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and Stem Cell Factor/kit Ligand Share Signaling Pathways in Hemopoietic Progenitors: A Potential Mechanism for Cooperative Induction of Chemotaxis1
Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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), the ligand for CXCR4, is a
chemokine that acts as a potent chemoattractant for hemopoietic
progenitor cells. Stem cell factor/kit ligand (SCF/KL),
an early acting cytokine, has recently been reported to enhance the
chemotaxis induced by SDF-1. However, very little is known about
downstream signaling events following these receptor-ligand
interactions. To investigate these events, we utilized a model
progenitor cell line, CTS, which expresses both the CXCR4 and
c-kit receptors. We observed strong Ca2+
mobilization and enhancement of chemotaxis following treatment with
SDF-1 or SCF/KL. A combination of these factors enhanced this
chemotaxis in CTS cells as well as in CD34+ bone marrow
cells. Prior treatment of CTS cells with pertussis toxin inhibited the
SDF-1-induced chemotaxis, suggesting that SDF-1 signaling
involves a pertussis-sensitive Gi-coupled protein. SDF-1
treatment resulted in a rapid phosphorylation of the focal adhesion
molecules RAFTK (related adhesion focal tyrosine kinase), paxillin, and
p130cas, which then declined within
minutes. SCF/KL alone or in combination with SDF-1 induced a rapid
and sustained effect on phosphorylation of these substrates. SDF-1
treatment resulted in a rapid and robust activation of p44/42
mitogen-activated protein kinase compared with the relatively weak and
delayed effect of SCF/KL treatment. Interestingly, a delayed but
sustained activation of mitogen-activated protein kinase activation was
observed when the factors were used in combination. Such cooperativity
in downstream signaling pathways may explain the enhanced chemotaxis of
progenitors observed with SDF-1 in combination with
SCF/KL. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine, the stromal cell-derived factor
(SDF-1),3 has been
described not only as a potent chemoattractant for lymphocytes and
monocytes, but also for human CD34+ hemopoietic progenitor
cells (3, 4). SDF-1 is a ligand for the CXCR4 receptor, which like
all chemokine receptors, is a seven-transmembrane surface structure
linked to G proteins (5, 6, 7). SDF-1, via CXCR4, has been shown to
mediate pertussis-sensitive chemotactic activity (4) and cytoplasmic
calcium flux in CD34+ bone marrow cells (3).
Stem cell factor/kit ligand (SCF/KL) is an early acting
cytokine (8, 9) that modulates growth of bone marrow progenitor cells,
megakaryocytes, and mast cells (10, 11). Its cognate receptor,
c-kit, belongs to the protein tyrosine kinase family. SCF/KL
functions by ligand-induced dimerization of this receptor, with
phosphorylation of the cytoplasmic tail and recruitment of substrates
with SH2 domains (12, 13). Synergy of SCF/KL with various growth
factors, including IL-1, IL-3, IL-7, thrombopoietin, and
erythropoietin, has been observed with respect to potentiation of
myeloid, lymphoid, megakaryocytic, and erythroid lineage colony
formation from purified primitive human and mouse hemopoietic
precursors (14, 15, 16, 17). Recently, an intriguing cooperative interaction
between SCF/KL and SDF-1 that enhances chemotaxis of
CD34+ hemopoietic progenitors has been shown (4). However,
the mechanism of this cooperativity has not been elucidated. To that
end, we studied the effects of SDF-1 and SCF/KL on chemotaxis and
signal-transduction pathways. We utilized the human hemopoietic
progenitor CTS cell line as a model since it expresses many of the
characteristics of primary marrow progenitors, including the CD34,
CD38, CXCR4, and c-kit receptors. Although SDF-1 signals
via the G protein-coupled CXCR4 receptor and SCF/KL via the protein
tyrosine kinase c-kit receptor, there appear to be common
downstream substrates where these pathways converge. Our studies show
that SDF-1 and SCF/KL treatment of CTS cells induced phosphorylation
of downstream substrates that are known to form focal adhesions, and
activation of p44/42 MAP kinase. These results provide a potential
mechanism to explain the apparent cooperativity of SDF-1 and SCF/KL
on the enhancement of CD34+ cell migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs to the related adhesion focal tyrosine kinase (RAFTK) were
generated using GST-fusion proteins by immunizing New Zealand rabbits,
as previously described (18). Serum R-4250 was chosen for further
studies based on its titer in ELISA. This antiserum does not
cross-react with focal adhesion kinase and recognizes both human and
murine forms of RAFTK. Abs to paxillin, JNK, p38 kinase, and rGST-c-Jun
amino-terminal protein (1–79 amino acids) were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine mAb (4G10)
was a generous gift from Dr. Brian Druker (Oregon Health Sciences
University, Portland, OR). Electrophoresis reagents were
obtained from Bio-Rad Laboratories (Hercules, CA). The protease
inhibitors leupeptin and -1 antitrypsin and all other reagents were
obtained from Sigma (St. Louis, MO). The nitrocellulose membrane was
obtained from Bio-Rad Laboratories. Indo-1 acetomethyl ester (Indo-1
AM) was purchased from Molecular Probes (Eugene, OR). Human rSDF-1
was purchased from R&D Systems (Minneapolis, MN). Human rSCF/KL was a
gift from Amgen (Thousand Oaks, CA). mAb to
p130cas was obtained from Transduction
Laboratories (Lexington, KY). Sodium azide-free mAbs to CXCR4 (12G5),
CCR5 (2D7), and matching isotype control Ab were obtained from
PharMingen (San Diego, CA).
Cell culture
The CTS hemopoietic cell line was grown at 37°C in 5% CO2 in RPMI 1640 with 10% FCS, 50 µg/ml penicillin, and 50 µg/ml streptomycin, as described previously (19).
Calcium flux assay
CTS cells were washed with RPMI 1640 (Life Technologies, Grand
Island, NY) and resuspended at 10 x 106 cells/ml in
RPMI. The cells were loaded with Indo-1 by adding 5 µl of working (1
µg/µl DMSO) Indo-1 solution to 10 x 106 cells
suspended in 1 ml of RPMI solution. They were then incubated for 45 min
at 37°C. Cells were diluted to a concentration of 1 x
106/ml, treated with the desired amounts of SDF-1 and/or
SCF/KL, and then analyzed for calcium mobilization by flow cytometry
(Coulter Electronics, Hialeah, FL), as described (20).
Stimulation of cells
Cells were washed twice with RPMI and resuspended at 10 x
106 cells/ml. Cells were then starved for 4 h at
37°C and stimulated with 25 nM SDF-1 and/or 50 ng/ml SCF/KL at
37°C for various time periods. After stimulation, cells were lysed in
modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mM
NaCl, 1 mM PMSF, 10 µg/ml of aprotinin, leupeptin and pepstatin, 10
mM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium
pyrophosphate). Total cell lysates (TCL) were clarified by
centrifugation at 10,000 x g for 10 min. Protein
concentrations were determined by protein assay (Bio-Rad Laboratories).
Cell lysis, immunoprecipitation, immunoblotting, kinase assays, and
autophosphorylation assays were conducted as described below.
Flow cytometry for cell surface expression
Cells (3 x 105) were washed three times with PBS and incubated with Abs to CD4, CD7, CD41a, FLK-1, CD117, FLT-1, CCR3, CXCR4, CCR5, CD38, or CD34 in PBS, 0.1% BSA at appropriate dilutions. After incubation at 4°C for 1 h, cells were washed two times and incubated with appropriately diluted secondary Abs conjugated to either FITC or phycoerythrin. Cells were washed twice and resuspended in 0.5 ml PBS, 0.1% BSA. Membrane fluorescence was analyzed using a flow cytometer (Coulter). Appropriate isotype control Abs were used as controls to adjust the background fluorescence of each reaction.
Immunoprecipitation and Western blot analysis
For immunoprecipitation studies, identical amounts of protein from each sample were clarified by incubation with protein A-Sepharose CL-4B (Pharmacia Biotech, Piscataway, NJ) for 1 h at 4°C. Following the removal of protein A-Sepharose by brief centrifugation, the solution was incubated with different primary Abs, as detailed below, for each experiment for 4 h at room temperature (RT) or overnight at 4°C. Isolation of the Ag-Ab complex was performed by adsorption for 2 h at 4°C with 50 µl of protein A-Sepharose (10% suspension). Nonspecific bound proteins were removed by washing the Sepharose beads three times with modified RIPA buffer and one time with PBS. Bound proteins were solubilized in 40 µl of 2x Laemmli buffer and further analyzed by immunoblotting. Samples were separated on 7.5% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk protein and probed with primary Ab for 3 h at RT or at 4°C overnight. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary Ab and the enhanced chemoluminescent (ECL) system (Amersham, Arlington Heights, IL). The mAb 4G10 (IgG2a) was used for Western blot analysis of phosphotyrosine protein.
MAP kinase assay
The in vitro MAP kinase assay was performed as described earlier
(21). The cell lysates were immunoprecipitated with ERK-1 and ERK-2
antisera and then washed twice with RIPA buffer and once in kinase
buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 5 mM
MnCl2, and 100 mM Na3VO4). For the
in vitro kinase assay, the immune complex was incubated in kinase
buffer containing myelin basic protein (Upstate Biotechnology, Lake
Placid, NY) and 5 µCi [
-32P]ATP at RT for 30 min.
The reaction was stopped by adding 4x SDS sample buffer and boiling
the sample for 5 min at 100°C. Proteins were then separated on 7.5%
SDS-PAGE and detected by autoradiography (22). Normal rabbit serum was
used as a negative control. Appropriate isotype control Abs were used
with each reaction to adjust the baseline of the flow cytometer.
JNK and p38 MAP kinase assays
The JNK assay was performed as described previously (23).
Briefly, cell lysates were immunoprecipitated with JNK Ab (Santa Cruz
Biotechnology). The immune complexes were washed twice with RIPA buffer
and once in kinase buffer (50 mM HEPES, pH 7.4, 10 mM
MgCl2, and 20 µM ATP). The complex was then incubated in
kinase buffer containing rGST c-Jun, 0.2 µg/ul (1–79 amino acids)
(Santa Cruz Biotechnology), and 5 µCi [-32P]ATP for
10 min at RT. The reaction was terminated by adding 2x SDS sample
buffer and boiling the sample for 5 min at 100°C. Proteins were
separated on 12% SDS-PAGE and detected by autoradiography. Rabbit IgG
was used as a negative control.
For the p38 MAP kinase assay, cell lysates from unstimulated or
stimulated cells were immunoprecipitated with anti-p38 MAP kinase
Ab (Santa Cruz Biotechnology). The immune complexes were then washed
twice with RIPA buffer and once in kinase buffer (50 mM HEPES, pH 7.4,
10 mM MgCl2, and 20 µM ATP). The complex was incubated in
kinase buffer containing 7 µg myelin basic protein (Upstate
Biotechnology) and 5 µCi [-32P]ATP for 20 min at
30°C. Proteins were separated on 15% SDS-PAGE and detected by
autoradiography. Rabbit IgG was used as a negative control.
Chemotaxis assay
The assay for chemotaxis was performed in 24-well plates
(Costar, Cambridge, MA) carrying 5-µm porosity inserts. Cells were
grown in RPMI 1640 medium, washed twice, and resuspended at 10 x
106 cells/ml in RPMI 1640 medium and H199 medium (1:1)
containing 0.5% BSA without any added serum. SDF-1 (10 nM) and/or
SCF/KL (25 ng/ml) were added to the wells, and cells were loaded onto
the inserts at 1 x 106/100 µl for each individual
assay. Chemotaxis medium added to the bottom wells without SDF-1 or
SCF/KL was used as a control. Cells migrating to the bottom well were
collected for counting on the flow cytometer after 4 h. Total
cells present in the 100-µl suspension used for loading were counted
as the total load to calculate the percentage of migration of cells to
the bottom wells. To see whether MIP-1-, MIP-1ß-, RANTES-, or
SDF-1-induced chemotaxis could be inhibited by prior incubation of
CTS cells with mAb to the CXCR4 or CCR5 receptor (2D7), CTS cells were
incubated with mAbs (2 µg/106 cells) to CXCR4 (12G5),
CCR5 (2D7), or isotype control Ab for 1 h at room temperature
before the chemotaxis assay was performed. All experiments were
performed in triplicate and were repeated at least three times.
Preparation of human bone marrow cells
Light-density bone marrow mononuclear cells were obtained from normal subjects after receiving their informed consent based on a protocol approved by our Institutional Review Board. Aspirates from donors were depleted of adherent cells, as previously described (24).
Isolation of CD34+ bone marrow cells by immunoadsorption
CD34+ cells were isolated by immunoadsorption using the CellPro (Bothell, WA) Ceprate LC system, according to the manufacturer’s instructions. After elution, cells were washed with Ca2+- and Mg2+-free PBS with 1% BSA and resuspended in the appropriate medium for the chemotaxis.
Chemotaxis assays for CD34+ bone marrow cells
Chemotaxis assays for CD34+ marrow cells were
performed in triplicate using 5-µm-pore filter transwell inserts, as
described above. The inserts were rinsed with migration medium
(complete -medium with 0.5% BSA), and the supernatant was aspirated
immediately before loading cells. A total of 1.5 x
105 CD34+ cells suspended in 100 µl migration
medium was loaded into each insert. The inserts were then transferred
to the wells consisting of 650 µl migration medium containing
SDF-1 and/or SCF/KL. The plates were incubated at 37°C in 5%
CO2 for 4 h. Following this incubation, the inserts
were removed and the cells that migrated to the bottom wells were
collected. Cells were washed, resuspended, and quantitated for viable
cells using the trypan blue exclusion method. These experiments were
repeated twice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FACS analysis of CTS cells showed that they expressed the expected
progenitor surface markers CD34 and CD38, as well as the chemokine
receptor CXCR4 and the c-kit receptor (Table I
).
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and SCF/KL stimulate Ca2+ flux in CTS cells
Ligand binding to chemokine receptors causes characteristic fluxes
in intracellular calcium. To verify that the CTS cells retain this
fundamental signaling property, we treated these cells with SDF-1
(12.5 or 25 nM) and/or SCF/KL (25 or 50 ng/ml) (Fig. 1). A strong dose-dependent calcium flux
was observed in response to SDF-1 within 40 s of treatment and
returned toward basal levels within 2 min. The SCF/KL effect on calcium
flux was also dose dependent, but delayed relative to SDF-1, and
extended over the 5-min period of the assay. Combinations of SDF-1
and SCF/KL at various concentrations showed a rapid and sustained
potent calcium mobilization (Fig. 1). The flux appeared to mirror the
additive effects of each ligand.
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and/or SCF/KL
Stimulation of CTS cells with SDF-1 and/or SCF/KL resulted in
enhanced migration of these cells as compared with the untreated cells
(Fig. 2A). Of particular note,
a four- to fivefold increase in migration was observed when both
cytokines were used in combination as compared with each alone.
SDF-1-induced migration was inhibited by prior treatment of CTS
cells with 100 ng/ml of pertussis toxin for 24 h. However,
SCF/KL-induced migration was not inhibited by similar treatment with
the pertussis toxin. This suggests that the pertussis-sensitive
Gi protein mediated the SDF-1 effect, but that SCF/KL
acted through a different pertussis-insensitive pathway.
Chemokines MIP-1, MIP-1ß, and RANTES, at a concentration of
25 nM, did not induce the migration of CTS cells (Fig. 2B).
However, in the presence of SCF/KL, a significant enhancement of the
chemotaxis of CTS cells was observed with all of these chemokines. mAbs
to CXCR4 or CCR5 did not block the chemotaxis of CTS cells induced by
SCF/KL, chemokines, or a combination of these factors (data not shown).
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To pursue the above observation on the model CTS cell line,
primary CD34+ bone marrow cells were stimulated with
SDF-1 and/or SCF/KL. This stimulation resulted in their enhanced
migration as compared with the untreated cells (Table II). Of particular note, a significant
increase in migration was observed when both of the factors were used
in combination as compared with each factor used alone (Table II).
These results in primary bone marrow progenitors confirmed our
observations in the model CTS cell line.
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and/or SCF/KLTo characterize the downstream signaling events in CTS cells expressing the chemokine receptor CXCR4, we first examined phosphorylation effects on RAFTK, a kinase that coordinates signals to the cytoskeleton and to the nuclear transcription apparatus.
CTS cells were stimulated with SDF-1 (25 nM) and/or SCF/KL (50
ng/ml). TCL were immunoprecipitated with anti-RAFTK Ab and analyzed
for RAFTK phosphorylation by blotting with anti-phosphotyrosine.
Rapid phosphorylation of endogenous RAFTK was observed in response to
SDF-1. Treatment with SCF/KL, or the combination of SCF/KL and
SDF-1, resulted in the rapid and sustained phosphorylation of RAFTK
as compared with treatment with SDF-1 alone (Fig. 3). Each immunoblot was reprobed with
RAFTK Ab to confirm that the phosphorylated protein was RAFTK. The
results demonstrated that in CTS cells, both SDF-1 and SCF/KL may
signal through RAFTK to downstream endogenous substrates, but with
different kinetics.
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and/or
SCF/KL
Since chemokines potently mediate cell migration, which involves
alterations in cytoskeletal elements, we assessed changes in paxillin,
a major cytoskeletal component of focal adhesions, following treatment
of CTS cells with SDF-1 and/or SCF/KL. We observed a rapid tyrosine
phosphorylation of paxillin following SDF-1 and/or SCF/KL treatment
(Fig. 4). SCF/KL-induced tyrosine
phosphorylation of paxillin appeared to be more potent as compared with
SDF-1 treatment alone. The kinetics of phosphorylation again
indicated a more sustained effect in the presence of SCF/KL.
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and/or SCF/KL
p130cas is a signaling molecule that
is known to participate in the formation of focal adhesion complexes
critical in cell adhesion and migration (25, 26, 27, 28). CTS cells were
stimulated with SDF-1 and/or SCF/KL, and the cell lysates were
immunoprecipitated with anti-p130cas Ab
and subjected to immunoblotting with anti-phosphotyrosine Ab. We
observed a strong and specific phosphorylation of
p130cas (Fig. 5) and the associated paxillin. As was
seen with paxillin, SCF/KL alone and in combination with SDF-1 had a
potent sustained effect on p130cas
phosphorylation. Upon reprobing the membrane, we found that the mAb
to p130cas reacted only weakly with
p130cas (data not shown), suggesting that
this mAb does not strongly recognize the denatured Ag. The same blot
was reprobed with anti-paxillin Ab and showed a specific band at
the expected molecular mass, indicating constitutive association of
paxillin with p130cas.
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and SCF/KL stimulate MAP kinase activity
The MAP kinase pathway is known to be important in modulating cell
proliferation (29, 30), cell cycle control (31, 32), and chemotaxis
(33, 34) in different cell types. Treatment of CTS cells with SDF-1
(25 nM) resulted in rapid and robust activation of MAP kinase compared
with untreated cells (Fig. 6).
Interestingly, there was relatively weak initial MAP kinase activation
when the two factors were combined. However, a delayed but significant
MAP kinase activation was observed over the 5-min assay period. This
effect was unique and not observed with either factor alone.
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Activation of the p38 MAP kinase and JNK kinase pathways resulted
in alterations of a number of transcription factors with modulation of
cell growth and migration. To assess whether these kinases also
participated in downstream signaling by SDF-1 and/or SCF/KL, TCL
from stimulated CTS cells were immunoprecipitated with anti-p38 MAP
kinase or anti-JNK kinase Abs and then subjected to an in vitro
kinase assay. We did not observe either increased p38 MAP kinase or JNK
kinase activity under these treatment conditions (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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binds to the
seven-transmembrane G protein-linked receptor CXCR4 (5, 35) and has
been reported to act as a chemoattractant for CD34+ marrow
cells (3, 4, 36). SCF/KL previously has been shown to be an early
acting growth factor (9, 10), and to mediate cell adhesion (11, 12, 37, 38, 39), particularly in its transmembrane form. We asked whether these
two cytokines that modulate hemopoietic progenitors might interact
functionally in modulating cell migration, and if so, by what
mechanism. During our studies, Kim and Broxmeyer (4) reported on the
cooperativity between these two cytokines in enhancing hemopoietic
progenitor chemotaxis. This study confirms their functional observation
and provides insight into how this phenomenon might occur via
modulation of signaling pathways.
We studied the effects of the combination of SDF-1 and SCF/KL on a
model CTS hemopoietic cell line. This cell line was chosen because it
expresses many of the surface receptors of primary marrow progenitors.
We first focused on early signaling events related to calcium
mobilization that are known to be essential in chemokine function. We
then examined changes in RAFTK, a midstream platform kinase that has
been reported to link various surface receptor stimuli to the
cytoskeletal apparatus and downstream to MAP kinase, JNK kinase, and
other pathways (18, 22). Focal adhesion components, including paxillin
and p130cas, were studied as examples of
molecules believed to be important in cell migration.
Our results demonstrate that SDF-1-induced migration of CTS cells
was inhibited by pertussis toxin, while SCF/KL-induced migration was
not (Fig. 2
A). This suggests that the initial signaling
cascades of SDF-1 and SCF/KL act through pathways that differ in
their dependence on Gi protein coupling. The results also
show that CTS cells expressing the CCR5 receptor did not migrate in
response to the CCR5 ligands MIP-1, MIP-1ß, or RANTES (Fig. 2B). However, in the presence of SCF/KL, all of these
factors induced a synergistically enhanced migration of CTS cells. This
finding is interesting because it suggests that - and ß-chemokines
may act in coordination with SCF/KL and other growth factors to enhance
chemotaxis and possibly other mechanisms in the stem cell population.
SDF-1 and/or SCF/KL each induced calcium flux in the CTS progenitor
cell line. Within 40 s following treatment with 12.5 or 25 nM
SDF-1, an increase in intracellular calcium was observed that then
declined sharply. SCF/KL at concentrations of 25 and 50 ng/ml induced a
relatively delayed calcium flux at 2 min, which was sustained through
the 5 min of incubation. This result suggested that although both
SDF-1 and SCF/KL act to increase cytosolic calcium, they differ in
terms of their time course of inducing the flux.
Stimulation of CTS cells with SDF-1 and/or SCF/KL resulted in an
increased tyrosine phosphorylation of RAFTK. The increase in tyrosine
phosphorylation of RAFTK following SDF-1 treatment occurred within
0.5 min and declined by 5 min. The kinetics appeared consistent with
the calcium flux observed upon SDF-1 treatment. SCF/KL treatment
also increased the tyrosine phosphorylation of RAFTK within 0.5 min;
however, the effect was prolonged and observable for 5 min. The
prolonged phosphorylation of RAFTK was consistent with the extended
calcium flux kinetics observed following treatment with SCF/KL.
RAFTK is emerging as an important component of focal adhesions
(18, 22, 39). We previously have shown that the phosphorylated form of
RAFTK associates with paxillin (22) in hemopoietic cells following
their activation by growth factors, including SCF/KL (40). We observed
that SDF-1 and/or SCF/KL stimulation resulted in the tyrosine
phosphorylation of paxillin in CTS cells. SCF/KL alone and in
combination with SDF-1 caused phosphorylation of paxillin to a
higher degree and for a more sustained time period as compared with its
phosphorylation by SDF-1 alone (Fig. 4). We noted similar kinetics
of SDF-1 and/or SCF/KL treatment on the phosphorylation of
p130cas and the associated paxillin (Fig. 5). We speculate that the observed sustained activation of RAFTK,
p130cas, and paxillin may result in the
formation of a more efficient focal adhesion that could contribute to
augmented migration when SDF-1 is present in conjunction with
SCF/KL. There may be other as yet unknown proteins that associate with
these molecules following SDF-1 and/or SCF/KL treatment that
modulate cytoskeletal proteins important in chemotaxis. Further studies
in CTS cells will address this possibility.
Of particular interest is our observation of the unique kinetics of MAP
kinase activation by the combination of SDF-1 and SCF/KL.
Stimulation of CTS cells with SDF-1 alone (25 nM) resulted in rapid
but transient enhancement in MAP kinase activity, whereas SCF/KL
treatment alone was slower and weaker in activating MAP kinase. A
delayed but sustained activation of MAP kinase was observed following
treatment of cells with both factors in combination. The early
activation of MAP kinase at 0.5 min as seen with SDF-1 treatment
alone was not observed after treatment with SCF/KL alone or both
factors in combination.
This suggests that MAP kinase activation, a relatively downstream event
in signaling, may be altered in such a way as to augment chemotaxis
with the combination of both factors compared with SDF-1 alone. The
significance of this finding on further downstream transcriptional
targets will be examined in future studies.
RAFTK has also been shown to mediate stress-induced c-Jun
amino-terminal kinase/stress-activated protein kinase (JNK/SAPK)
activation in neuronal cells (41). CCR5, a ß-chemokine receptor, was
reported recently by us to signal via JNK activation (22). However, we
did not observe any increase in JNK activity upon stimulation of CTS
cells with SDF-1 and/or SCF/KL (data not shown), suggesting
important differences between - and ß-chemokine receptor pathways.
Another recently discovered pathway mediating transcriptional
activation is via the p38 MAP kinases. These kinases are activated by
physical and chemical stresses as well as by bacterial LPSs and various
cytokines (42, 43, 44, 45, 46, 47). p38 MAP kinases play an important role in the
phosphorylation and activation of transcription factors, including CHOP
(a mammalian nuclearprotein), ELK-1 (a nuclear target of ERK), and
activating transcription factor-2 (ATF-2) (45, 46). We also did not
observe any increase in p38 MAP kinase activity upon stimulation of CTS
cells with SDF-1 and/or SCF/KL. These data again indicate
potentially important differences between - and
ß-chemokine-mediated effects in different cell types.
Our findings provide new information on the signal-transduction
pathways utilized by the -chemokine receptor CXCR4, and demonstrate
how its ligand SDF-1 may act in conjunction with SCF/KL to modulate
cell signaling in early hemopoietic cells, particularly with regard to
focal adhesion elements and downstream MAP kinase activation.
Activation of various shared signaling substrates under the influence
of chemokines and growth factors and changes in their kinetics, as
shown in this study, may be important in determining mobilization and
other physiologic functions of progenitors in the hemopoietic
microenvironment.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerome E. Groopman, Chief, Division of Experimental Medicine, Harvard Institutes of Medicine-BIDMC, 4 Blackfan Circle, Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: SDF-1, stromal cell-derived factor; ERK, extracellular signal-related kinase; GST, glutathione-S-transferase; JNK, c-Jun amino-terminal kinase; MAP, mitogen-activated protein; MIP, macrophage-inflammatory protein; RAFTK, related adhesion focal tyrosine kinase; RT, room temperature; SCF/KL, stem cell factor/kit ligand; TCL, total cell lysates.
Received for publication February 26, 1998. Accepted for publication May 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. Nobile, M. Lind, F. Miro, K. Chemin, M. Tourret, G. Occhipinti, S. Dogniaux, S. Amigorena, and C. Hivroz Cognate CD4+ T-cell-dendritic cell interactions induce migration of immature dendritic cells through dissolution of their podosomes Blood, April 1, 2008; 111(7): 3579 - 3590. [Abstract] [Full Text] [PDF]
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 S. Basu, N. T. Ray, S. J. Atkinson, and H. E. Broxmeyer Protein Phosphatase 2A Plays an Important Role in Stromal Cell-Derived Factor-1/CXC Chemokine Ligand 12-Mediated Migration and Adhesion of CD34+ Cells J. Immunol., September 1, 2007; 179(5): 3075 - 3085. [Abstract] [Full Text] [PDF]
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W. Tan, D. Martin, and J. S. Gutkind The G{alpha}13-Rho Signaling Axis Is Required for SDF-1-induced Migration through CXCR4 J. Biol. Chem., December 22, 2006; 281(51): 39542 - 39549. [Abstract] [Full Text] [PDF]
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H. Bonig, G. V. Priestley, and T. Papayannopoulou Hierarchy of molecular-pathway usage in bone marrow homing and its shift by cytokines Blood, January 1, 2006; 107(1): 79 - 86. [Abstract] [Full Text] [PDF]
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 K. M. Roundy, G. Spangrude, J. J. Weis, and J. H. Weis Partial rescue of B cells in microphthalmic osteopetrotic marrow by loss of response to type I IFNs Int. Immunol., November 1, 2005; 17(11): 1495 - 1503. [Abstract] [Full Text] [PDF]
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 E. Vasyutina, J. Stebler, B. Brand-Saberi, S. Schulz, E. Raz, and C. Birchmeier CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells Genes & Dev., September 15, 2005; 19(18): 2187 - 2198. [Abstract] [Full Text] [PDF]
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M. Burger, T. Hartmann, M. Krome, J. Rawluk, H. Tamamura, N. Fujii, T. J. Kipps, and J. A. Burger Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells Blood, September 1, 2005; 106(5): 1824 - 1830. [Abstract] [Full Text] [PDF]
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 L. J. Bendall, R. Baraz, J. Juarez, W. Shen, and K. F. Bradstock Defective p38 Mitogen-Activated Protein Kinase Signaling Impairs Chemotaxic but not Proliferative Responses to Stromal-Derived Factor-1{alpha} in Acute Lymphoblastic Leukemia Cancer Res., April 15, 2005; 65(8): 3290 - 3298. [Abstract] [Full Text] [PDF]
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S. Fukuda, H. E. Broxmeyer, and L. M. Pelus Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1{alpha}(CXCL12)/CXCR4 axis Blood, April 15, 2005; 105(8): 3117 - 3126. [Abstract] [Full Text] [PDF]
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Y. Le, M. Honczarenko, A. M. Glodek, D. K. Ho, and L. E. Silberstein CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase Activation and Segregation into Membrane Domains Is Modulated by Regulator of G Protein Signaling 1 in Pro-B Cells J. Immunol., March 1, 2005; 174(5): 2582 - 2590. [Abstract] [Full Text] [PDF]
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A. M. Fischer, J. C. Mercer, A. Iyer, M. J. Ragin, and A. August Regulation of CXC Chemokine Receptor 4-mediated Migration by the Tec Family Tyrosine Kinase ITK J. Biol. Chem., July 9, 2004; 279(28): 29816 - 29820. [Abstract] [Full Text] [PDF]
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M. Lenoir, B. Djerdjouri, and A. Perianin Stroma Cell-Derived Factor 1{alpha} Mediates Desensitization of Human Neutrophil Respiratory Burst in Synovial Fluid from Rheumatoid Arthritic Patients J. Immunol., June 1, 2004; 172(11): 7136 - 7143. [Abstract] [Full Text] [PDF]
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R. Reca, D. Mastellos, M. Majka, L. Marquez, J. Ratajczak, S. Franchini, A. Glodek, M. Honczarenko, L. A. Spruce, A. Janowska-Wieczorek, et al. Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1 Blood, May 15, 2003; 101(10): 3784 - 3793. [Abstract] [Full Text] [PDF]
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A. Z. Fernandis, R. P. Cherla, and R. K. Ganju Differential Regulation of CXCR4-mediated T-cell Chemotaxis and Mitogen-activated Protein Kinase Activation by the Membrane Tyrosine Phosphatase, CD45 J. Biol. Chem., March 7, 2003; 278(11): 9536 - 9543. [Abstract] [Full Text] [PDF]
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E. Y. Choe, E. S. Schoenberger, J. E. Groopman, and I.-W. Park HIV Nef Inhibits T Cell Migration J. Biol. Chem., November 22, 2002; 277(48): 46079 - 46084. [Abstract] [Full Text] [PDF]
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S. R. Vlahakis, A. Villasis-Keever, T. Gomez, M. Vanegas, N. Vlahakis, and C. V. Paya G Protein-Coupled Chemokine Receptors Induce Both Survival and Apoptotic Signaling Pathways J. Immunol., November 15, 2002; 169(10): 5546 - 5554. [Abstract] [Full Text] [PDF]
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T. Kijima, G. Maulik, P. C. Ma, E. V. Tibaldi, R. E. Turner, B. Rollins, M. Sattler, B. E. Johnson, and R. Salgia Regulation of Cellular Proliferation, Cytoskeletal Function, and Signal Transduction through CXCR4 and c-Kit in Small Cell Lung Cancer Cells Cancer Res., November 1, 2002; 62(21): 6304 - 6311. [Abstract] [Full Text] [PDF]
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J. Libura, J. Drukala, M. Majka, O. Tomescu, J. M. Navenot, M. Kucia, L. Marquez, S. C. Peiper, F. G. Barr, A. Janowska-Wieczorek, et al. CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion Blood, September 18, 2002; 100(7): 2597 - 2606. [Abstract] [Full Text] [PDF]
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 A. Ptasznik, E. Urbanowska, S. Chinta, M. A. Costa, B. A. Katz, M. A. Stanislaus, G. Demir, D. Linnekin, Z. K. Pan, and A. M. Gewirtz Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells J. Exp. Med., September 2, 2002; 196(5): 667 - 678. [Abstract] [Full Text] [PDF]
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Y. Lee, A. Gotoh, H.-J. Kwon, M. You, L. Kohli, C. Mantel, S. Cooper, G. Hangoc, K. Miyazawa, K. Ohyashiki, et al. Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines Blood, May 29, 2002; 99(12): 4307 - 4317. [Abstract] [Full Text] [PDF]
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I. B. Mazo, E. J. Quackenbush, J. B. Lowe, and U. H. von Andrian Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow Blood, May 13, 2002; 99(11): 4182 - 4191. [Abstract] [Full Text] [PDF]
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 T. Hideshima, D. Chauhan, T. Hayashi, K. Podar, M. Akiyama, D. Gupta, P. Richardson, N. Munshi, and K. C. Anderson The Biological Sequelae of Stromal Cell-derived Factor-1{alpha} in Multiple Myeloma Mol. Cancer Ther., May 1, 2002; 1(7): 539 - 544. [Abstract] [Full Text] [PDF]
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K. Balabanian, A. Foussat, L. Bouchet-Delbos, J. Couderc, R. Krzysiek, A. Amara, F. Baleux, A. Portier, P. Galanaud, and D. Emilie Interleukin-10 modulates the sensitivity of peritoneal B lymphocytes to chemokines with opposite effects on stromal cell-derived factor-1 and B-lymphocyte chemoattractant Blood, January 15, 2002; 99(2): 427 - 436. [Abstract] [Full Text] [PDF]
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E. A. Sweeney, H. Lortat-Jacob, G. V. Priestley, B. Nakamoto, and T. Papayannopoulou Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells Blood, January 1, 2002; 99(1): 44 - 51. [Abstract] [Full Text] [PDF]
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T. Nanki, K. Nagasaka, K. Hayashida, Y. Saita, and N. Miyasaka Chemokines Regulate IL-6 and IL-8 Production by Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis J. Immunol., November 1, 2001; 167(9): 5381 - 5385. [Abstract] [Full Text] [PDF]
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 B. Dubois, C. Massacrier, and C. Caux Selective attraction of naive and memory B cells by dendritic cells J. Leukoc. Biol., October 1, 2001; 70(4): 633 - 641. [Abstract] [Full Text] [PDF]
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 R. S. Klein, J. B. Rubin, H. D. Gibson, E. N. DeHaan, X. Alvarez-Hernandez, R. A. Segal, and A. D. Luster SDF-1{alpha} induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells Development, June 1, 2001; 128(11): 1971 - 1981. [Abstract] [Full Text] [PDF]
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X.-F. Zhang, J.-F. Wang, E. Matczak, J. Proper, and J. E. Groopman Janus kinase 2 is involved in stromal cell-derived factor-1{alpha}-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells Blood, June 1, 2001; 97(11): 3342 - 3348. [Abstract] [Full Text] [PDF]
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M. Majka, A. Janowska-Wieczorek, J. Ratajczak, M. A. Kowalska, G. Vilaire, Z. K. Pan, M. Honczarenko, L. A. Marquez, M. Poncz, and M. Z. Ratajczak Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis Blood, December 15, 2000; 96(13): 4142 - 4151. [Abstract] [Full Text] [PDF]
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J. A. Burger, N. Tsukada, M. Burger, N. J. Zvaifler, M. Dell'Aquila, and T. J. Kipps Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1 Blood, October 15, 2000; 96(8): 2655 - 2663. [Abstract] [Full Text] [PDF]
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N. Yanai, N. Matsui, T. Furusawa, T. Okubo, and M. Obinata Sphingosine-1-phosphate and lysophosphatidic acid trigger invasion of primitive hematopoietic cells into stromal cell layers Blood, July 1, 2000; 96(1): 139 - 144. [Abstract] [Full Text] [PDF]
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T. Nanki and P. E. Lipsky Cutting Edge: Stromal Cell-Derived Factor-1 Is a Costimulator for CD4+ T Cell Activation J. Immunol., May 15, 2000; 164(10): 5010 - 5014. [Abstract] [Full Text] [PDF]
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J.-F. Wang, I.-W. Park, and J. E. Groopman Stromal cell-derived factor-1alpha stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C Blood, April 15, 2000; 95(8): 2505 - 2513. [Abstract] [Full Text] [PDF]
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G. Venkatakrishnan, R. Salgia, and J. E. Groopman Chemokine Receptors CXCR-1/2 Activate Mitogen-activated Protein Kinase via the Epidermal Growth Factor Receptor in Ovarian Cancer Cells J. Biol. Chem., March 15, 2000; 275(10): 6868 - 6875. [Abstract] [Full Text] [PDF]
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 S. A. Rempel, S. Dudas, S. Ge, and J. A. Gutiérrez Identification and Localization of the Cytokine SDF1 and Its Receptor, CXC Chemokine Receptor 4, to Regions of Necrosis and Angiogenesis in Human Glioblastoma Clin. Cancer Res., January 1, 2000; 6(1): 102 - 111. [Abstract] [Full Text]
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A. J. Naiyer, D.-Y. Jo, J. Ahn, R. Mohle, M. Peichev, G. Lam, R. L. Silverstein, M. A.S. Moore, and S. Rafii Stromal Derived Factor-1-Induced Chemokinesis of Cord Blood CD34+ Cells (Long-Term Culture-Initiating Cells) Through Endothelial Cells Is Mediated by E-Selectin Blood, December 15, 1999; 94(12): 4011 - 4019. [Abstract] [Full Text] [PDF]
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J. A. Burger, M. Burger, and T. J. Kipps Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells Blood, December 1, 1999; 94(11): 3658 - 3667. [Abstract] [Full Text] [PDF]
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J. M. Watson, T. W. Harding, V. Golubovskaya, J. S. Morris, D. Hunter, X. Li, J. S. Haskill, and H. S. Earp Inhibition of the Calcium-dependent Tyrosine Kinase (CADTK) Blocks Monocyte Spreading and Motility J. Biol. Chem., January 26, 2001; 276(5): 3536 - 3542. [Abstract] [Full Text] [PDF]
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