(Received for publication, August 7, 1996, and in revised form, December 30, 1996)
From the Divisions of Human Immunology and
¶ Haematology, Hanson Centre for Cancer Research, IMVS, Adelaide,
5000 S.A., Australia, the ** Fred Hutchinson Cancer Research Center,
Seattle, Washington 98104, and the
Institute of Molecular and Cellular
Biosciences, University of Tokyo, Tokyo 113, Japan
The granulocyte-macrophage colony-stimulating
factor (GM-CSF) analog E21R induces apoptosis of hemopoietic cells. We
examined the GM-CSF receptor subunit requirements and the signaling
molecules involved. Using Jurkat T cells transfected with the GM-CSF
receptor we found that both receptor subunits were necessary for
E21R-induced apoptosis. Specifically, the 16 membrane-proximal residues
of the subunit were sufficient for apoptosis. This sequence could be replaced by the corresponding sequence from the interleukin-2 receptor common
subunit, identifying this as a conserved cytokine motif necessary for E21R-induced apoptosis. Cells expressing the
subunit and truncated
c mutants showed that the 96 membrane-proximal residues of
c were sufficient for apoptosis. E21R, in contrast to
GM-CSF, did not alter tyrosine phosphorylation of
c, suggesting that
receptor-associated tyrosine kinases were not activated. Consistent
with this, E21R decreased the mitogen-activated protein kinase ERK
(extracellular signal-regulated kinase). E21R-induced apoptosis was
independent of Fas/APO-1 (CD95) and required
interleukin-1
-converting enzyme (ICE)-like proteases. In contrast,
Bcl-2, which protects cells from growth factor deprivation-induced cell
death, did not prevent this apoptosis. These findings demonstrate the
GM-CSF receptor and ICE-like protease requirements for E21R-induced
apoptosis.
The multifunctional human cytokine granulocyte-macrophage
colony-stimulating factor (GM-CSF)1
regulates the function and viability of a wide range of hemopoietic cells (for review, see Ref. 1). GM-CSF exerts its biological effects
through binding to its high affinity, heterodimeric receptor complex
composed of a low affinity GM-CSF-specific subunit (GMR-
) and a
common
subunit (
c) shared with the receptors for interleukin-3 (IL-3) and IL-5 (2). Amino acid residue 21 in the GM-CSF molecule is
essential for binding to its GM-CSF receptor
c complex (3). Substitution of glutamic acid for arginine at this position in GM-CSF
resulted in a GM-CSF analog (E21R) that bound to the low affinity
GMR-
, was devoid of high affinity binding to the GM-CSF receptor
c complex, and effectively antagonized GM-CSF in binding experiments and functional assays (4). Furthermore, E21R induced apoptosis (programmed cell death) in hemopoietic cells expressing the
GM-CSF receptor (5). Importantly, E21R directly induced apoptosis in the absence of any preexisiting GM-CSF, and this appeared to be an active process since inhibition of protein
phosphorylation, transcription, and protein synthesis rescued the cells
from E21R-induced apoptosis (5). Moreover, E21R induced apoptosis even
if the survival factors granulocyte colony-stimulating factor or stem cell factor were added to the cells. However, addition of IL-3 blocked
E21R-induced apoptosis, indicating that ligand-mediated engagement of
c is central for maintaining cell viability (5). These results
raised the question of the role played by each GM-CSF receptor subunit
in E21R-induced apoptosis.
GMR- is the major binding subunit of the GM-CSF receptor complex and
plays a role in biological signaling. Mapping of the cytoplasmic domain
of the GMR-
subunit using truncated receptor mutants suggested that
the membrane-proximal region (upstream of residue 382) is important for
growth of BaF3 cells (6-9). We wanted to study if the same regions
were involved in E21R-induced apoptosis.
The c alone does not bind GM-CSF, but confers high affinity binding
of GM-CSF when coexpressed with the low affinity GMR-
subunit (10).
Moreover,
c is crucial for signal transduction generated by the
GM-CSF receptor (10, 11). Studies of
c mutants with cytoplasmic
truncations coexpressed with wild-type GMR-
in BaF3 cells revealed
two regions critical for signaling. The membrane-proximal region
(upstream of residue 517) is involved in phosphorylation,
proliferation, and viability (12-14). The distal region (between
residues 626 and 763) is essential for activation of signaling proteins
including ras and mitogen-activated protein (MAP) kinases (14) and
rescue from growth factor deprivation-induced apoptosis (15). Although
c is essential for survival, its role in E21R-induced apoptosis is
not known. The absence of high affinity binding of E21R (presumably the
result of a lack of
c contact) suggested that binding to GMR-
was
sufficient and that
c was not required for E21R-induced
apoptosis.
Ligand binding of the GM-CSF receptor c complex leads to
phosphorylation of
c, the JAK2 kinase, and MAP kinases (8, 14, 16).
Certain stimuli such as activation of Fas/APO-1 (CD95), osmotic shock,
and UV irradiation cause an increase in the activity of the MAP kinase
c-Jun-NH2-terminal-kinase (JNK) compared with the
extracellular signal-regulated kinase (ERK), and this imbalance has
been associated with apoptosis (17-21). As GM-CSF can activate the
MAP-kinases, it was of interest to see if activation of JNK and ERK is
associated with E21R-induced apoptosis.
Although various external stimuli can initiate apoptosis through
different signaling cascades, some of these converge at the level of
one or several interleukin-1-converting enzyme (ICE) and related
cysteine proteases (for review, see Ref. 22). It has not been
established whether ICE and ICE-like proteases are involved in
E21R-induced apoptosis.
In this study we sought to determine (i) the requirements of the
GMR- and
c subunits in E21R-induced apoptosis; (ii) the role
played by JNK and ERK; (iii) whether E21R activates the Fas/APO-1 (CD95) receptor; and (iv) if ICE-like proteases were essential for
E21R-induced apoptosis. We show that the membrane-proximal regions of
both GMR-
and
c are required for E21R-induced apoptosis. This
process does not involve tyrosine phosphorylation of
c, but
treatment with E21R alters the ERK/JNK balance. The apoptosis-inducing surface receptor Fas/APO-1 (CD95) is not activated with E21R. Furthermore, we provide evidence for involvement of ICE-like proteases following E21R treatment. Finally, the physiological cell death inhibitor Bcl-2 does not inhibit apoptosis by E21R, indicating that the
molecular mechanism of this pathway is distinct from that induced by
growth factor deprivation.
Permission was approved by ethics committees to collect blood from leukemic patients.
CellsPeripheral blood was collected from patients with untreated acute myeloid leukemia (AML). Primary blast cells expressing functional heterodimeric GM-CSF receptors were isolated and cultured as described previously (5). These AML cells did not express mRNA for GM-CSF, and they were devoid of endogenous GM-CSF production (5). We also studied Jurkat T cells. This human cell line grows autonomously and was maintained in RPMI medium supplemented with 10% fetal calf serum without exogenous growth factors. Nonstimulated Jurkat T cells do not produce detectable GM-CSF mRNA or protein.
Transfection of Jurkat T CellsDNA constructs were
introduced into Jurkat T cells by electroporation using a Gene Pulser
(Bio-Rad, North Ryde, NSW, Australia). We cotransfected 2 × 107 cells in 0.3 ml of RPMI medium with 3 µg of GMR-
constructs, 7 µg of
c constructs, and 10 µg of apoptosis
inhibitor expression constructs, and at 960 microfarads and 270 V.
The generation of the cDNAs and the corresponding vectors for the
GMR- and
c mutants have been described elsewhere (7, 8, 23, 24).
These receptor cDNA constructs were transiently transfected into
the Jurkat T cells before they were sorted and studied after 48 h.
Plasmids encoding RSV/Bcl-2, pCXN2/CrmA, or pcDNA3/p35 were
introduced into Jurkat T cells, and stable clones were selected for
resistance to neomycin (1.5 mg/ml, CrmA and p35) or hygromycin (0.5 mg/ml, Bcl-2). After selection for antibiotic resistance over the next
15 days, we transiently transfected the cells with both GMR- and
c cDNA as outlined above.
The methods for
immunoprecipitation of the c subunit and Western blotting of
phosphorylated
c have been detailed recently (25). Briefly, AML
cells (107/sample) were stimulated with either GM-CSF (10 ng/ml, Genetics Institute, Cambridge, MA) or E21R (1 µg/ml, BresaGen,
Adelaide, Australia) for selected time periods before they were lysed,
and
c was immunoprecipitated with our anti-
c monoclonal antibody 4F3. Immunoprecipitated
c was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing
conditions.
In separate experiments, AML cells were metabolically labeled with
32PO4 (1 µCi/107cells, BresaGen)
overnight prior to cytokine stimulation. Gels with immunoprecipitated
c were analyzed with a PhosphoImager (Molecular Dynamics, Sunnyvale,
CA).
In other experiments we specifically examined tyrosine phosphorylation
of immunoprecipitated c using an anti-phosphotyrosine monoclonal
antibody (3-365-10, Boehringer Mannheim, Frankfurt, Germany) and the
ECL detection kit (Amersham, Little Chalfont, U. K.).
To measure the JNK/ERK
activity we used a kinase method described previously (26).
Essentially, AML cells (107/sample) were cultured with
either GM-CSF (10 ng/ml), E21R (10 µg/ml), or subjected to 30 min of
osmotic stress using D-sorbitol (0.6 M, Sigma,
Castle Hill, NSW, Australia). The cells were lysed, and the supernatant
was immunoprecipitated with either an anti-JNK1 or an anti-ERK1
monoclonal antibody (1 µg/ml, Pharmigen, San Diego, CA).
Immunoprecipitates were next adsorbed to protein A-Sepharose before
washing in lysis buffer and in kinase buffer. The immunocomplexes were
then resuspended in kinase buffer supplemented with 20 µCi of
[-32P]ATP (BresaGen) and 20 µg of either the fusion
protein c-Jun (1-169)-GST (Upstate Biotechnology, Inc., Lake Placid,
NY) or myelin basic protein (Sigma). The kinase reaction was performed for 30 min at 30 °C before it was terminated with an equal volume of
Laemmli sample buffer. The phosphorylation of products was examined
after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5%)
using the PhosphoImager.
To test whether the Fas/APO-1 (CD95) or tumor
necrosis factor (TNF) receptors were activated upon adding E21R, we
cultured AML cells for 48 h with E21R (1 µg/ml) with or without
a soluble Fas receptor (anti-Fas, 20 µg/ml) or a soluble TNF receptor
(TNFR, 20 µg/ml) (27). As positive controls we measured, after
24 h, either the viability of Jurkat T cells cultured with an
anti-CD3 monoclonal antibody (OKT3, 100 µg/ml) with or without
anti-Fas (20 µg/ml), or the viability of the human laryngeal
carcinoma cell line Hep2 (28) cultured with TNF- (50 ng/ml) with or
without anti-TNFR (20 µg/ml).
We used the ICE-inhibitor Tyr-Val-Ala-Asp-chloromethylketone (YVAD-CMK; Bachem, Switzerland) or the alkylating agent iodoacetamide (Sigma) to block protease activity. Dexamethasone (0.1 mM, Sigma) was used to induce apoptosis of Jurkat T cells with or without overexpression of Bcl-2.
Determination of ApoptosisThe degradation of chromosomal DNA into low molecular weight fragments was displayed on 1.2% agarose gels after an overnight incubation with lysis buffer followed by extraction with organic solutions as described elsewhere (29).
We quantitated the number of apoptotic cells by either measuring the reduced binding of propidium iodide to DNA using an EPICS-Profile II Flow Cytometer (Coulter Electronics, Hialeah, FL) as outlined (30) or by determination of trypan blue exclusion.
All
human, primary, hemopoietic cells express both the GMR- and
c. To
establish the individual requirements of GMR-
and of
c for
E21R-induced apoptosis, we expressed either or both receptor subunits
in Jurkat T cells since these cells do not have endogenous expression
of the GM-CSF receptor. Using monoclonal antibodies and flow cytometry
we isolated the GM-CSF receptor-positive cells and confirmed the
expression of the GMR-
or the
c mutants and that the levels of
expression were similar. Furthermore, the expression levels remained
constant over the next 40 h (data not shown). E21R (10 µg/ml)
induced apoptosis only in cells expressing both receptor subunits and
in a dose-dependent manner (Fig.
1A) with a maximal effect seen after 30 h (Fig. 1B). Cells expressing either receptor subunit alone
remained fully viable. We identified key features of apoptosis (22)
such as condensation of the chromatin and decreases in size of the
nuclei and cells upon addition of E21R by examination of electron
micrographs (Fig. 2, A and B) and
demonstration of fragmented DNA (Fig. 2C). Since Jurkat T cells grow in a GM-CSF-independent manner these experiments further illustrate the active nature of the E21R-induced apoptosis.
To study in more detail the cytoplasmic regions of the GMR- and
c
involved in E21R-induced apoptosis, we expressed a series of mutant
receptors in Jurkat T cells (Fig. 3). In cells
expressing wild-type GMR-
and
c mutants having up to 255 amino
acids deleted (
763,
626), E21R (10 µg/ml) induced apoptosis in a manner similar to cells expressing
the wild-type GM-CSF receptor complex (Fig. 4). Cells
with mutant
544 showed intermediate decline in
viability, whereas cells carrying the
517 and
455 mutants were resistant to E21R treatment. Addition
of wild-type GM-CSF alone had no impact on the viability of these cells
(data not shown). Notably, the chimeric receptor composed of the
extracellular domain of
c and the full-length cytoplasmic domain of
the IL-2 receptor
subunit was unable to signal apoptosis in
response to E21R (Fig. 4). Thus, the membrane-proximal region of the
cytoplasmic domain of
c appears important for E21R-induced
apoptosis.
With a similar approach using cells expressing wild-type c and
truncated GMR-
mutants (Fig. 3), we determined the cytoplasmic region of the GMR-
subunit required for E21R-induced apoptosis. Fig.
5A shows that a region spanning 16 amino
acids downstream of the transmembrane domain was necessary for E21R (10 µg/ml) to induce apoptosis. Interestingly, a GMR-
/IL-2 receptor
common
subunit chimera allowed E21R-induced apoptosis, indicating
that the cytoplasmic region of the IL-2 receptor common
subunit can substitute for GMR-
in this effect (Fig. 5B). Consistent
with the need for the membrane-proximal region of the receptor, the GMR-
/CPROX chimera prevented E21R-induced apoptosis (Fig.
5B). Addition of wild-type GM-CSF alone had no impact on the
viability of the cells (data not shown).
E21R Induces Apoptosis Independent of Phosphorylation of
We next examined whether phosphorylation of c accompanied
E21R-induced apoptosis. We could not detect any alteration in the level
of total phosphorylation of
c immunoprecipitated from AML cells
metabolically labeled with 32P and treated with E21R (1 µg/ml; Fig. 6A). GM-CSF (10 ng/ml), on the
other hand, markedly enhanced phosphorylation of
c. This observation
was confirmed and extended with immunoprecipitated
c and an
anti-phosphotyrosine immunoblot. Whereas GM-CSF caused a rapid and
clear increase in tyrosine phosphorylation of
c, E21R did not (Fig.
6B). Tyrosine phosphorylation of
c is therefore apparently not essential for E21R-induced apoptosis. Neither did we
observe any detectable phosphorylation of the GMR-
subunit upon
addition of E21R (data not shown). Moreover, in separate coimmunoprecipitation experiments using anti-
c antibody for
immunoprecipitation and anti-JAK2 antibody for Western blotting, we
noted that JAK2 was intrinsically associated with
c and that neither
GM-CSF nor E21R altered the levels of JAK2 preassociation up to the
30-min time point. Furthermore, GM-CSF, but not E21R, induced
phosphorylation of JAK2 (data not shown).
E21R Suppresses ERK Activity without Affecting JNK Activity
To assess whether MAP kinases are associated with
E21R-induced apoptosis, we measured the activities of
immunoprecipitated ERK1 and JNK1 in a kinase assay. It is evident from
Fig. 7 that E21R (1 µg/ml) prominently decreased ERK1
activity without affecting JNK1 activity in AML cells. These data
reflect the actual activity of the MAP kinases and not their
concentrations since we could not detect any alteration in the total
amount of either ERK1 or JNK using immunoprecipitation followed by
Western blotting (data not shown).
E21R Induces Apoptosis Independent of the Fas/APO-1 (CD95) or TNF Receptors
We studied E21R-induced apoptosis of AML cells cultured
with soluble Fas and soluble TNF receptors (Fig.
8A). These soluble receptors did not affect
the decline in AML cell viability upon addition of E21R (1 µg/ml),
whereas anti-Fas inhibited Jurkat T cells from dying following
activation of the Fas/APO-1 (CD95) system with an anti-CD3
monoclonal antibody, and anti-TNFR inhibited TNF--induced
apoptosis of Hep2 cells (Fig. 8B).
E21R-induced Apoptosis Is Dependent on ICE-like Proteases
ICE-like proteases participate in the induction of cell
death in response to several different stimuli (22, 31). Apoptosis mediated by several members of the family of ICE-like proteases has
been shown to be inhibited by the cysteine protease inhibitor iodoacetamide and the ICE-inhibitor YVAD-CMK and the viral proteins CrmA and p35 (for review, see Ref. 22). We used these inhibitors to
test whether E21R induced apoptosis via ICE-like proteases. Indeed,
addition of iodoacetamide or YVAD-CMK dose dependently blocked
E21R-induced apoptosis in Jurkat T cells (Fig.
9A) and in AML cells (data not shown).
Treatment with YVAD-CMK or iodoacetamide alone had no impact on cell
viability (Fig. 9A). Jurkat T cells overexpressing either
CrmA or p35 and the wild-type GM-CSF c receptor complex were
rescued from E21R-induced apoptosis (Fig. 9B). However,
overexpression of the oncogene product Bcl-2, an apoptosis-inhibitor
(32), failed to block E21R-induced apoptosis, although it prevented
dexamethasone-induced apoptosis of these cells (Fig.
9C).
We show here that apoptosis induced by the GM-CSF analog E21R
requires the coexpression of GMR- and
c on the cells. Data obtained with the truncated GMR-
mutants revealed that a short membrane-proximal region was crucial for E21R to induce apoptosis. This
notion was strengthened further with data obtained from chimeric GMR-
receptor constructs containing either full-length or regions of
the cytoplasmic IL-2 receptor
common subunit. Interestingly, the
membrane-proximal region of the cytoplasmic domain of GMR-
contains
a proline-rich motif termed box 1 which is conserved among the IL-3R
and IL-5R
subunits, and the common IL-2 receptor
subunit (24).
Deletions within this region in the human GMR-
, and in both the
human and murine IL-5R
, abolished proliferation and protein
phosphorylation (7, 33, 34), demonstrating its pivotal role in cell
growth regulation. Our data pose the intriguing and novel possibility
that this region might also be involved in eliciting a death signal
under certain conditions.
Using a series of truncated c mutants, we found that a
membrane-proximal region spanning approximately 100 amino acids of the
cytoplasmic domain is sufficient for E21R-induced apoptosis. This
particular region has been shown previously to be important for
proliferation of BaF3 cells (8), but it does not include the more
distal region involved in promoting cell survival (15). Moreover,
E21R-induced apoptosis specifically required this membrane-proximal region of
c since the chimeric receptor containing full-length cytoplasmic domain of the IL-2 receptor
subunit, whose amino acid
sequence shares no significant homology with
c, failed to signal
apoptosis in response to E21R.
Stimulation with GM-CSF results in phosphorylation of both c and the
associated JAK2 kinase (8, 16), and tyrosine phosphorylation of the
more distal residue 750 of
c is important for maintaining viability
of BaF3 cells (12). Whereas GM-CSF induced a rapid increase in both
total phosphorylation and tyrosine phosphorylation of
c, E21R was
without any effect. Consistent with this finding is the observation
that E21R, in contrast to GM-CSF, did not activate JAK2. Apparently,
c propagates an E21R-initiated death signal at least without
requiring
c tyrosine phosphorylation, which is consistent with our
previous observation that the tyrosine kinase inhibitor genistein did
not prevent E21R-induced apoptosis (5).
An increase in the activity of the MAP kinase JNK concomitant with a decreased ERK activity has been observed in various cells undergoing apoptosis (17-21). A novel finding in the present study is that E21R-induced apoptosis leads to an increased ratio of JNK to ERK activity because of a decrease in ERK activation. This altered balance in MAP kinase activity might interfere with the activity of transcription factors governing gene expression involved in the control of the cell death effector machinery.
ICE-like proteases play a key role in the execution of apoptosis (for review, see Ref. 22). YVAD-CMK and the thiol protease inhibitor iodoacetamide both block apoptosis by inhibiting one or more of the ICE family members (22). We found that both of these inhibitors abrogated E21R-induced apoptosis. These findings were confirmed and extended using the viral proteins CrmA and p35. Overexpression of either inhibitor blocked E21R-induced apoptosis. In contrast, Bcl-2, an oncogene product that inhibits the apoptotic pathway probably upstream of cysteine protease-activation (35), had no effect.
Among the apoptosis-inducing events most thoroughly studied are the
Fas-related apoptosis and cell death caused by a lack of growth factors
(26, 32, 36). Our data indicate that the apoptotic pathway triggered by
E21R binding to the GM-CSF receptor c complex is probably
different from Fas involvement. First, we could not detect surface
expression of the Fas ligand on primary AML cells (data not shown).
Second, a soluble Fas receptor did not prevent E21R-induced apoptosis.
Third, E21R did not affect the expression and activation of the
serine/threonine kinase FAST and the nuclear RNA-binding protein TIA1
(data not shown) involved in Fas-mediated apoptosis of Jurkat T
cells (36). Fourth, the cytoplasmic domains of GMR-
and
c do not
possess any significant homology to the death domains identified in the
Fas/APO1 and TNF receptors (37, 38).
Although Bcl-2 can prevent apoptosis following growth factor deprivation in various cell types (32, 39), the data presented here indicate that the mechanism of E21R-induced apoptosis is distinct from growth factor deprivation. This is based on the inability of overexpression of Bcl-2 to rescue Jurkat T cells (Fig. 9B) or primary AML cells (data not shown) treated with E21R despite being able to rescue Jurkat T cells from dexamethasone-induced apoptosis (Fig. 9C). In addition, the protein kinase C inhibitor staurosporine blocked E21R-induced apoptosis of AML cells (5), but it failed to suppress death caused by growth factor deprivation (data not shown). Collectively, our data suggest that the death signal initiated with E21R ultimately leads to activation of one or more ICE-like cysteine proteases, and in a Fas/APO-1 (CD95)/Bcl-2-independent manner.
We suggest that a tyrosine-dephosphorylated state of c might be
important for E21R-induced apoptosis. Possibly E21R disrupts a
preformed, productive GM-CSF receptor (5), and events downstream of the
receptor might alter the balance among the activity of the MAP kinases
JNK and ERK leading to gene expression involved in triggering
apoptosis, such as activators of ICE-like proteases. Future studies are
required to define the exact mechanism by which the GM-CSF receptor
elicits the death signal within the cell and the identity of any
resulting gene products.
We thank Dr. A. Strasser for comments on the
manuscript. The cDNA for the truncated GMR- mutants was from A. Kraft. The cDNA for Bcl-2 was from Dr. D. Vaux, CrmA from Dr. D. Pickup, and p35 from Dr. V. Dixit. The supplies of GM-CSF were from
Genetics Institute, E21R from BresaGen, and soluble Fas and TNF
receptors from Dr. P. Krammer.