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Blood, 15 June 2002, Vol. 99, No. 12, pp. 4307-4317
CHEMOKINES
Enhancement of intracellular signaling associated with
hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in
synergy with other cytokines
Younghee Lee,
Akihiko Gotoh,
Hyung-Joo Kwon,
Minute You,
Lisa Kohli,
Charlie Mantel,
Scott Cooper,
Giao Hangoc,
Keisuke Miyazawa,
Kazuma Ohyashiki, and
Hal E. Broxmeyer
From the Departments of Microbiology/Immunology,
Medicine, Biochemistry and Molecular Biology, and the Walther Oncology
Center, Indiana University School of Medicine, and the Walther Cancer
Institute, Indianapolis; and First Department of Internal Medicine,
Tokyo Medical University, Nishishinjuku, Shinjuku-ku, Tokyo, Japan.
 |
Abstract |
Stromal cell-derived factor 1 (SDF-1/CXCL12) is a multifunctional
cytokine. We previously reported that myelopoiesis was enhanced in
SDF-1 transgenic mice, probably due in part to SDF-1 enhancement of myeloid progenitor cell (MPC) survival. To understand signaling pathways involved in this activity, we studied the effects on factor-dependent cell line MO7e cells incubated with SDF-1 alone or
in combination with other cytokines. SDF-1 induced transient activation of extracellular stress-regulated kinase (ERK1/2), ribosomal S6 kinase (p90RSK) and Akt, molecules implicated in cell
survival. Moreover, ERK1/2, p90RSK, and Akt were synergistically activated by SDF-1 in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor (SLF), or
thrombopoietin (TPO). Similar effects were seen after pretreatment of
MO7e cells with SDF-1 followed by stimulation with the other
cytokines, suggesting a priming effect of SDF-1. Nuclear factor- B
(NF-B) did not appear to be involved in SDF-1 actions, alone or
in combination with other cytokines. These intracellular effects were
consistent with enhanced myeloid progenitor cell survival by SDF-1
after delayed addition of growth factors. SDF-1 alone supported
survival of highly purified human cord blood CD34+++ cells,
less purified human cord blood, and MO7e cells; this effect was
synergistically enhanced when SDF-1 was combined with low amounts of
other survival-promoting cytokines (GM-CSF, SLF, TPO, and FL). SDF-1
may contribute to maintenance of MPCs in bone marrow by enhancing cell
survival alone and in combination with other cytokines.
(Blood. 2002;99:4307-4317)
© 2002 by The American Society of Hematology.
| |
Introduction |
Blood cell production is maintained by a balance
between growth and death, processes regulated in part by cytokine-cell
interactions. Hematopoietically relevant cytokines have been identified
and their implications in proliferation and survival
studied.1-4
Chemokines are part of a family of cytokines having chemotactic
activities; they mediate effects by binding 7 transmembrane domain
receptors associated with heterotrimeric Gi proteins.5-8 The human chemokine system includes more than 50 chemokines and 18 chemokine receptors.7-9 Many chemokines bind more than one receptor, and receptors generally bind more than one chemokine. However, stromal cell-derived factor 1 (SDF-1; CXCL12) is a CXC chemokine apparently interacting with only one receptor,
CXCR4.10-13 This single chemokine-single receptor is
supported by the nearly identical phenotypes of SDF-1 /
and CXCR4/ mice including impaired
hematopoiesis.10,11 SDF-1 protects against human
immunodeficiency virus (HIV) infection by competing with gp120 for
binding to CXCR4 and by down-regulation of the receptor.14
Studies have implicated SDF-1 in the migration of myeloid progenitor
cells (MPCs) and stem cells.5-7,15-18 SDF-1 is
constitutively expressed in specific lymphoid or nonlymphoid tissues,
in contrast to inflammatory chemokines expressed in inflamed
tissues.9 SDF-1 is also involved in differentiation,
proliferation, and survival of various cellular systems. SDF-1 was
originally cloned from a stromal cell line as a pre-B cell
growth-stimulating factor.19 SDF-1 acts with
thrombopoietin (TPO) to enhance development of megakaryocytic
progenitor cells.20 Stromal cells or cytokines in synergy
with SDF-1 support survival of human leukemic B-cell precursors.21 Blood-derived nurselike cells protect
chronic lymphocytic leukemia B cells from spontaneous apoptosis through SDF-1.22 We reported that myelopoiesis is enhanced in
SDF-1 transgenic mice, and SDF-1 modulates myelopoiesis by
regulating progenitor cell survival and inhibitory effects of
myelosuppressive chemokines.23
A number of intracellular pathways have been implicated in cell
survival. Phosphoinositol 3-kinase (PI3K)/protein kinase B (PKB)(Akt) and mitogen-activated protein kinase (MAPK)/p90 ribosomal S6
kinase (RSK) pathways are 2 major survival pathways induced by
cytokines.24,25 After activation of specific growth factor receptors, PI3K is recruited to the inner surface of the plasma membrane and generates phosphoinositol-3,4,5-triphosphate
(PIP3).26,27 The PI3K-generated phospholipids act by
multiple mechanisms that cooperate to regulate Akt
activity.24 Akt is phosphorylated and activated by
phospholipid-dependent kinase 1 (PDK1). Targets of Akt include BAD,
caspase-9, forkhead family transcription factors, and the nuclear
factor-B (NF-B) regulator inhibitor of kB (IB) kinase
(IKK).24 Akt also phosphorylates cyclic adenosine
monophosphate (cAMP) responsive element binding protein (CREB), which
increases binding of CREB to CREB-binding protein and enhances
CREB-mediated transcription.28 These Akt activities
modulate proapoptotic or antiapoptotic proteins through transcriptional
or posttranscriptional modes.24 RSKs are mediators of ERK
signal transduction.29 The RSKs are serine/threonine
kinases with 2 functional kinase domains activated in sequential manner
by a series of phosphorylation.29 RSK activity is
regulated by ERK as well as PDK1.30,31 RSK phosphorylates
a number of substrates including c-Fos, NFB/IB, BAD, CREB,
histone H3, and Myt1 in different cell situations, which direct
transcriptional activation of immediate early genes and promote cell
survival.25 Studies on the PI3K/Akt and ERK/RSK pathways
offer some insight into the potential for cross-talk in
survival/antiapoptotic mechanisms.7
Because cells in vivo are likely subjected to multiple cytokines that
can influence growth and survival, it is probable that the combined
actions of several cytokines will be of physiologic relevance. Here we
demonstrate that SDF-1 in synergy with other cytokines has the
capacity to enhance survival of primary MPCs and the factor-dependent
myeloid cell line MO7e subjected to delayed addition of growth factors;
this is associated with synergistic stimulation in MO7e cells of
intracellular signaling pathways implicated in mediating
survival/antiapoptotic effects.
| |
Materials and methods |
Cells and cell culture
MO7e cells were cultured in RPMI 1640 medium with 20% fetal
bovine serum (FBS; Hyclone, Logan, UT), 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 ng/mL human granulocyte-macrophage colony-stimulating factor (GM-CSF). This cell line and its
characteristics have been described elsewhere.3,32,33
Before stimulation with cytokines, cells were factor starved for 16 to
18 hours in RPMI 1640 supplemented with 0.5% bovine serum albumin
(BSA). For intracellular signaling studies, factor-starved cells were
incubated with SDF-1 alone or in combination with GM-CSF, Steel
factor (SLF; stem cell factor), or TPO. When inhibitors were used,
cells were preincubated for 1 hour at 37°C before factor stimulation.
Heparinized human marrow cells were collected from healthy volunteers
with informed consent. Heparinized human cord blood was collected from
healthy, full-term neonates according to institutional guidelines.
Mononuclear cells were separated by density gradient centrifugation on
Ficoll Hypaque (1.077 g/mL; Pharmacia, Piscataway, NJ).
CD34+ cells were positively selected by MACS
CD34+ isolation kit (Miltenyi Biotec, Auburn, CA; purity
was more than 90% in each experiment). More highly purified
CD34+++ cells (containing the top 20% highest
CD34-expressing cells;  98% pure CD34+) were isolated
by FACS.34
Cytokines and antibodies
Recombinant human (rhu) GM-CSF and rhu Flt3 ligand (FL) were
provided by Immunex (Seattle, WA). Rhu TPO was from Genentech (Emeryville, CA). Rhu erythropoietin (Epo) was purchased from Amgen
(Thousand Oaks, CA). Rhu SLF, rhu SDF-1, rhu macrophage inflammatory
protein 1 (MIP-1), and rhu regulated on activation, normal T
cell-expressed and secreted (RANTES) were purchased from R & D Systems
(Minneapolis, MN). PC-5-conjugated APO2.7 monoclonal antibody (mAb)
was obtained from Immunotech (Marseille, France). Fluorescein
isothiocyanate (FITC)-conjugated mAb to c-kit and purified mAb to
c-mpl were purchased from Becton Dickinson (Bedford, MA) and Genzyme
(Cambridge, MA), respectively. Phycoerythrin (PE)-conjugated mAb to
CXCR4 was from BD Pharmingen (San Diego, CA). Antibodies to
ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), phospho-Elk-1 (Ser383), phospho-p90RSK (Ser381), Akt, phospho-Akt (Ser473) were from Cell Signaling Technology (Beverly, MA). Anti-p90RSK (Rsk-1) was from Santa
Cruz Biotechnology (Santa Cruz, CA).
Cell lysate preparation
MO7e cells were washed twice with phosphate-buffered saline
(PBS). The cell pellet was resuspended in lysis buffer (20 mM Tris-HCl,
pH 8.0, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride,
0.15 U/mL aprotonin, 10 mM EDTA, 10 µg/mL leupeptin, 100 mM NaF, 2 mM
Na3VO4, and 1% NP-40) and incubated for 30 minutes on ice. Insoluble fractions were removed by centrifugation at 14 000g for 10 minutes, and the supernatants were frozen at
 20°C. Protein concentration of the lysate was measured by
bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL).
Western blotting
Equal amounts of protein were loaded on sodium dodecyl
sulfate-polyacrylamide gel, subjected to electrophoresis (SDS-PAGE), and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). Membranes were blocked in Tris-buffered saline containing 0.05% Tween 20 and 2% BSA for 1 hour at room temperature, and incubated with appropriate primary antibody for 1 to 2 hours. Immunoreactive proteins were detected by horseradish peroxidase-conjugated secondary antibody and an enhanced
chemiluminescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ).
To reprobe with another primary antibody, membranes were incubated in
striping buffer (62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol,
and 2% SDS) for 30 minutes at 50°C, washed, and then used for
further study.
Immunoprecipitation and MAPK activity assay
Cell lysates were immunoprecipitated with anti-phospho-ERK1/2
mAb. Immunoprecipitates were washed twice with lysis buffer and then
twice with kinase buffer (25 mM Tris, pH 7.5, 5 mM
 -glycerolphosphate, 2 mM dithiothreitol [DTT], 0.1 mM
Na3VO4, 10 mM MgCl2).
Immunoprecipitates were resuspended in 50 µL kinase buffer containing
200 µM adenosine triphosphate (ATP) and 2 µg GST-Elk-1 fusion
protein, and then incubated at 30°C for 30 minutes. Reactions were
terminated by adding SDS-PAGE sample buffer, and boiled samples were
separated by 10% SDS-PAGE. Phosphorylated GST-Elk-1 fusion protein
was visualized by immunoblotting with anti-phospho-Elk-1 antibody.
Immunofluorescent staining
Factor-starved MO7e cells were incubated with SDF-1 alone or
with other cytokines, fixed using PBS containing 4% paraformaldehyde for 10 minutes, and permeabilized with PBS containing 0.1% Triton X-100 for 10 minutes. To investigate the cellular localization of
NF-B, samples were treated with a mAb against human NF-B p65
(Santa Cruz Biotechnology, 1:100) for 1.5 hours. After extensive washing in PBS, samples were further incubated with FITC-conjugated donkey antimouse IgG (Jackson Immunotech Laboratory, 3:400) for 1 hour.
Nuclei were stained by 5 µg/mL Hoechst no. 33258 (Sigma, St Louis,
MO). After extensive washing, samples were examined by laser scanning
confocal microscopy (Carl Zeiss, Thornwood, NY).
Primary hematopoietic progenitor cell assays
Magnetic bead-separated human cord blood CD34+
(103 cells/mL), FACS-sorted CD34+++ (100-150 cells/mL), or MO7e cells (103 cells/mL) were plated without
or with single or multiple cytokines at time 0 hour. For human cord
blood progenitor cells plated in 0.9% methylcellulose culture medium
with 30% FBS, the combination of rhu GM-CSF (10 ng/ml), rhu
interleukin 3 (IL-3; 10 ng/mL), and rhu SLF (50 ng/mL) or rhu Epo (1 U/mL) plus rhu FL (100 ng/mL) was used as a maximally
potent combination of cytokines for stimulation of colonies.
Plates were incubated at 5% CO2 and 5% O2 in
a humidified atmosphere and scored for progenitor cell-derived
colonies 14 days after addition of the maximally stimulatory growth
factors. These assays have been described in detail
elsewhere.35
Apoptosis assay after growth factor withdrawal
Factor-starved MO7e or CD34+ marrow cells were
incubated in serum-free media with either 100 ng/mL SDF-1, 10 ng/mL
SLF, or the combination of these 2 cytokines. After 4 days, cells were stained with PC-5-conjugated APO2.7 mAb and analyzed by flow cytometry (EPICS XL, Coulter, Miami, FL). The antigen defined by this antibody (7A6 antigen) is a 38-kd protein localized to the membrane of mitochondria and is involved in the molecular cascade of
apoptosis.36,37 The expression of 7A6 antigen is
preferentially detected on apoptotic cells, but not on the normal cell
surface or digitonin-permeabilized cells. Expression of 7A6 antigen
represents an early event of apoptosis.36
Statistical analysis
Colony results are expressed as mean ± SD from triplicate
plates for each experiment. Statistical significance was determined using Student t test.
| |
Results |
SDF-1 triggers activation of ERK, p90RSK, and Akt
We reported that myelopoiesis was enhanced in SDF-1 transgenic
mice, and SDF-1 modulated myelopoiesis by regulating progenitor cell
survival and the inhibitory effects of myelosuppressive
chemokines.23 To understand possible molecular mechanisms
involved in this survival-enhancing activity of SDF-1, we used the
factor-dependent cell line MO7e,3,32,33 which expresses
the SDF-1 receptor, CXCR4. First, we incubated the cells with SDF-1
alone at an optimal concentration (100 ng/mL) and checked for
activation of signaling molecules by Western blotting using
phosphospecific antibodies. As shown in Figure
1A, SDF-1 induced activation of ERK1/2
as reported in other cells.38-40 Additionally, p90RSK, a
crucial downstream effector molecule of ERK, was activated. Here, we
used antibody recognizing phosphorylated p90RSK(Ser 381), which
represents its activation by ERK.29 Because activation of
Akt correlates with its phosphorylation at residues Thr308 and
Ser473,24 we used antibody recognizing phosphorylated
Akt(Ser473) to check if Akt, a downstream molecule of PI3K, was
activated by SDF-1. SDF-1 also activated Akt, consistent with
results in other cells.38-41
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| Figure 1.
Activation of Akt, ERK1/2, and p90RSK in MO7e cells
after stimulation with SDF-1.
(A) MO7e cells were incubated with 100 ng/mL SDF-1 for the indicated
time periods. (B) MO7e cells were preincubated in the presence of
dimethyl sulfoxide (DM) vehicle control or LY 294002 (LY, 30 µM),
wortmannin (W, 100 nM), PD 98059 (PD, 25 µM), or rapamycin
(R [also called sirolimus], 10 nM) for 1 hour, and stimulated with
100 ng/mL SDF-1 for 5 minutes. Cell lysates were analyzed by Western
blotting with phosphospecific antibodies to Akt (Ser473), ERK1/2
(Thr202/Tyr204), or p90RSK (Ser381). Amounts of p90RSK (A) or Akt (B)
are shown on the bottom panel of each as a loading control. This
experiment was performed 3 times with similar results.
|
|
We used pathway-specific inhibitors to determine if activation of ERK
was sensitive to MEK1 inhibitor PD98059 (50 µM) and Akt activation
was sensitive to PI3K inhibitors, LY 294002 (30 µM) or wortmannin
(100 nM; Figure 1B). Wortmannin reduced ERK activation, but another
PI3K inhibitor LY 294002 did not. Considering the inhibitory effects of
wortmannin on MAPK activation,42 and the greater
specificity of LY 294002 for PI3K, which acts on the ATP binding site
of this enzyme,43 we consider it reasonable that PI3K
contributes little if anything to the MEK1/ERK activation induced by
SDF-1.
SDF-1 in combination with other cytokines synergistically
activates MAPK/p90RSK and Akt
To determine if SDF-1 has synergistic activity in combination
with other cytokines, we stimulated MO7e with SDF-1 alone, other
cytokines alone, or SDF-1 plus other cytokines, and checked for
activation of ERK, p90RSK, and Akt. Here, we used optimal concentrations of cytokines such as GM-CSF (10 ng/mL), SLF (50 ng/mL),
or TPO (50 ng/mL).
We found that GM-CSF alone, SLF alone, and TPO alone activated ERK and
the combined stimulation with SDF-1 plus either of these other
cytokines triggered a synergistic activation of ERK (Figure
2). Because stimulation of ERK activation
by SDF-1 showed much faster kinetics of signaling than those by the
other cytokines, we could not detect much activation by 15 or 30 minutes after stimulation with SDF-1. This is consistent with
previous observations that signaling pathways from the G-protein
coupled CXCR4 receptor are activated faster than those from growth
factor receptors.44-46 Activation of p90RSK, a downstream
molecule of ERK, showed similar patterns of response to SDF-1 alone
or in combination with GM-CSF, SLF, or TPO (Figure 2). GM-CSF alone,
SLF alone, or TPO alone induced activation of Akt in MO7e cells, and
SDF-1 in combination with these cytokines induced synergistic
activation of Akt (Figure 3).
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| Figure 2.
Synergistic activation of ERK1/2 and p90RSK by SDF-1
in combination with other cytokines.
MO7e cells were incubated for the indicated time periods with SDF-1
(100 ng/mL), GM-CSF (10 ng/mL; GM), SLF (50 ng/mL), or TPO (50 ng/mL)
each alone, or with the combination of SDF-1 plus one of these
cytokines. Cell lysates were analyzed by Western blotting with
phosphospecific antibodies to ERK1/2 (Thr202/Tyr204) or p90RSK
(Ser381). The amount of p90RSK is shown as a loading control in the
bottom panels. (A) SDF-1 plus GM-CSF. (B) SDF-1 plus SLF. (C)
SDF-1 plus TPO. (D,E) Columns represent relative band intensities of
phospho-ERK (D) and phospho-RSK (E) ± SD from 3 experiments.
*P < .05 (greater than additive).
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| Figure 3.
Synergistic activation of Akt in MO7e cells by SDF-1 in combination
with other cytokines.
Cell lysates were analyzed by Western blotting with phosphospecific
antibodies to Akt (Ser473). The amount of total Akt is shown as a
loading control in the bottom panels. (A) SDF-1 plus GM-CSF. (B)
SDF-1 plus SLF. (C) SDF-1 plus TPO. (D) Columns represent
relative band intensities of phospho-Akt ± SD from 3 experiments.
*P < .05 (greater than additive).
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To confirm the synergism of SDF-1 with these other cytokines, we
evaluated signaling events using different concentrations of SDF-1
and the other cytokines. The concentrations of cytokines used were 100 or 10 ng/mL SDF-1; 10, 1, or 0.1 ng/mL GM-CSF; 50, 10, or 1 ng/mL
SLF; and 10 or 1 ng/mL TPO. The results in Figure
4 demonstrate clear synergistic
activation of ERK. As shown later, the lower concentrations of
cytokines do not alone enhance survival of primary MPCs or MO7e
colony-forming cells. However, low concentrations of SDF-1 with low
concentrations of GM-CSF, SLF, or TPO do enhance the survival of these
cells.
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| Figure 4.
Synergistic activation of ERK1/2 by SDF-1 in combination with
nonproliferative concentrations of other cytokines.
MO7e cells were stimulated for 15 minutes with SDF-1 alone, another
cytokine alone, or SDF-1 plus another cytokine at the indicated
concentrations. Cell lysates were analyzed by Western blotting using
phosphospecific antibodies to ERK1/2 (Thr202/Tyr204). The membrane was
reblotted with antibody recognizing p90RSK to show equal loading. (A)
SDF-1 plus GM-CSF. (B) SDF-1 plus SLF. (C) SDF-1 plus TPO. (D)
Columns represent relative band intensities of phospho-ERK ± SD
from 3 experiments with similar results. *P < .05
(greater than additive).
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To determine if SDF-1 influenced surface expression of other
cytokine receptors, we checked the level of the c-kit (SLF
receptor) and c-mpl (TPO receptor) gene products by flow
cytometry analysis after treatment of cells with SDF-1 (100 ng/mL)
for 30 and 60 minutes. SDF-1 did not change the expression levels of
c-kit or c-mpl as assessed by mean fluorescence intensity (data not shown). To exclude the possibility that one of the cytokines may have
affected the surface expression level of CXCR4, we treated MO7e cells
with GM-CSF for 1 hour and 24 hours and checked the expression level of
surface CXCR4 by flow cytometry. We did not see any change of CXCR4
expression level induced by GM-CSF as assessed by mean fluorescence
intensity (data not shown).
Priming with SDF-1 induces enhancement of survival
signaling
To determine if the other cytokines had to be given
simultaneously with SDF-1 to detect synergistic enhancement in
intracellular signaling, we pretreated MO7e cells for 30 minutes with
SDF-1 and then added one of the other cytokines prior to analysis of effects on ERK activation. We confirmed activation of ERK by using an
in vitro kinase assay with ERK1/2 immunoprecipitates (Figure 5A). Because SDF-1-induced ERK
activation kinetics are very fast (Figure 1A), we did not detect ERK
activation by SDF-1 alone at this time point. SLF alone induced ERK
activation, and SLF given 30 minutes after SDF-1 induced clear
synergism of ERK activation (Figure 5Ai). We detected the same
pattern of synergistic activation of ERK when GM-CSF was added to the
cells 30 minutes after SDF-1 (Figure 5Aii). To check if this effect
was unique to SDF-1, we pretreated MO7e cells with other chemokines
(MIP-1 or RANTES) instead of SDF-1, and monitored for their
possible effects on ERK activity. Neither of these 2 chemokines
enhanced the ERK activation induced by subsequent addition of SLF
(Figure 5Aii). Pretreatment with SDF-1 induced synergistic
activation of ERK even when SLF was added 1 hour after SDF-1 when
ERK activation by SDF-1 alone could not be detected (Figure 5Aiii).
Enhanced activation of ERK was sensitive to pretreatment of the cells
with the MEK1 inhibitor PD98059 (Figure 5Aiv). To see if Akt was also
synergistically activated after pretreatment with SDF-1, we checked
phosphorylation levels of Akt. As shown in Figure 5B, Akt activation
was significantly enhanced by SLF after pretreatment with SDF-1, but
not after pretreatment with MIP-1 or RANTES. MO7e cells do express
the receptors for MIP-1 and RANTES, CCR1 and CCR3 (data not shown). Pretreatment of cells with SDF-1 for 30 minutes and subsequent treatment with GM-CSF or TPO also resulted in synergistic enhancement of ERK and Akt activation (data not shown). Therefore, we conclude that
SDF-1 activates ERK as well as Akt in synergy with other cytokines
and that SDF-1 may act as a priming agent to sensitize the cells to
the actions of these other cytokines.
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| Figure 5.
Enhanced activation of MAPK and Akt signaling by pretreatment of MO7e
cells with SDF-1.
Factor-starved MO7e cells were preincubated with or without 100 ng/mL of either SDF-1, MIP-1, or RANTES for 30 minutes or the
indicated periods of time (Aiii), followed by treatment with 10 ng/mL
SLF or 1 ng/mL GM-CSF for 5 minutes. In some experiments, cells were
pretreated with PD98059 for 1 hour before SLF treatment (Aiv). (A) ERK
immunoprecipitates obtained from total cell lysate using
anti-phospho-ERK mAb were subjected to MAPK activity assay as
described in "Materials and methods." Recombinant active MAPK (20 ng) was included as a positive control (Ai). These results are
representative of 3 independent experiments. (B) Phosphorylation levels
of Akt were determined by Western blotting with phosphospecific
antibodies to Akt (Ser473). The amount of total Akt is shown as a
loading control in the bottom panels of B. This is a representative of
3 separate experiments with similar results.
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SDF-1 alone or SDF-1 in combination with other cytokines does
not trigger nuclear localization of NF-B in MO7e cells
Previously, SDF-1 was reported to activate NF-B in other
cells.38,47 Activated NF-B is known to translocate into
the nucleus.48 To examine the localization of NF-B, we
made use of immunofluorescence staining with antibody against human
NF-B p65 and confocal microscopy. As seen in Figure
6A, tumor necrosis factor- (TNF-;
10 ng/mL) induced very clear translocation of NF-B into the nucleus
within 15 minutes, but we did not detect any significant change induced
by SDF-1 alone, GM-CSF alone, or SDF-1 plus GM-CSF in MO7e cells.
SLF alone, TPO alone, or SDF-1 in combination with SLF or TPO did
not cause the translocation of NF-B to the nucleus either (Figure
6B). The same results were obtained up to 2 hours after stimulation.
Additionally, we checked NF-B DNA binding activity by gel shift
assay using NF-B consensus binding site, and the results obtained
from 30 minutes and 1 hour time points after stimulation were in
agreement with confocal microscopy data; no activation of NF-B was
noticed (data not shown). Therefore, it is likely that NF-B
activation is not involved in the survival-enhancing activity of MO7e
cells induced by SDF-1 alone or in synergy with these other
cytokines.
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| Figure 6.
No effect of SDF-1 or other cytokines on translocation of NF-B.
Factor-starved MO7e cells were stimulated with SDF-1 alone, other
cytokines alone, or combinations of cytokines for 15 minutes, and the
cellular localization of NF-B was determined by confocal microscopy
after staining with a mAb against human NF-B p65. Nuclei were
stained by Hoechst no. 33258. Cells treated with TNF- (15 minutes) are shown as a positive control for NF-B nuclear
translocation. Magnification, × 100.
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SDF-1 synergistically enhances survival in vitro of primary
myeloid progenitor and MO7e cells in the context of delayed growth
factor addition with low concentrations of other cytokines
Because we had detected synergistic activation of intracellular
signaling pathways known to be associated with cell survival and
antiapoptotic effects after treatment of MO7e cells with SDF-1 plus
other cytokines, we checked the survival-enhancing activities of these
cytokines on primary myeloid progenitor cells as well as on MO7e cells.
To assess survival-enhancing effects of SDF-1 and other cytokines,
we performed colony-forming assays after delayed addition of growth
factors. In brief, we incubated the cells with and without the test
cytokines at time 0 and then added maximally potent combinations of
growth factors used to stimulate colony formation to the plates at 24 and 48 hours later.
We previously determined that SDF-1 at 1 to 1000 ng/mL did not
stimulate colony or cluster formation of primary granulocyte-macrophage colony forming units (CFU-GMs), erythroid burst-forming units (BFU-Es)
or granulocyte-erythrocyte-macrophage-megakaryocyte colony forming
units (CFU-GEMMs) from human or murine bone marrow, human cord blood,
or MO7e cells. Moreover, SDF-1 at these concentrations did not
enhance colony formation stimulated by Epo, GM-CSF, or IL-3 each alone
or in the absence or presence of SLF or FL (data not shown).
A number of cytokines have been shown to enhance the survival of
hematopoietic progenitor cells in vitro that have been subjected to
withdrawal and subsequent delayed addition of growth factors. These
survival enhancing cytokines include but are not limited to
IL-1,49 IL-6,50 FL,51
SLF,51 and TPO.2 Moreover, the combinations
of IL-1 plus IL-650 or FL and SLF51
synergized in this effect when used at concentrations below which each
acted alone to enhance survival of myeloid progenitors. Based on our above data on intracellular effects, we hypothesized that SDF-1 would
synergize with other cytokines to enhance survival of primary myeloid
progenitor cells in human cord blood and of MO7e cells subjected to
delayed addition of growth factors that would stimulate these cells to
form colonies in semisolid medium. One hundred and fifty FACS-sorted
CD34+++ human cord blood cells were plated in
methylcellulose culture medium with varying concentrations of SDF-1,
GM-CSF, TPO, SLF, or FL alone, or the combination of SDF-1 plus
varying concentrations of GM-CSF, TPO, SLF, or FL, prior to addition of
maximally stimulating concentrations of Epo (1 U/mL) plus GM-CSF (10 ng/mL) with IL-3 (10 ng/mL) and SLF (50 ng/mL) at 24 hours compared to
at time 0. As shown in Figure 7 at high,
but not lower concentrations of each cytokine alone, survival
enhancement of CFU-GMs and CFU-GEMMs was noticed. We did not evaluate
BFU-Es in these studies because we previously showed that the addition
of SLF to cultures with Epo plus colony-stimulating factors will
stimulate CFU-GM and CFU-GEMM but not BFU-E colonies.52
Low concentrations of SDF-1 with low concentrations of either
GM-CSF, TPO, SLF, or FL, that each alone did not enhance survival of
CFU-GMs or CFU-GEMMs, synergized with each other to enhance the
survival of these very highly enriched populations of progenitor cells
in which more than 1 of 2 cells plated in the presence of optimal
concentration of the combination of growth factors formed a CFU-GM or
CFU-GEMM colony. Similar results were seen in one other experiment
using CD34+++ cord blood cells, one experiment using
bead-separated CD34+ cord blood cells (> 90%
CD34+ cells with > 15% optimal cloning efficiency at
1000 cells/mL) and 2 experiments using Ficol-Hypaque separated low
density cord blood cells plated at 2.5 × 104 cells/mL.
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| Figure 7.
Influence of SDF-1, GM-CSF, TPO, SLF, and FL, alone
and in combination on the survival of CFU-GMs and CFU-GEMMs in
FACS-sorted CD34+++ cord blood cells subjected to delayed
addition of a combination of maximally stimulating growth factors.
Human CD34+++ cells were plated at time 0 in the absence
and presence of various concentrations of SDF-1, GM-CSF, TPO, SLF,
FL, or SDF-1 plus either GM-CSF, TPO, SLF, or FL. The combination of
rhu Epo (1 U/mL), rhu GM-CSF (10 ng/mL), rhu IL-3 (10 ng/mL), and rhu
SLF (50 ng/mL), a maximally potent combination of cytokines to
stimulate colony formation, was added to the plates at either time 0 or
24 hours and cultures were scored for CFU-GM and CFU-GEMM colonies 14 days after the addition of the maximally stimulating cytokines. Results
are given as colonies ± SD (with the percent survival given in
parentheses). (A) Significant increase in survival compared to control
plates at the same time of delayed growth factor addition
(P < .001). (B) Significant increase in survival
compared to control plates at same time of delayed growth factor
addition (P < .01). (C) Significant increase in survival
compared to control plates at same time of delayed growth factor
additions (P < .05). (D) Significantly greater survival
than either cytokine alone and additive to slightly less than additive
effects at the same time of delayed growth factor addition
(P < .05). (E) Significantly greater survival than with
either cytokine alone and greater than additive effect at the same time
of delayed growth factor addition (P < .01). The addition
of either SDF-1, GM-CSF, TPO, SLF, FL, or the combination of SDF-1 plus
these cytokines along with the maximally stimulating combination of
Epo, GM-CSF, IL-3, and SLF at time 0 had no significant effect
(P > .05) compared to control medium added with the
maximally stimulating combination of growth factors at time 0 (data not
shown).
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As shown in Figure 8, SDF-1 synergized
with low concentrations of GM-CSF, TPO, and SLF to enhance survival of
MO7e colony-forming cells subjected to 24- and 48-hour delayed growth
factor addition. These results were representative of 2 additional
studies with MO7e cells. Because MO7e cells do not express Flt3, the
receptor for FL, we could not evaluate FL effects on these cells nor
the signaling events triggered by SDF-1 plus FL or FL alone. Neither SDF-1 nor the low concentrations of the other cytokines used for
cell survival stimulated colony formation by MO7e cells when used alone
or together (data not shown).
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| Figure 8.
Influence of SDF-1, GM-CSF, TPO, and SLF on survival of MO7e
colony-forming cells (CFCs) after growth factor withdrawal.
MO7e cells were plated at 103 cells/mL in agar culture
medium at time 0 with control medium or the amounts of SDF-1,
GM-CSF, TPO, or SLF, or SDF-1 plus either GM-CSF, TPO, or SLF shown.
The combination of GM-CSF (10 ng/mL) plus SLF (50 ng/mL), a maximal
concentration of factors to stimulate MO7e colony formation, was added
to these plates at either time 24 or 48 hours. Results are shown as
colonies ± SD (with the percent survival compared to time 0 culture plated with control medium shown in parentheses). Statistical
differences between groups are noted by the same letters as for the
results in Figure 7. The addition of either SDF-1, GM-CSF, TPO, SLF, or
SDF-1 plus these cytokines along with the maximally stimulating
combination of GM-CSF plus SLF at time 0 had no significant effect
(P > .05) compared to control medium added with the
maximally stimulating combination of growth factors at time 0 (data not
shown).
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We performed apoptosis assays to see if the enhanced survival read-outs
of the colony-forming cell assays correlated with antiapoptotic
activity. Here, we assessed apoptosis by measuring APO2.7+
cells expressing the 7A6 antigen, which is involved in the molecular cascade of apoptosis.36 As shown in Figure
9, SDF-1 alone slightly suppressed
apoptosis in response to growth factor withdrawal in human
CD34+ cells (Figure 9A) as well as in MO7e (Figure 9B)
cells. SLF alone induced more apoptosis inhibition, and SDF-1 plus
SLF significantly enhanced suppression of apoptosis of these cells
compared to SDF-1 alone or SLF alone. When we treated MO7e cells
with MIP-1 (Figure 9B) or RANTES (data not shown) instead of
SDF-1, there was no significant effect suggesting that the survival
enhancing/antiapoptotic activity we noted is not a common property of
chemokines.
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| Figure 9.
Effect of SDF-1 on apoptosis induced by growth factor
withdrawal.
Factor-starved CD34+ cells prepared from human bone marrow
(A) or MO7e cells (B) were further incubated in serum-free media in the
presence of either 100 ng/mL SDF-1 alone, 10 ng/mL SLF alone, or the
combination of these 2 cytokines. After 4 days, cells were stained with
PC-5-conjugated APO2.7 mAb and analyzed by flow cytometry. (A) Values
in histogram represent percent of APO2.7+ cells. This is
representative of 3 separate experiments. (B) Columns represent the
average percent of APO2.7+ cells ± SD of 3 separate
experiments. *P < .01 versus SLF alone.
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Discussion |
It is known that SDF-1 is an important chemokine in the bone
marrow microenvironment. It has been implicated in migration of MPCs
and stem cells through its chemotactic activity,5-7,15-18 events consistent with abnormalities in the marrow of both
SDF-1/ and CXCR4/
mice.10,11 We postulated that SDF-1 might have other
activities in addition to that of chemotaxis. Because
SDF-1/ mice die perinatally, we made SDF-1
transgenic mice and found that these mice had enhanced myelopoiesis
with a higher MPC cell cycling status and a greater absolute number of
MPCs per femur and spleen compared to their littermate controls. This
did not appear to be due to SDF-1 activities in inducing the
proliferation of MPCs. Rather, we found that SDF-1 had
survival-enhancing effects on MPCs in vitro.23 SDF-1
has previously been shown to induce,53,54 protect against
apoptosis,55,56 or to have no effects on cell survival.57 These contradictory results might be due to
differences in cell types or experimental conditions.
In this present study, we have additionally found that SDF-1 can
synergize with other cytokines at very low concentrations in the
survival enhancement of primary MPCs subjected to the delayed addition
of growth factors. Our results revealed that SDF-1 enhanced survival
of purified human CD34+++ and CD34+ cord blood
CFU-GMs and CFU-GEMMs in synergy with other cytokines such as GM-CSF,
FL, SLF, or TPO. We obtained similar results with the
factor-dependent cell line MO7e. Strikingly, the effective concentrations of other cytokines used in combination with SDF-1 were as low as 1 ng/mL for FL, 0.01 ng/mL for GM-CSF, 1 ng/mL for SLF,
and 1 ng/mL for TPO. Therefore, SDF-1 clearly had survival-enhancing activity that synergized with these other cytokines. Based on our own
observations, SDF-1 did not change the expression level of other
cytokine receptors such as c-kit and c-mpl and GM-CSF did not change
the expression level of CXCR4 in MO7e cells; this latter observation is
in agreement with a previous report using monocytes.58
Therefore, we believe that the synergistic survival-enhancing activity
of SDF-1 likely results from enhanced intracellular signaling rather
than effects on cytokine receptor expression.
Because we observed the same results using primary human MPCs as we did
using MO7e cells in the survival-enhancing activity of SDF-1, we
felt that biochemical data from the MO7e cell line could serve as a
model system to understand some of the intracellular signaling
mechanisms that might be involved in these effects. We checked
activation of 2 survival-related signal transduction pathways, the
PI3K/Akt and MAPK/RSK pathways, using phosphospecific antibodies
against Akt/PKB, ERK1/2, or RSK in MO7e cells after stimulation with
SDF-1 alone or SDF-1 in combination with other cytokines such as
GM-CSF, SLF, and TPO. These 2 pathways were activated by stimulation
with SDF-1 alone and inhibited by PI3K inhibitors and a MEK1
inhibitor, respectively. These results imply that PI3K and MAPK
pathways activated by SDF-1 are independent of each other. Combined
stimulation with SDF-1 plus another of the cytokines we used here
activated these 2 pathways synergistically. These results were in
agreement with biologic effects showing enhanced survival by SDF-1
alone and SDF-1 in synergy with other cytokines in primary cells as
well as MO7e cells. Interestingly, when MO7e cells were stimulated with
other cytokines after pretreatment with SDF-1, synergistic
activation of ERK and Akt was still evident, even 1 hour after
pretreatment with SDF-1. In contrast, when we checked ERK activation
after stimulation with SDF-1 alone for 30 minutes, we could not
detect significant activity. This suggests that SDF-1 and the other
cytokines trigger signal transduction pathways in agreement with their
unique biologic activities. Furthermore, SDF-1 may prime or
sensitize cells to be more responsive to other cytokines for cell
survival enhancement. We have previously noted that SDF-1 blocks
suppression of murine MPC proliferation in vitro by CC chemokines
MIP-1, CK-11, TECK, and MCP-1, CXC chemokines IL-8 and PF4, and
the C chemokine lymphotactin.23 MPCs from SDF-1
transgenic, but not littermate control, mice were insensitive to
inhibition by these chemokines.23 We speculate that a
priming effect of SDF-1 may somehow also contribute to these
phenomena, at least, in part.
Nuclear factor-B is a ubiquitous heterodimeric transcription factor,
and IB binds NF-B and keeps it localized to the
cytoplasm.48 Phosphorylation of IB targets it for
ubiquitination and proteosome-mediated degradation, which frees NF-B
from IB, allowing NF-B nuclear translocation and activation of
target genes. It was previously reported that SDF-1 activated
NF-B in the CTS cell line38 and
megakaryocytes,47 and information from the literature
suggests that both the PI3K/Akt and MAPK/RSK pathways can activate
NF-B.24,25 Therefore, we checked NF-B activation in
MO7e cells after stimulation with SDF-1 alone or in combination with
other cytokines. NF-B activation and localization to nucleus was
detected after stimulation with TNF-, but not after stimulation with
other cytokines such < |
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Abstract
Introduction