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Hematologic Malignancy,
Immunology, and
Molecular Oncology Programs, H. Lee Moffitt Cancer Center and Research Institute,
Department of Internal Medicine/Veterans Administration Hospital, and
¶ Department of Biochemistry and Molecular Biology at the University of South Florida College of Medicine, Tampa, FL 33612
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our laboratory and other investigators have identified that the GM-CSF-mediated survival pathway in PMN is linked to activation of the Src family tyrosine kinase, Lyn (2). We demonstrated that GM-CSF treatment resulted in the rapid activation of Lyn kinase activity but not Hck or Fgr. Antisense oligonucleotides to Lyn prevented the antiapoptotic activity of GM-CSF. GM-CSF elicits its response by binding to surface receptors on the cell that activate a cascade of intracellular signaling events, including both serine/threonine and tyrosine phosphorylation (8). Phospholipase A2 and phosphatidylinositol 3-kinase (PI 3-kinase) activation are important in the activation of further downstream targets such as protein kinase B/AKT and transcription factors such as cAMP response element binding protein (CREB) (9, 10, 11, 12). In addition to Lyn tyrosine kinase, c-Fps/Fes and JAK family tyrosine kinases are also activated by GM-CSF in PMN (2, 13, 14, 15, 16). JAK2 phosphorylation occurs in response to GM-CSF in PMN that reportedly activates the STAT family proteins STAT1, STAT3, and STAT5 (14, 17, 18). Other than the involvement of Lyn tyrosine kinase, little is known about the signaling pathways involved in the survival signaling of PMN. PI 3-kinase activation has also been linked to antiapoptotic activity in PMN. The target proteins that are regulated by these signaling pathways that control cell survival are poorly understood. Interestingly, we and others have demonstrated that key antiapoptotic and cell cycle regulatory proteins such as Bcl-2, Bcl-XL, p53, cdc2, and Rb fail to be expressed in either freshly isolated or GM-CSF-treated PMN (2, 19, 20, 21, 22, 23, 24). Therefore, the role of other Bcl-2 family proteins is of interest. This study has identified that Mcl-1, an antiapoptotic protein structurally related to Bcl-2, is an important downstream element in GM-CSF-mediated cell survival signaling of PMN by both the STAT and PI 3-kinase signaling pathways. We found that Mcl-1 is expressed constitutively in human PMN, and treatment with GM-CSF induced Mcl-1 protein expression (21, 23, 25, 26). More importantly, we provide the first evidence that both the STAT3 and PI 3-kinase pathways cooperatively regulate cell survival activity of GM-CSF through Mcl-1 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocyte buffy coats were obtained from normal volunteers of the Southwest Florida Bloodbank (Tampa, FL). Buffy coats were diluted with PBS (BioWhittaker, Walkersville, MD), and PBMC were separated from PMN by centrifugation on Ficoll-Hypaque (Pharmacia, Piscataway, NJ). The PMN layer was further purified by hypotonic lysis of contaminating red blood cells and washed with PBS. PMN were shown to be at least 95% pure by flow cytometry (data not shown). The freshly isolated PMN were treated with 1000 U/ml GM-CSF (Immunex, Seattle, WA) for the indicated times at 37°C and used in apoptosis assays or washed with ice-cold PBS and lysed for EMSA and Western blot analysis.
Treatment with pharmacologic inhibitors and antisense STAT3
PMN were isolated, and pharmacologic inhibitors were added. The compounds used in these experiments included AG-490 (Calbiochem, La Jolla, CA), wortmannin (New England Biolabs, Beverly, MA), and LY294002 (Calbiochem) that were added 1 h before GM-CSF treatment. PMN were cultured with GM-CSF for the indicated duration before lysis for Western blot analysis. PMN from the same donor were also placed in 24-well plates for 42 h with and without GM-CSF for analysis of apoptosis. Antisense STAT3 (5'-ACT CCA ACT GCC CTC CTG CT-3') or control oligonucleotide (5'-TCT GGC AAA GTG TCA GTA TG-3') (Isis Pharmaceuticals, Carlsbad, CA) was added at the concentrations indicated. Oligonucleotides were synthesized using phosphorothioate chemistry and 2'-O-methoxyethyl modification (underlined bases) were used to increase stability, as previously described (27). Antisense STAT3 or control oligonucleotide was added to the cells under serum-free conditions. Preincubation was continued for 6 h and then 5% FCS and GM-CSF were added. The cells were further incubated for 18 h and then lysed for Western blot analysis or prepared for apoptosis assays and cell viability staining.
Cell lysate preparation and EMSA
Nuclear and cytoplasmic lysates for EMSA were prepared as
previously described, and aliquots of the supernatant were stored at
-70°C (28). For use as positive controls, a similar
protocol was used to isolate cytoplasmic and nuclear extracts from U266
multiple myeloma cells, PMN stimulated with 1000 U/ml IFN-
, and
SKBR-3 breast cancer cells treated with epidermal growth factor, as
previously described (28). EMSA was performed by
incubating cytoplasmic lysates (20 µg of total protein) or nuclear
extracts (5 µg of total protein) with incubation buffer containing
100 mM HEPES (pH 7.9), 500 mM KCl, and 10 mM EDTA. For cold competition
or Ab supershifting experiments, excess cold probe (100x molar
ratio) or Ab was added to the incubation reaction for 30 min at room
temperature in a 10-µl reaction volume. Blocking or supershifting
polyclonal rabbit Abs to STAT1 (clone C136), STAT3 (clone H190), STAT5B
(pan-STAT5, clone C17), and STAT5A (clone L20) were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). The following oligonucleotide
probes and the complementary sequences were synthesized and allowed to
anneal in equal molar amounts prior to labeling and use in binding
assays: hSIE (5'-CTTCATTTCCCGTAAATCCCTA-3'
(29), MGFe
(5'-AGATTTCTAGGAATTCAA-3' (30),
and nonrelated DNA FIRE (5'-GTCCCCCGGCCGGGGAGGCGCT-3'
(28), all from Life Technologies (Grand Island, NY). The
oligonucleotide probes (2 pmol) were labeled with
[
-32P]dATP and dCTP and the DNA protein complexes were
separated on a nondenaturing 5% polyacrylamide gel in 0.25x
Tris-borate-EDTA buffer.
Western immunoblotting and immunoprecipitation
Freshly isolated PMN (1 x 107) were lysed in 1
ml buffer composed of 50 mM Tris-Cl, pH 7.6, 5 mM EDTA, 150 mM NaCl,
0.5% Nonidet P-40, 0.5% Triton X-100 containing 1 µg/ml leupeptin,
aprotinin, and antipain, 1 mM sodium orthovanadate, and 0.5 mM PMSF
(all obtained from Sigma, St. Louis, MO). The total protein was
estimated, and 50 µg of denatured protein was analyzed on a 10%
SDS-polyacrylamide gel. Western immunoblotting was performed with the
following Ab dilutions: anti-Mcl-1 (Santa Cruz Biotechnology),
anti-Bax (rabbit polyclonal; PharMingen, San Diego, CA),
anti-PO4AKT (clone 9271L; New England Biolabs),
anti-totalAKT/PKB (catalog no. 06-558; Upstate Biotechnology, Lake
Placid, NY), and anti-STAT3 (clone H190; Santa Cruz Biotechnology)
all at a dilution of 1/1000; anti-
-actin (Sigma) at 1/2500
dilution; anti-JAK2 (Santa Cruz Biotechnology) at 1/200;
anti-phosphoSTAT3 (clone 9131S; New England Biolabs) and
anti-phosphotyrosine (clone 4G10; Upstate Biotechnology) at 1/500
dilution. The blots were then incubated with anti-rabbit or
anti-mouse IgG-conjugated HRP (Amersham, Arlington Heights, IL) and
visualized by ECL (Amersham). The gels were scanned by densitometry and
the signal was quantified using the ImageQuant program (Molecular
Dynamics, Sunnyvale, CA).
Immunoprecipitation was performed by the addition of 25 µl of protein A- or protein G-Sepharose beads to cell lysates from 2.5 x 107 PMN. The immune complexes were washed twice with 50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 0.5% NP-40 containing 1 µg/ml leupeptin, aprotinin, antipain, and 0.5 mM PMSF followed by one wash with 50 mM Tris-Cl, pH 7.6, 150 mM NaCl (buffer A) and 1 wash with Tris-Cl, pH 6.7 (buffer B). The complexes were removed by boiling in Laemmli SDS-PAGE sample loading buffer and analyzed on a 10% SDS-PAGE gel.
RNase protection assay (RPA)
Cell pellets were lysed in TRIzol reagent, and total RNA was isolated according to the manufacturer’s protocol (Life Technologies). RNA was quantified at OD260–280 and aliquoted at -70°C. Probes were synthesized using the Bcl-2 family, multi-probe template set hAPO-2 (PharMingen), and 10 µg RNA per sample was prepared for electrophoresis using In Vitro Transcription Kit (PharMingen). The probes were resolved on a 5% denaturing polyacrylamide gel, dried, and autoradiographed. The image was quantified by densitometry using ImageQuant software.
Apoptosis assay
Freshly isolated PMN in complete medium were placed in 24-well tissue culture plates at a cell density of 1 x 106 cells/ml in 0.5 ml per well in the presence or absence of GM-CSF. Pharmacologic inhibitors were added for the time indicated in each experiment, and the cells were washed in sample wash buffer (PharMingen) and stained with either annexin-V-FITC alone (PharMingen) or in combination with PI according to manufacturer’s recommendation. In experiments where PI was used, it was added as an indicator of viability and no distinction was made between intermediate and late apoptosis. Cells that stained positively for annexin-V-FITC were considered apoptotic. Data acquisition and analysis was performed by the Flow Cytometry Core Facility at the H. Lee Moffitt Cancer Center, (Tampa, FL).
Cell viability
PMN were treated with pharmacologic inhibitors as described for the apoptosis assay. After treatment, cells were washed in RPMI 1640 medium and resuspended at a concentration of 2 x 105 cells/0.1 ml, and cytospins were made in duplicate. The slides were stained with modified Wright Giemsa. Cells demonstrating condensation of the nuclei were considered apoptotic; each slide was examined by two individuals with one person blinded.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and other investigators have demonstrated that
GM-CSF delays the apoptotic cell death of PMN (1, 2, 6, 9). A hallmark of apoptosis is the appearance of
phosphatidylserine in the external plasma membrane that can be detected
by binding to annexin-V-FITC by flow cytometry. Although
externalization of phosphatidylserine has been described in
medium-cultured PMN, the kinetics has not been determined with GM-CSF
treatment (31). We first examined the expression of
phosphatidylserine and found that it was highly expressed in
PMN undergoing apoptosis, and the binding of annexin-V-FITC
was reduced by treatment with GM-CSF at 42 h (Fig. 1
). This quantatitive method was used to
describe the level of apoptosis in further experiments and confirmed by
microscopic examination.
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The Janus kinase (JAK)-family of tyrosine kinases includes JAK1,
JAK2, JAK3, and TYK2 (32, 33). Of the JAK family proteins,
JAK2 was shown to be activated by GM-CSF in PMN (12, 17, 18). The downstream targets for activated JAK family tyrosine
kinases are members of the STAT family transcription factors
(14, 15, 16, 17). We hypothesized that JAK-STAT signaling
contributed to cell survival due to the previously published results
linking STAT-activation to the transcriptional regulation of
antiapoptotic proteins (34). Importantly, IL-6 activation
of STAT3 in multiple myeloma cells was demonstrated to protect from
apoptosis by transcriptional regulation of Bcl-XL
(34). Also, STAT activation is strongly associated with
tumorogenesis of oncoproteins such as v-src and
v-abl (28, 35, 36). We evaluated whether the
STAT proteins STAT1, STAT3, and STAT5 were activated in GM-CSF-treated
PMN by EMSA. Using a 32P-labeled probe that has been
demonstrated to recognize both STAT5 (MGFe) and STAT1 homodimers, we
found robust activation of STAT5-DNA binding activity that was competed
by cold MGFe but not a nonrelated DNA sequence (FIRE) (Fig. 2a). We then performed
supershift analyses of IL-2, GM-CSF, and IFN--treated PMN extracts
with Abs to STAT1, STAT3, STAT5B (pan-STAT5), and STAT5A (specific to
STAT5A) (Fig. 2b). IL-2 and GM-CSF treatment induced
STAT5-like DNA binding activity. Anti-STAT1 and anti-STAT3 Abs failed
to eliminate or supershift the complex, respectively. A pan-STAT5 Ab
that recognizes STAT5A and STAT5B supershifted the entire complex
activated by IL-2. A partial supershift with anti-STAT5A Abs
confirmed the presence of both activated STAT5A and B in the complex of
IL-2-activated PMN (Fig. 2b, lanes 7–10).
Extracts from GM-CSF-treated PMN were only supershifted by
anti-STAT5B Ab, indicating that the DNA binding activity contained
STAT5B (Fig. 2b, lane 15). STAT5B was previously
shown by others to be activated by GM-CSF in neutrophils
(18).
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c) that binds STAT3:3
homodimers, STAT1:3 heterodimers, and STAT1:1 homodimers. GM-CSF
activated a complex that bound this probe, and by supershift analyses
the presence of STAT3 and, to a lesser extent, STAT1 was
identified. In contrast, the activity that predominated in
IFN--treated PMN was STAT1. These data are in agreement with those
of other investigators, which demonstrated activation of STAT1 and
STAT3 using Western immunoblotting with phosphorylation-specific Abs
(17, 18). Reversal of GM-CSF antiapoptotic activity by AG-490
The role of STAT activation in GM-CSF-mediated delay in apoptosis
was evaluated using the JAK-selective tyrphostin pharmacologic
inhibitor, AG-490 (37). AG-490 was demonstrated to be a
selective inhibitor of both JAK2 and JAK3 kinase activity and thereby
reduce STAT activation (38). Increased phosphorylation of
JAK2 was detected in immunoprecipitates of GM-CSF-treated cells that
was reduced with increasing doses of AG-490 pretreatment (Fig. 3a). Furthermore, we also
found that STAT3 phosphorylation by GM-CSF was inhibited by AG-490
(Fig. 3b). To determine if JAK/STAT activation is related to
GM-CSF-delayed apoptosis, we performed apoptosis assays using
GM-CSF-treated PMN from the same donor after 42 h. Indicative of
GM-CSF-mediated antiapoptotic activity, there was a reduction in the
percentage of apoptotic cells with GM-CSF treatment in comparison to
medium-cultured cells (Fig. 3c, p < 0.05 by
2 analysis). In contrast, PMN treated with GM-CSF and
AG-490 demonstrated enhanced cell death in comparison to GM-CSF-treated
PMN (Fig. 3c, panel 4, p < 0.05 by
2 analysis). These data support involvement of the
JAK/STAT pathway in GM-CSF-mediated delayed apoptosis. Interestingly,
AG-490 treatment only partially reversed the GM-CSF-mediated protection
from apoptosis in PMN. Increasing the doses of AG-490 to 100 µM
progressively increased the amount of apoptosis in the presence of
GM-CSF (Fig. 3d). However, the amount of apoptosis remained
statistically less than medium control even at doses of 200 µM AG-490
(data not shown; p < 0.05 by 2
analysis). These data suggest that other pathways contribute to
GM-CSF-induced cell survival.
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A prominent feature of PMN apoptosis is condensation of
multi-lobular nuclei to small condensed nuclear bodies easily
identified by light microscopy after cell staining (2, 5, 6, 7). To confirm that the results obtained by annexin-V-FITC
binding correlated with apoptotic morphology, we performed experiments
with GM-CSF treatment in the presence of AG-490 and wortmannin and
calculated the percentage of apoptotic cells by microscopic
examination. Typical PMN apoptotic morphology was observed in the
majority of medium cultured cells (86%), whereas GM-CSF treatment had
more PMN with normal multi-lobular nuclei and only 19% apoptotic cells
(Fig. 3e). AG-490 reversed the GM-CSF prevention of
apoptosis by 
50%. We consistently observed fewer apoptotic cells in
GM-CSF-treated PMN as assessed by morphology than by flow cytometry.
These results are anticipated because phosphotidylserine positivity
detected by annexin-V binding occurs before changes in nuclear
morphology. However, the effects of AG-490 and wortmannin treatment
were similar as assessed using both techniques.
Antisense STAT3 reduced GM-CSF-mediated cell survival
By EMSA DNA-binding studies, we found that STAT1, STAT3, and
STAT5B were activated in response to GM-CSF in PMN. Others have
previously demonstrated similar results for STAT activation but the
biological significance was not determined (14, 17, 18).
An antisense oligonucleotide approach to reduce intracellular STAT3
protein expression is an effective means of dissecting the direct
contribution of this protein to GM-CSF-mediated cell survival
(39). The STAT3 oligonucleotide treatment in comparison to
control oligonucleotide partially but not completely reduced STAT3
expression in medium and GM-CSF-cultured PMN by Western blot analysis
(Fig. 4a). In parallel assays,
the PMN treated with GM-CSF and antisense STAT3 displayed increased
annexin-V-FITC staining (Fig. 4b) and reduced viability
(Fig. 4c) in comparison to PMN cultured for 24 h with
control oligonucleotide. Therefore, using a method that specifically
targeted the STAT3 protein, we confirmed that STAT3 signaling
participates in GM-CSF-mediated cell survival.
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Apoptosis is tightly regulated by Bcl-2 family proteins, some of
which function to suppress apoptosis (Bcl-2, Bcl-xL, Mcl-1,
Bfl-1/A1, and A20) and others that potentiate apoptosis (Bax, Bad, Bid,
Bok, Bcl-xs) (40, 41, 42, 43, 44, 45, 46, 47, 48). Our laboratory and others have
previously shown that PMN fail to constitutively express Bcl-2 and
Bcl-xL antiapoptotic proteins (2, 20, 21, 22, 23, 24, 25).
In contrast, Mcl-1 protein was shown to be constitutively expressed and
inducible by GM-CSF, IL-1, sodium butryate, and LPS
(21). We first examined the level of mRNA expression of
several Bcl-2-family members by RPAs after 1, 8, and 18 h of
GM-CSF treatment. Here, the antiapoptotic proteins constitutively
expressed in PMN were Bfl-1/A1, Bcl-w (data not shown), and Mcl-1 (Fig. 5a). These data are in
agreement with previously published results for constitutive expression
of antiapoptotic proteins in PMN (21, 22, 23, 49). Bfl-1/A1 is
known to be inducible by some but not all survival enhancing
treatments. In our study, GM-CSF treatment resulted in the induction of
both Bcl-x and Mcl-1 mRNA but not Bfl-1/A1 or Bcl-w (Fig. 5a). We found that Bcl-2 mRNA was not expressed
constitutively or induced by GM-CSF. We then examined the expression of
Bcl-xL and Mcl-1 proteins by Western blot analysis. After
treatment with GM-CSF, Mcl-1 protein expression was increased in
comparison to -actin (Fig. 5b); Bcl-xL
protein was not detected (Fig. 5b). Continued incubation of
PMN with GM-CSF for 18 h did not result in detectable
Bcl-xL protein induction (data not shown).
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The relative ratio and dimerization partnerships of antiapoptotic
and proapoptotic Bcl-2-family proteins are critical determinants of
life and death (50). Several experimental models of Mcl-1
overexpression has confirmed that this protein protects cells against
apoptotic cell death (51, 52, 53, 54). Protection against
Bax-mediated cell death occurred when Mcl-1 was overexpressed in a
yeast two hybrid system (55). Therefore, we evaluated
whether association between Mcl-1 and Bax could be detected by
coimmunoprecipitation in PMN. Using a mouse mAb to Bax for
immunoprecipitation of untreated or GM-CSF (18 h)-treated PMN, we
detected Mcl-1 in Bax immunoprecipitates by Western immunoblotting with
a rabbit polyclonal anti-Mcl-1 Ab (Santa Cruz Biotechnology) (Fig. 6). In the presence of GM-CSF, there was
a 3-fold increased amount of Mcl-1 coimmunoprecipitated with Bax as
determined by densitometry. The relative rise in Mcl-1 expression and
enhanced association with Bax after GM-CSF treatment supports our
hypothesis that Mcl-1 plays an important role in GM-CSF-delayed
apoptosis.
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Induction of Mcl-1 in BaF3 cells treated with IL-3 or GM-CSF has
been linked to the activation of PI 3-kinase/AKT (10, 12, 55, 56). Therefore, we examined the level of Mcl-1 expression in the
presence of GM-CSF alone and in combination with PI 3-kinase
inhibitors. GM-CSF induced rapid phosphorylation of AKT that was
inhibited by wortmannin and LY294002 pretreatment, suggesting that the
PI 3-kinase/AKT pathway was activated (Fig. 7a). We found that a dose of
100 nM wortmannin and 25 µM LY294002 completely reversed the AKT
phosphorylation as determined by densitometry. Additionally, we found
that increased doses of both wortmannin and LY294002 reduced the
GM-CSF-mediated induction of Mcl-1 protein expression after 24 h.
The relative densitometry measurements obtained in comparison to
-actin were determined with the following data obtained: medium
(0.13), GM-CSF (0.42), GM-CSF with wortmannin 50 (0.34), 100 (0.23),
and 200 nM (0.2), GM-CSF with LY294002 25 (.13) and 50 µM (0.07)
(Fig. 7a). We found that the expression of Bax was unchanged
by any of these treatments. Activation of the PI 3-kinase/AKT pathway
has often been linked to survival signaling in PMN (19).
Using wortmannin in combination with GM-CSF, we observed increased
apoptosis by annexin-V binding (Fig. 7b) and microscopic
examination (Fig. 7c) in contrast to GM-CSF-treated PMN.
Also, treatment of PMN with increasing doses of wortmannin or LY294002
progressively increased the percentage of apoptotic cells in
GM-CSF-treated PMN (Fig. 7d). These data suggest that not
only the STAT3 signaling pathway but also the PI 3-kinase pathway
regulate GM-CSF-delayed apoptosis. One downstream effector of the PI
3-kinase pathway is the induction of antiapoptotic Mcl-1
expression.
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The role of STAT activation in Mcl-1 expression was evaluated
using AG-490. We used doses of AG-490 known to inhibit both JAK2
phosphorylation and GM-CSF-mediated apoptosis. We then evaluated the
level of Mcl-1 protein after incubation with AG-490 or an equal volume
of DMSO (vehicle control) (Fig. 8). There
was a progressive decline in Mcl-1 protein expression in PMN cultured
in medium consistent with rapid degradation of Mcl-1 previously linked
to the presence of a "PEST" sequence in the protein (Fig. 8, lanes 1 and 4) (48). However, the
amount of Mcl-1 protein was higher in GM-CSF-treated extracts at both
time points with a partial reduction in Mcl-1 protein in AG-490-treated
cell extracts (Fig. 8). These data are consistent with the involvement
of the JAK/STAT pathway in GM-CSF-induced Mcl-1 expression in addition
to that of PI 3-kinase.
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Using either AG-490 or wortmannin alone, we consistently observed
only partial inhibition of Mcl-1 induction and only partial reversal of
the cell survival advantage mediated by GM-CSF. Furthermore, the STAT
and PI 3-kinase pathways regulate similar biological functions in PMN
suggesting that they may participate together. Therefore, we examined
whether the two pathways functioned cooperatively by using suboptimal
doses of both AG-490 and wortmannin. Using either drug alone, we
observed a partial reversal of GM-CSF-reduced apoptosis (Fig. 9, a and b). Under
these conditions, morphologic examination showed a mixture of
nonapoptotic PMN as well as PMN with early findings of apoptosis. In
contrast, however, when both drugs were added concurrently with GM-CSF,
the findings were similar to cells cultured without GM-CSF for 42
h, containing almost all apoptotic PMN (Fig. 9b). Mcl-1
protein expression was greatly reduced in the cells treated with both
AG-490 and wortmannin after 24 h. In contrast, the levels of Bax
and -actin were unchanged by GM-CSF or drug treatments. These data
are consistent with a cooperative role for PI 3-kinase and JAK/STAT in
the regulation of Mcl-1 expression and GM-CSF survival signaling
in PMN.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that the STAT pathway is an important determinant of GM-CSF antiapoptotic signaling in PMN. Our data using the AG-490 JAK-selective pharmacological inhibitor as well as an antisense approach support this conclusion. In particular, the reversibility of survival signaling by antisense oligonucleotide for STAT3 is highly supportive of a role for this protein in neutrophil survival. There is considerable evidence to link STAT activation with cellular transformation and oncogenesis. However, the downstream effectors mediating this phenomenon are not clearly delineated. In multiple myeloma, the mechanism of STAT3-mediated survival is its transcriptional regulation of the Bcl-xL antiapoptotic protein. However, we were unable to detect Bcl-xL protein in PMN even after incubation with GM-CSF. Instead, our data suggest that the prolonged survival of PMN after GM-CSF treatment results from STAT upregulation of antiapoptotic Mcl-1. In PMN, we have demonstrated the first link between STAT activation and a biologic function. Enhanced coimmunoprecipitation of Mcl-1 with proapoptotic Bax lends additional supportive data for the involvement of Mcl-1 in GM-CSF-mediated antiapoptotic signaling.
We also demonstrated that the PI 3-kinase/AKT pathway contributed to GM-CSF-mediated delayed apoptosis in PMN. These results are similar to the effects of GM-CSF observed in BaF3 cells, in which a pathway involving PI 3-kinase activation of AKT was critical for survival (56, 57). A well-defined target of AKT phosphorylation is the Ser136 site of the proapoptotic protein Bad (47). Traditionally, phosphorylation results in the binding of Bad to the 14-3-3 protein that interupts the association between Bad and Bcl-xL or Bcl-2. Increased amounts of Bcl-xL and Bcl-2 are then free to bind Bax and prevent its proapoptotic activity. Interestingly, others have shown Bad phosphorylation in response to GM-CSF in human neutrophils; however, Bcl-xL and Bcl-2 are not present in PMN (19). These results suggest the possibility that Bad might interact with Mcl-1. However, yeast two-hybrid analyses using Mcl-1 and Bad failed to demonstrate a viability response suggesting that Mcl-1 and Bad are incapable of dimerization. It is conceivable that biologically relevant partnerships such as Mcl-1 and Bad may only occur in some cell types and fail to be detected in yeast two-hybrid studies. Future experiments will determine the role of Bad phosphorylation and its possible association with Mcl-1 in PMN.
In our experiments, we found that GM-CSF signaling led to increased expression of the antiapoptotic protein Mcl-1. Similar to the results of yeast two-hybrid and FDC-P1 cells overexpressing Mcl-1, we were able to identify a heterodimeric complex of Mcl-1 and Bax by coimmunoprecipitation experiments (52). These are important novel findings that provide evidence of Mcl-1/Bax heterodimerization from human cell extracts without overexpression. In contrast, coimmunoprecipitation experiments in 32D myeloid leukemia cells failed to detect Mcl-1/Bax heterodimers by coimmunoprecipitation even with overexpression (58). Although not studied here, GM-CSF may also contribute to accumulation of the Mcl-1 protein by stabilization of the protein. However, from the data presented here, we can conclude that PI 3-kinase/AKT-mediated induction of Mcl-1 protein expression acts as an additional mechanism to positively regulate cell survival in PMN.
Increased PMN survival after GM-CSF treatment has also been attributed to activation of mitogen-activated protein kinase (MAPK), p42/44 extracellular signal-related kinase-1 (2, 19). Based upon studies by Klein et al., no additive inhibition of apoptosis delay occurred when LY294002 and a MAPK kinase (MEK) inhibitor that blocks activation of MAPK (PD098059) were added concurrently (19). These results suggest that pathways distinct from PI 3-kinase are involved in MAPK prevention of apoptosis.
Other investigators have demonstrated that AKT phosphorylation of a transcription factor complex containing CREB was involved in the up-regulation of Mcl-1 gene expression in BaF3 cells (57). Interestingly, an unidentified STAT-like transcription factor was also involved in IL-3-mediated mcl-1 inducibility. Using heterologous studies of the murine Mcl-1 promoter, these authors showed that both an SIE-STAT-like element and a CREB site were necessary to confer complete IL-3 inducibility. The human mcl-1 promoter is yet to be cloned to confirm the results of the murine promoter studies. However, we have found that STAT3 can directly regulate the murine mcl-1 promoter in NIH3T3 cells overexpressing v-src (our unpublished observations). With the confirmed results of transcriptional regulation, these data are strong evidence for cooperation between the JAK/STAT and PI 3-kinase pathways with regard to GM-CSF-inducible Mcl-1 protein expression in this biologic setting. Additional experiments are required to identify the mechanism of STAT and PI 3-kinase cooperation. These data are also consistent with a role for Mcl-1 in GM-CSF-mediated pro-survival activity, although a direct link must still be established. Using antisense oligonucleotides to Mcl-1, Moulding et al. recently showed that Mcl-1 is essential for prevention of apoptosis during differentiation of U937 cells (59). Our data provide further insight into key intracellular events that are necessary for GM-CSF-mediated regulation of apoptosis in human neutrophils.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. P. K. Epling-Burnette, Hematologic Malignancy Program, MRC Room 2068 f and g, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: burnetpk{at}moffitt.usf.edu
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte(s); PI, propidium iodide; PI 3-kinase, phosphatidylinositol 3-kinase; NP-40, Nonidet P-40; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; CREB, cAMP response element binding protein; SIE, SIS-inducible element; RPA, RNase protection assay.
Received for publication October 13, 2000. Accepted for publication March 19, 2001.
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References |
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 C. Yang, J. H. Schwab, A. J. Schoenfeld, F. J. Hornicek, K. B. Wood, G. P. Nielsen, E. Choy, H. Mankin, and Z. Duan A novel target for treatment of chordoma: signal transducers and activators of transcription 3 Mol. Cancer Ther., September 1, 2009; 8(9): 2597 - 2605. [Abstract] [Full Text] [PDF]
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 A. Chakravarti, M.-A. Raquil, P. Tessier, and P. E. Poubelle Surface RANKL of Toll-like receptor 4-stimulated human neutrophils activates osteoclastic bone resorption Blood, August 20, 2009; 114(8): 1633 - 1644. [Abstract] [Full Text] [PDF]
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 D. L. Hodge, J. Yang, M. D. Buschman, P. M. Schaughency, H. Dang, W. Bere, Y. Yang, R. Savan, J. J. Subleski, X.-M. Yin, et al. Interleukin-15 Enhances Proteasomal Degradation of Bid in Normal Lymphocytes: Implications for Large Granular Lymphocyte Leukemias Cancer Res., May 1, 2009; 69(9): 3986 - 3994. [Abstract] [Full Text] [PDF]
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 B. B. Aggarwal, R.V. Vijayalekshmi, and B. Sung Targeting Inflammatory Pathways for Prevention and Therapy of Cancer: Short-Term Friend, Long-Term Foe Clin. Cancer Res., January 15, 2009; 15(2): 425 - 430. [Abstract] [Full Text] [PDF]
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S. A. Didichenko, N. Spiegl, T. Brunner, and C. A. Dahinden IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils Blood, November 15, 2008; 112(10): 3949 - 3958. [Abstract] [Full Text] [PDF]
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 M. Katsube, T. Kato, M. Kitagawa, H. Noma, H. Fujita, and S. Kitagawa Calpain-mediated regulation of the distinct signaling pathways and cell migration in human neutrophils J. Leukoc. Biol., July 1, 2008; 84(1): 255 - 263. [Abstract] [Full Text] [PDF]
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H. Hu, Y. Shikama, I. Matsuoka, and J. Kimura Terminally differentiated neutrophils predominantly express Survivin-2{alpha}, a dominant-negative isoform of Survivin J. Leukoc. Biol., February 1, 2008; 83(2): 393 - 400. [Abstract] [Full Text] [PDF]
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K. Dvorak, M. Chavarria, C. M. Payne, L. Ramsey, C. Crowley-Weber, B. Dvorakova, B. Dvorak, H. Bernstein, H. Holubec, R. E. Sampliner, et al. Activation of the Interleukin-6/STAT3 Antiapoptotic Pathway in Esophageal Cells by Bile Acids and Low pH: Relevance to Barrett's Esophagus Clin. Cancer Res., September 15, 2007; 13(18): 5305 - 5313. [Abstract] [Full Text] [PDF]
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G. Seumois, M. Fillet, L. Gillet, C. Faccinetto, C. Desmet, C. Francois, B. Dewals, C. Oury, A. Vanderplasschen, P. Lekeux, et al. De novo C16- and C24-ceramide generation contributes to spontaneous neutrophil apoptosis J. Leukoc. Biol., June 1, 2007; 81(6): 1477 - 1486. [Abstract] [Full Text] [PDF]
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R. Blomgran, L. Zheng, and O. Stendahl Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization J. Leukoc. Biol., May 1, 2007; 81(5): 1213 - 1223. [Abstract] [Full Text] [PDF]
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 L. M. Greene, L. Kelly, V. Onnis, G. Campiani, M. Lawler, D. C. Williams, and D. M. Zisterer STI-571 (Imatinib Mesylate) Enhances the Apoptotic Efficacy of Pyrrolo-1,5-Benzoxazepine-6, a Novel Microtubule-Targeting Agent, in Both STI-571-Sensitive and -Resistant Bcr-Abl-Positive Human Chronic Myeloid Leukemia Cells J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 288 - 297. [Abstract] [Full Text] [PDF]
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C. F. Fortin, A. Larbi, O. Lesur, N. Douziech, and T. Fulop Jr. Impairment of SHP-1 down-regulation in the lipid rafts of human neutrophils under GM-CSF stimulation contributes to their age-related, altered functions J. Leukoc. Biol., May 1, 2006; 79(5): 1061 - 1072. [Abstract] [Full Text] [PDF]
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C. A. Lindemans, P. J. Coffer, I. M. M. Schellens, P. M. A. de Graaff, J. L. L. Kimpen, and L. Koenderman Respiratory Syncytial Virus Inhibits Granulocyte Apoptosis through a Phosphatidylinositol 3-Kinase and NF-{kappa}B-Dependent Mechanism J. Immunol., May 1, 2006; 176(9): 5529 - 5537. [Abstract] [Full Text] [PDF]
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 J. G. Clohessy, J. Zhuang, J. de Boer, G. Gil-Gomez, and H. J. M. Brady Mcl-1 Interacts with Truncated Bid and Inhibits Its Induction of Cytochrome c Release and Its Role in Receptor-mediated Apoptosis J. Biol. Chem., March 3, 2006; 281(9): 5750 - 5759. [Abstract] [Full Text] [PDF]
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S. D. Kobayashi, J. M. Voyich, A. R. Whitney, and F. R. DeLeo Spontaneous neutrophil apoptosis and regulation of cell survival by granulocyte macrophage-colony stimulating factor J. Leukoc. Biol., December 1, 2005; 78(6): 1408 - 1418. [Abstract] [Full Text] [PDF]
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E. Sakamoto, F. Hato, T. Kato, C. Sakamoto, M. Akahori, M. Hino, and S. Kitagawa Type I and type II interferons delay human neutrophil apoptosis via activation of STAT3 and up-regulation of cellular inhibitor of apoptosis 2 J. Leukoc. Biol., July 1, 2005; 78(1): 301 - 309. [Abstract] [Full Text] [PDF]
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S. Francois, J. El Benna, P. M. C. Dang, E. Pedruzzi, M.-A. Gougerot-Pocidalo, and C. Elbim Inhibition of Neutrophil Apoptosis by TLR Agonists in Whole Blood: Involvement of the Phosphoinositide 3-Kinase/Akt and NF-{kappa}B Signaling Pathways, Leading to Increased Levels of Mcl-1, A1, and Phosphorylated Bad J. Immunol., March 15, 2005; 174(6): 3633 - 3642. [Abstract] [Full Text] [PDF]
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 P. Boutet, D. Boulanger, L. Gillet, A. Vanderplasschen, R. Closset, F. Bureau, and P. Lekeux Delayed Neutrophil Apoptosis in Bovine Subclinical Mastitis J Dairy Sci, December 1, 2004; 87(12): 4104 - 4114. [Abstract] [Full Text] [PDF]
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Y. Kotone-Miyahara, K. Yamashita, K.-K. Lee, S. Yonehara, T. Uchiyama, M. Sasada, and A. Takahashi Short-term delay of Fas-stimulated apoptosis by GM-CSF as a result of temporary suppression of FADD recruitment in neutrophils: evidence implicating phosphatidylinositol 3-kinase and MEK1-ERK1/2 pathways downstream of classical protein kinase C J. Leukoc. Biol., November 1, 2004; 76(5): 1047 - 1056. [Abstract] [Full Text] [PDF]
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 L. E. Kilpatrick, S. Sun, and H. M. Korchak Selective regulation by {delta}-PKC and PI 3-kinase in the assembly of the antiapoptotic TNFR-1 signaling complex in neutrophils Am J Physiol Cell Physiol, September 1, 2004; 287(3): C633 - C642. [Abstract] [Full Text] [PDF]
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T. Kato, E. Sakamoto, H. Kutsuna, A. Kimura-Eto, F. Hato, and S. Kitagawa Proteolytic Conversion of STAT3{alpha} to STAT3{gamma} in Human Neutrophils: ROLE OF GRANULE-DERIVED SERINE PROTEASES J. Biol. Chem., July 23, 2004; 279(30): 31076 - 31080. [Abstract] [Full Text] [PDF]
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M. Derouet, L. Thomas, A. Cross, R. J. Moots, and S. W. Edwards Granulocyte Macrophage Colony-stimulating Factor Signaling and Proteasome Inhibition Delay Neutrophil Apoptosis by Increasing the Stability of Mcl-1 J. Biol. Chem., June 25, 2004; 279(26): 26915 - 26921. [Abstract] [Full Text] [PDF]
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 Y.-H. Chung, N.-h. Cho, M. I. Garcia, S.-H. Lee, P. Feng, and J. U. Jung Activation of Stat3 Transcription Factor by Herpesvirus Saimiri STP-A Oncoprotein J. Virol., June 15, 2004; 78(12): 6489 - 6497. [Abstract] [Full Text] [PDF]
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N. A. Maianski, D. Roos, and T. W. Kuijpers Bid Truncation, Bid/Bax Targeting to the Mitochondria, and Caspase Activation Associated with Neutrophil Apoptosis Are Inhibited by Granulocyte Colony-Stimulating Factor J. Immunol., June 1, 2004; 172(11): 7024 - 7030. [Abstract] [Full Text] [PDF]
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 O. Moshynska, K. Sankaran, P. Pahwa, and A. Saxena Prognostic Significance of a Short Sequence Insertion in the MCL-1 Promoter in Chronic Lymphocytic Leukemia J Natl Cancer Inst, May 5, 2004; 96(9): 673 - 682. [Abstract] [Full Text] [PDF]
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A. Bouchard, C. Ratthe, and D. Girard Interleukin-15 delays human neutrophil apoptosis by intracellular events and not via extracellular factors: role of Mcl-1 and decreased activity of caspase-3 and caspase-8 J. Leukoc. Biol., May 1, 2004; 75(5): 893 - 900. [Abstract] [Full Text] [PDF]
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P. K. Epling-Burnette, J. S. Painter, P. Chaurasia, F. Bai, S. Wei, J. Y. Djeu, and T. P. Loughran Jr Dysregulated NK receptor expression in patients with lymphoproliferative disease of granular lymphocytes Blood, May 1, 2004; 103(9): 3431 - 3439. [Abstract] [Full Text] [PDF]
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K. Dvorakova, C. M. Payne, L. Ramsey, H. Holubec, R. Sampliner, J. Dominguez, B. Dvorak, H. Bernstein, C. Bernstein, A. Prasad, et al. Increased Expression and Secretion of Interleukin-6 in Patients with Barrett's Esophagus Clin. Cancer Res., March 15, 2004; 10(6): 2020 - 2028. [Abstract] [Full Text] [PDF]
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Z. Hu and M. M. Sayeed Suppression of mitochondria-dependent neutrophil apoptosis with thermal injury Am J Physiol Cell Physiol, January 1, 2004; 286(1): C170 - C178. [Abstract] [Full Text] [PDF]
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K. Brocke-Heidrich, A. K. Kretzschmar, G. Pfeifer, C. Henze, D. Loffler, D. Koczan, H.-J. Thiesen, R. Burger, M. Gramatzki, and F. Horn Interleukin-6-dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family-independent survival pathway closely associated with Stat3 activation Blood, January 1, 2004; 103(1): 242 - 251. [Abstract] [Full Text] [PDF]
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L. J. Crossley Neutrophil activation by fMLP regulates FOXO (forkhead) transcription factors by multiple pathways, one of which includes the binding of FOXO to the survival factor Mcl-1 J. Leukoc. Biol., October 1, 2003; 74(4): 583 - 592. [Abstract] [Full Text] [PDF]
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M. Choi, S. Rolle, M. Wellner, M. C. Cardoso, C. Scheidereit, F. C. Luft, and R. Kettritz Inhibition of NF-{kappa}B by a TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis Blood, September 15, 2003; 102(6): 2259 - 2267. [Abstract] [Full Text] [PDF]
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H. Liu, Y. Ma, S. M. Cole, C. Zander, K.-H. Chen, J. Karras, and R. M. Pope Serine phosphorylation of STAT3 is essential for Mcl-1 expression and macrophage survival Blood, July 1, 2003; 102(1): 344 - 352. [Abstract] [Full Text] [PDF]
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N. A. Maianski, D. Roos, and T. W. Kuijpers Tumor necrosis factor alpha induces a caspase-independent death pathway in human neutrophils Blood, March 1, 2003; 101(5): 1987 - 1995. [Abstract] [Full Text] [PDF]
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T. Hasegawa, K. Suzuki, C. Sakamoto, K. Ohta, S. Nishiki, M. Hino, N. Tatsumi, and S. Kitagawa Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia Blood, February 1, 2003; 101(3): 1164 - 1171. [Abstract] [Full Text] [PDF]
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Y. Ma, W. D. Cress, and E. B. Haura Flavopiridol-induced Apoptosis Is Mediated through Up-Regulation of E2F1 and Repression of Mcl-1 Mol. Cancer Ther., January 1, 2003; 2(1): 73 - 81. [Abstract] [Full Text] [PDF]
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 S. Saba, G. Soong, S. Greenberg, and A. Prince Bacterial Stimulation of Epithelial G-CSF and GM-CSF Expression Promotes PMN Survival in CF Airways Am. J. Respir. Cell Mol. Biol., November 1, 2002; 27(5): 561 - 567. [Abstract] [Full Text] [PDF]
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K. Yasui, Y. Sekiguchi, M. Ichikawa, H. Nagumo, T. Yamazaki, A. Komiyama, and H. Suzuki Granulocyte macrophage-colony stimulating factor delays neutrophil apoptosis and primes its function through Ia-type phosphoinositide 3-kinase J. Leukoc. Biol., November 1, 2002; 72(5): 1020 - 1026. [Abstract] [Full Text] [PDF]
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I. M. Pedersen, S. Kitada, L. M. Leoni, J. M. Zapata, J. G. Karras, N. Tsukada, T. J. Kipps, Y. S. Choi, F. Bennett, and J. C. Reed Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1 Blood, August 13, 2002; 100(5): 1795 - 1801. [Abstract] [Full Text] [PDF]
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 R. Kettritz, M. Choi, W. Butt, M. Rane, S. Rolle, F. C. Luft, and J. B. Klein Phosphatidylinositol 3-Kinase Controls Antineutrophil Cytoplasmic Antibodies--Induced Respiratory Burst in Human Neutrophils J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1740 - 1749. [Abstract] [Full Text] [PDF]
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 G. Z. Rassidakis, R. Lai, T. J. McDonnell, F. Cabanillas, A. H. Sarris, and L. J. Medeiros Overexpression of Mcl-1 in Anaplastic Large Cell Lymphoma Cell Lines and Tumors Am. J. Pathol., June 1, 2002; 160(6): 2309 - 2310. [Full Text] [PDF]
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R. Buettner, L. B. Mora, and R. Jove Activated STAT Signaling in Human Tumors Provides Novel Molecular Targets for Therapeutic Intervention Clin. Cancer Res., April 1, 2002; 8(4): 945 - 954. [Abstract] [Full Text] [PDF]
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C. Yu, G. Krystal, L. Varticovksi, R. McKinstry, M. Rahmani, P. Dent, and S. Grant Pharmacologic Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase/Mitogen-activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-expressing Human Leukemia Cells Cancer Res., January 1, 2002; 62(1): 188 - 199. [Abstract] [Full Text] [PDF]
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J. G. Kupfner, J. J. Arcaroli, H.-K. Yum, S. G. Nadler, K.-Y. Yang, and E. Abraham Role of NF-{kappa}B in Endotoxemia-Induced Alterations of Lung Neutrophil Apoptosis J. Immunol., December 15, 2001; 167(12): 7044 - 7051. [Abstract] [Full Text] [PDF]
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