSF-1 stimulation (33) and have been
shown to transactivate the bcl-x promoter (reviewed in Ref.
34). When STAT5a and Ets2 are cotransfected, -fold inductions actually
decrease, suggesting that STAT5a inhibits Ets2 transactivation capacities. This inhibition could be due to competition for
sites due to steric hindrance of STAT5a or by affecting the capacity of
an unidentified cofactor to interact with Ets2. These results reinforce
the crucial role of Ets2 and PU.1 in bcl-x regulation. Jin
et al. (6) reported that primary peritoneal macrophages obtained from To summarize, we have shown that ets2 and
bcl-xL expression is tightly regulated by CSF-1 in
primary bone marrow-derived cells as they differentiate to BMM or when
terminally differentiated BMM undergo apoptosis induced by CSF-1
starvation. The induction of bcl-xL is not limited
to CSF-1 signaling since priming and triggering signals independent of
CSF-1 also induce ets2, PU.1, and bcl-xL.
We show that transient expression of both Ets2 and PU.1 in
CSF-1-depleted BAC1.2F5 macrophages increases cell survival. We also
demonstrate that both proteins function together to inhibit apoptosis
since transiently expressing a transcriptionally inactive PU.1 mutant
in CSF-1-starved BAC1.2F5 macrophages constitutively expressing Ets2
results in decreased cell viability. Finally, we show that Ets2 and
PU.1 can functionally replace Bcl-xL in inhibiting
apoptosis induced by the pro-apoptotic protein Bax. This is the first
report showing that bcl-x gene regulation involves two
members of the same transcription factor family (PU.1 and Ets2) and
that these proteins function in synergy to activate the transcription
of the bcl-x gene, resulting in the synthesis of the
anti-apoptotic Bcl-xL protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(100 units/ml) or LPS (10 µg/ml) was added
to macrophages for 4 h.
PU.1 and 5 µg
of pRK-
ets2 (for description of plasmids, see below) as
previously described (7). BAC1.2F5 cells constitutively expressing Ets2
(BACets2.1D) were electroporated with 10 µg of pRK5 or
pRK-
PU.1. After electroporation, the cells were plated
with CSF-1 in duplicate dishes until they became adherent. Dishes were
washed two times in PBS, and then the cells were cultivated in
Dulbecco's modified Eagle's medium and 10% fetal calf serum with or
without 20% conditioned medium as a source of CSF-1. The number
of viable cells was determined by trypan blue exclusion.
1-238Ets2), Ets1, and PU.1/Spi.1 were cloned into pRK5 (20)
to generate pRK-ets2, pRK
1-238ets2,
pRK-ets1, and pRK-PU.1, respectively, as
previously described (7, 17). A hemagglutinin (HA) epitope tag was
inserted upstream of the first ATG codon of Ets2,
Ets2, Ets1, or
PU.1.
PU.1 was constructed by deleting sequences corresponding to
the transactivation domains found in the first 144 amino acids. An HA
tag was inserted upstream of the newly created ATG codon. 293 cells
were transfected by the calcium phosphate coprecipitation method in
96-well dishes by adding cells in suspension to pXP-Bcl-xPr
(45 ng) and to different concentrations of pRK-ets1,
pRK-PU.1, or pRK-
PU.1 (4-256 ng) or of
pRK5-ets2 or pRK5
1-238ets2 (0.25-256 ng). In
these experiments, 5 ng of pCMV-
gal was used as an internal control
for transfection efficiency. For experiments performed in 12-well
dishes, 293 cells or NIH3T3 cells were transfected by the calcium
phosphate coprecipitation method or with LipofectAMINE Plus (Life
Technologies, Inc.), respectively, using 200 ng of the reporter
construct in the presence of varying amounts of pRK5, pRK-ets2, or pRK-PU.1 as indicated in the
figure legends with 20 ng of pCMV-
gal as an internal control for
transfection efficiency as described above. One-half of the lysate was
used to quantify transfected protein levels by Western analysis, and
the other half was used to measure luciferase and
-galactosidase
activities. For transfections in 24-well dishes, 293 cells or NIH3T3
cells exogenously expressing the CSF-1R were transfected by the calcium phosphate coprecipitation method or with LipofectAMINE Plus,
respectively, as described above for 12-well dishes.
gal
as described (17). STAT activity was measured using the full-length
pXP-Bcl-xPr reporter construct in the presence of STAT3 or
STAT5a cloned into pRK5. For dimerization and activation of STAT
proteins, NIH3T3-cfms cells were cotransfected with
pXP-Bcl-xPr in 24-well dishes with varying amounts of
STAT3, STAT5a, and Ets2. 12 h after transfection, the cells were
placed in low serum conditions (0.5% fetal calf serum) for 24 h
ad then stimulated with CSF-1 for 24 h.
-galactosidase (Tropix Inc., Galactolight) activities as described
by the manufacturers. For 96-well dishes, the luciferase and
-galactosidase activities were read on a MicrobetaTrilux 1450 luminescence counter (Wallac). All luciferase activities were corrected
according to pCMV-
gal used as an internal control for transfection efficiency.
80 °C with Dupont Quanta Fast
intensifying screens.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Proliferation and Northern analysis of
BMM. Primary macrophages were obtained by treating bone
marrow-derived precursor cells with 12 or 120 ng/ml CSF-1 in the
presence of 0.1 ng/ml leukemia inhibitory factor for 5 days. Viable
cells were counted by trypan blue exclusion (A), and total
RNA was isolated (B). Northern analysis was
performed using the following cDNAs as probes:
bcl-xL, ets2, PU.1, lysozyme M
(lysM), and S26 as a control for loading. The sizes of the
corresponding transcripts in kilobases (kb) are
indicated.
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Fig. 2.
Morphology, cell number, and
Bcl-xL expression of primary bone marrow-derived
macrophages maintained in the absence or presence of CSF-1.
Primary macrophages were obtained by treating bone marrow-derived cells
with CSF-1 (Control BMM). Once differentiation was observed,
macrophages were either starved of CSF-1 (0 ng) or maintained
with decreasing amounts of CSF-1 (120, 12, or 6 ng/ml). Cells were then
photographed 24 or 36 h later (magnification × 200)
(A). Cell numbers were determined by trypan blue exclusion
and are graphically represented in B.
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Fig. 3.
CSF-1 starvation of primary bone
marrow-derived macrophages induces programmed cell death by
apoptosis. Fully differentiated primary macrophages were
maintained with or starved of CSF-1 for 24 h. Cells were labeled
with annexin V and 4,6-diamidino-2-phenylindole (DAPI) as a
control for nuclear staining of intact cells and were photographed
(magnification × 100).
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Fig. 4.
Bcl-xL protein and
bcl-xL mRNA expression correlates with
primary macrophage cell survival and activation of functional
competence. A, total cell lysates were obtained from primary
macrophages starved of CSF-1 for 24 h and then restimulated with
decreasing CSF-1 concentrations (120, 12, 6, or 0 ng/ml) for 24 h.
After migration and transfer, Bcl-xL, but not
Bcl-xS, was detected using a Bcl-xL/S-specific
antibody. p42 MAPK levels are shown as a control for sample loading and
transfer. B, primary BMM were untreated
(nontreated) or were treated with IFN- (100 units/ml) or
LPS (10 µg/ml) for 4 h, and total RNA was isolated. After
migration and transfer, the membrane was hybridized to
bcl-xL, ets2, PU.1,
IP10, and S26 as probes. kb, kilobases.
and LPS Induce the Up-regulation of bcl-xL,
ets2, and PU.1 Transcripts--
Macrophage cytocidal activity for
killing neoplastic cells or microorganisms requires both priming and
triggering signals. Priming and triggering by IFN-
and LPS,
respectively, rapidly down-regulate CSF-1R expression even in the
presence of CSF-1 (26, 27). In addition, IFN-
and LPS
up-regulate bcl-xL, whose expression
depends on de novo protein synthesis (14). To determine
whether the up-regulation of bcl-xL correlates with
PU.1 and ets2 expression independent of CSF-1, we
treated primary macrophages with IFN-
and LPS. As shown in Fig.
4B, a 4-h treatment with IFN-
or LPS up-regulated the
expression of bcl-xL, ets2, and
PU.1. IP10 was used as a positive control of an
mRNA abundantly induced after IFN-
or LPS treatment (28). These
results demonstrate that bcl-xL, ets2,
and PU.1 are coexpressed in macrophages not only after a
CSF-1 growth, differentiation, and survival stimulus, but also upon
activation of macrophage functions independent of CSF-1.
and LPS, which
down-regulates CSF-1 signaling (26, 27), also up-regulates
ets2, PU.1, and bcl-xL
expression. Because PU.1 (17) and Ets2 (17, 29) individually
transactivate the bcl-x promoter, and PU.1 and Ets2 were
present when we detected the bcl-xL transcript, we
asked whether these transcription factors could compete or work
together in activating bcl-x transcription. Human 293 cells
were used in these studies for two reasons. First, it is not possible
to transiently transfect primary macrophages due to their rapid cell
death following addition of DNA to these cells (30). Second, no
endogenous Ets2 or PU.1 was detected by Western analysis (data not
shown) in 293 cells, thereby eliminating potential contributions from
endogenously expressed proteins.
Ets2, Ets2,
PU.1, and PU.1 were expressed at comparable levels in
transiently transfected 293 cells (Fig.
5A). Visualized in Fig.
5B (left panels) are the levels of the tagged
Ets2 and PU.1 proteins following transfection of human 293 cells. In
these experiments, the levels of transfected HA-PU.1 decreased as the levels of transfected HA-Ets2 were increased to keep the exogenously added amounts of total Ets proteins constant (transfected Ets DNAs at
400 ng). Corresponding transactivation studies from the same
transfected samples are also shown using the 5'-regulatory sequences of
the bcl-x gene upstream of the luciferase gene as the
reporter (Fig. 5B). The -fold inductions were higher when both Ets2 and PU.1 were equally expressed (200 ng of each DNA) than
when either protein was expressed alone, but at twice the amount
(400 ng of DNA) (see Fig. 5B). To verify that this
observation is valid in other cell systems, similar experiments were
performed using murine NIH3T3 cells (Fig. 5B, right
panels). Similar results were obtained using NIH3T3 cells, in
which Ets2 and PU.1 transactivated the bcl-x promoter better
(8-fold) than Ets2 (3-fold) or PU.1 (2-fold) alone. These experiments
indicate that, under constant levels of EBS activity, transcriptional
activation is more efficient when both PU.1 and Ets2 are present. In
other words, keeping the exogenous levels of Ets proteins constant,
PU.1 and Ets2 transactivate the bcl-x promoter better than
PU.1 or Ets2 alone in two different cell types from two different
species.
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Fig. 5.
Western analysis of transfected Ets
proteins. A, shown is HA-tagged protein expression
following transfection in 293 cells. Cells were lysed in Laemmli
buffer, and lysates were electrophoresed on a 10-15% polyacrylamide
gel. B, HA-tagged Ets2 and PU.1 were transfected in either
human 293 (left panels) or murine NIH3T3 (right
panels) cells in duplicate at 400 µg/ml alone or together at 200 µg/ml each in 12-well dishes. Cells were split into two dishes.
One-half of the cells were lysed and processed as described for
A. After transfer, the polyvinylidene difluoride
membrane was stained with Amido Black as a control for loading and
immunoblotted with an anti-HA antibody. The second half of each sample
was used to measure luciferase activities.
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Fig. 6.
Transactivation studies of Ets2 and PU.1 on
the bcl-x promoter. 293 cells in 96-well dishes
were cotransfected with the full-length bcl-x promoter
construct (Bcl-xPr) and increasing concentrations of
pRK-ets2 (0.25-256 ng), pRK-PU.1 (4-256 ng), or
both together. Transcriptional activities reported as relative
bcl-x promoter activity are compared with
Bcl-xPr in the absence of exogenous transactivator,
arbitrarily set to 1. pCMV- gal was used in all experiments
as an internal control for transfection efficiency. This is one
representative experiment out of five different experiments, all giving
similar results.
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Fig. 7.
Transactivation studies of Ets2 and Ets1 or
of Ets1 and PU.1 on the bcl-x promoter. 293 cells
in 96-well dishes were cotransfected with the full-length
bcl-x promoter construct (Bcl-xPr) and either
with increasing concentrations of pRK-ets2 (0.25-256 ng),
pRK-ets1 (4-256 ng), or both together (A)
or with increasing concentrations of pRK-ets1 (0.25-256
ng), pRK-PU.1 (4-256 ng), or both together
(B) as described in the legend to Fig. 6.
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Fig. 8.
Transactivation studies of Ets2 with Fos and
Jun or with STAT5a on the bcl-x promoter.
A, the bcl-x promoter construct
(Bcl-xPr) and either pRK-ets2 or
pRK-fos and pRK-jun or all together were
transfected in quadruplicate using 293 cells in 24-well dishes.
Experiments were repeated three times. Luciferase activities were
measured as described in the legend to Fig. 6. B,
pRK-STAT5a, pRK-ets2, or both together were
transfected in quadruplicate using NIH3T3 cells exogenously expressing
the CSF-1R in the absence or presence of CSF-1. Experiments were
repeated three times. Transcriptional activities were determined as
described in the legend to Fig. 6.
Ets2.
Ets2 bound to DNA, but remained
inactive since its transactivation domain is absent. In agreement with
the results obtained in Fig.
9A, the transactivation
capacities of PU.1 alone increased with increasing concentrations of
PU.1. However, these capacities were not significantly affected by
increasing concentrations of
Ets2 (Fig. 9A). These results show that
Ets2 does not interfere negatively with the potential of PU.1 to transactivate the bcl-x promoter, but
that the transactivation domain of Ets2 is required for its synergistic response with PU.1.
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Fig. 9.
Transactivation studies of
Ets2 and PU.1 or of Ets2 and
PU.1 on the bcl-x promoter.
293 cells in 96-well dishes were cotransfected with the full-length
bcl-x promoter construct (Bcl-xPr) and either
with increasing concentrations of pRK-
ets2 (0.25-256
ng), pRK-PU.1 (4-256 ng), or both together
(A) or with increasing concentrations of
pRK-ets2 (0.25-256 ng), pRK-
PU.1 (4-256 ng),
or both together (B) as described in the legend to
Fig. 6.
PU.1. Cotransfection of
PU.1 with Ets2 demonstrated that
increasing concentrations of Ets2 resulted in increased luciferase activities of the bcl-x reporter construct, yet no synergy
was observed with
PU.1 (Fig. 9B). Taken together, these
results show that the transactivation domains of both Ets2 and PU.1 are
necessary for the synergy observed between these two proteins.
PU.1 and
Ets2
mutants. In a second set of experiments, electroporations of BACets2.1D
cells with a control empty expression plasmid or with
PU.1 were
performed. Electroporated cells were split into two dishes and were
first cultured with CSF-1 to allow cells to adhere to culture dishes. Cells were washed in PBS and then cultured in either the presence or
absence of CSF-1. 48 h post-transfection, the numbers of viable cells were determined by trypan blue exclusion.
PU.1, and cell viability was determined 24 and
48 h after transfection. No difference in cell numbers was observed, demonstrating that the effect of
PU.1 is specific to macrophages and
is not due to the toxicity of
PU.1 in other cell types (data not
shown).
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Fig. 10.
Ets2 and PU.1 function together to inhibit
CSF-1 depletion-induced apoptosis of macrophages. A,
BAC1.2F5 macrophages were electroporated either with Ets2 and PU.1 or
with transactivation-inactive mutants Ets2 and
PU.1. BACets2.1D
cells were electroporated with a control plasmid (Control)
or with
PU.1. Cells were split, allowed to adhere to culture dishes
with CSF-1, and then either starved of (
) or treated with (+) CSF-1
(CM) for 24 h. Cell viability was calculated using
trypan blue exclusion. B, the percentages of decreases in
the number of viable cells upon CSF-1 depletion are indicated.
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Fig. 11.
Ets2 and PU.1 are as effective as
Bcl-xL in protecting cells against Bax-induced
apoptosis. The proportion of apoptotic cells was determined by
annexin V labeling, and the results from FACS analysis are graphically
represented as the percentage of annexin V-positive cells. 293 cells
were transfected with an empty expression plasmid (Control);
with a Bax expression plasmid (3 µg); with Bax and Bcl-xL
expression plasmids (3 µg of each); or with Bax, PU.1, and Ets2
expression plasmids (3 µg each). FACS analysis showed that
Bax-induced apoptosis (empty peaks corresponding to annexin
V-positive cells) was inhibited by Bcl-xL or by PU.1 and
Ets2 (shaded peaks shifted to the left corresponding to
annexin V-negative cells).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and LPS is independent of CSF-1 signaling (26, 27). IFN-
and
LPS induce Bcl-xL expression in peritoneal macrophages (14)
and increase PU.1 DNA binding in tissue macrophages (36). In this
report, we show that IFN-
and LPS induces bcl-xL, ets2, and PU.1 in BMM.
ets2 transgenic animals die rapidly by
apoptosis upon removal of CSF-1, but the mechanism by which Ets2
functions to prevent cell death was not characterized. This biological
observation may be explained molecularly by our results showing that a
decrease in activity would be due to the lack of synergy between Ets2
and PU.1 regulating the activation of bcl-x expression.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by European Communities Grant ERBFMBICT972684
and by the Foundation pour la Recherche Medicale.
§ Supported by Ministère de L'Education Nationale de La Recherche et de la Technologie.
¶ Supported by Association pour la Recherche contre le Cancer Grant 9691. To whom correspondence should be addressed. Tel. and Fax: 33-4-92-07-64-13; E-mail: boulukos@unice.fr.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M008270200
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ABBREVIATIONS |
---|
The abbreviations used are:
CSF-1, colony-stimulating factor-1;
CSF-1R, colony-stimulating factor-1
receptor;
PBS, phosphate-buffered saline;
IFN-, interferon-
;
LPS, lipopolysaccharide;
HA, hemagglutinin;
STAT, signal transducer and
activator of transcription;
MAPK, mitogen-activated protein kinase;
FACS, fluorescence-activated cell sorting;
BMM, bone-derived
macrophages;
USF-1, upstream stimulatory factor-1;
FIP, Fos-interacting
protein;
GAS, interferon-
activation sequence;
EBS, Ets-binding
sites.
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REFERENCES |
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