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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2505-2513
CHEMOKINES
Stromal cell-derived factor-1 stimulates tyrosine
phosphorylation of multiple focal adhesion proteins and induces
migration of hematopoietic progenitor cells: roles of
phosphoinositide-3 kinase and protein kinase C
Jian-Feng Wang,
In-Woo Park, and
Jerome E. Groopman
From the Divisions of Experimental Medicine and Hematology/Oncology,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston,
MA.
 |
Abstract |
The stromal cell-derived factor-1 (SDF-1) is an alpha chemokine that
binds to the CXCR4 receptor. Knock-out studies in mice demonstrate that
this ligand-receptor pair is essential in hematopoiesis. One function
of SDF-1 appears to be the regulation of migration of hematopoietic
progenitor cells. We previously characterized signal transduction
pathways induced by SDF-1 in human hematopoietic progenitors and
found tyrosine phosphorylation of focal adhesion components, including
the related adhesion focal tyrosine kinase (RAFTK), the adaptor
molecule p130 Cas, and the cytoskeletal protein paxillin. To better
understand the functional role of signaling molecules connecting the
CXCR4 receptor to the process of hematopoietic migration, we studied
SDF-1-mediated pathways in a model hematopoietic progenitor cell
line (CTS), as well as in primary human bone marrow CD34+
cells. We observed that several other focal adhesion components, including focal adhesion kinase (FAK) and the adaptor molecules Crk and
Crk-L, are phosphorylated on SDF-1 stimulation. Using a series of
specific small molecule inhibitors, both protein kinase C (PKC) and
phosphoinositide-3 kinase (PI-3K) appeared to be required for
SDF-1-mediated phosphorylation of focal adhesion proteins and the
migration of both CTS and primary marrow CD34+ cells,
whereas the mitogen-activated protein kinases ERK-1 and -2 were not.
These studies further delineate the molecular pathways mediating
hematopoietic progenitor migration and response to an essential
chemokine, SDF-1.
(Blood. 2000;95:2505-2513)
© 2000 by The American Society of Hematology.
| |
Introduction |
Stromal cell-derived factor-1 (SDF-1) was initially
characterized as a pre-B-cell stimulatory factor and cloned from mouse bone marrow stromal cells.1-4 SDF-1 is a CXC chemokine,
secreted constitutively from several cell types.5 There are
2 isoforms of SDF-1,  and  , which are generated by differential
splicing from a single gene.2 SDF-1 has been characterized
as a highly efficient chemotactic factor for T cells,
monocytes,6,7 pre-B cells,8 dendritic
cells9 and hematopoietic progenitor cells,10-12 and binds to the CXCR4 receptor. Like other chemokine receptors, CXCR4 is a 7-transmembrane surface structure linked to G
proteins.7,13,14 The chemotactic effect of SDF-1 on
hematopoietic progenitor cells has been shown to be mediated by the
CXCR4 receptor. Targeted disruption of either the SDF-1 or CXCR4 gene
is lethal in mice and is accompanied by many severe defects, including
the absence of both lymphoid and myeloid hematopoiesis in the fetal
bone marrow.15,16 More recently, it was found that SDF-1
and the CXCR4 receptor play a critical role in the engraftment of
hematopoietic stem cells to bone marrow.17 These results
indicate that SDF-1/CXCR4 can regulate hematopoiesis by modulation of
migration and homing of hematopoietic stem and progenitor cells.
Despite the increasingly prominent role of SDF-1/CXCR4 in the
regulation of hematopoietic progenitor cells, there is scant information on the signal transduction pathways induced by SDF-1/CXCR4. SDF-1, binding to CXCR4, mobilizes calcium and reorganizes the actin
structure. The chemotactic activity of SDF-1 is blocked by pertussis
toxin.6,10,18,19 These results indicate that the CXCR4
receptor transmits signals through the Gi-protein.
Various cytokines that induce migration modulate the formation and
function of focal adhesions.20 These adhesions are
cytoskeletal structures that form adherent contacts with the
extracellular matrix. Formation of such focal adhesions and
reorganization of the actin cytoskeleton have been shown to be
associated with the phosphorylation of focal adhesion
components.21,22 For example, the tyrosine phosphorylation
of focal adhesion kinase (FAK),23,24 and of the adaptor
proteins p130 Cas,25,26 Crk,25,27 and paxillin28,29 are directly associated with cell migration
induced by cytokines or chemokines.
To characterize the signal transduction pathways associated with
SDF-1-induced migration in hematopoietic progenitor cells, we first
studied primitive bone marrow CD34+ cells and then a human
hematopoietic progenitor cell line, CTS, which has been found to
express a robust level of the CXCR4 receptor.18 CTS has
been characterized as a multipotential progenitor cell line that can
differentiate into either myeloid or lymphoid lineages.30 We previously showed that SDF-1 stimulates migration and induces calcium flux and the tyrosine phosphorylation of related adhesion focal
tyrosine kinase (RAFTK), paxillin, and p130 Cas in CTS
cells.18 Here, we extend our previous study and report that
SDF-1 induces the tyrosine phosphorylation of multiple adhesion
proteins. In addition to RAFTK, paxillin, and p130 Cas, we observed
that FAK, Crk, and Crk-L are also phosphorylated on SDF-1
stimulation. With respect to the functional roles of signaling
molecules, both phosphoinositide-3 kinase (PI-3) and protein kinase C
(PKC), but not p44/42 ERK, appeared to be required for the
SDF-1-induced tyrosine phosphorylation of focal adhesion proteins,
and for the migration of CTS cells and of human bone marrow
CD34+ progenitor cells.
| |
Materials and methods |
Reagents and antibodies
Recombinant SDF-1 was purchased from R&D Systems (Minneapolis,
MN). Rabbit anti-RAFTK antibody (R-4250) was generated as described
previously.31,32 Anti-FAK polyclonal antibody was generated
from New Zealand white rabbits immunized with a bacterially expressed
GST-fusion protein containing the C-terminal (750-end aa residues) of
FAK cDNA, and was tested to react specifically with FAK. Rabbit
anti-PLC- , and anti-Crk-L polyclonal antibodies were obtained from
Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The mouse
antiphosphotyrosine monoclonal antibody (mAb) 4G10 was a generous gift
from Dr Brian Druker (University of Oregon, Portland, OR), and rabbit
anti-PI-3 kinase polyclonal antibodies were purchased from Upstate
Biotechnology, Inc (Lake Placid, NY). Anti-Crk, anti-p130 Cas,
anti-paxillin, and anti-phosphotyrosine mAb (PY20) were purchased from
Transduction Laboratories (Lexington, KY). Normal rabbit serum and
purified normal rabbit IgG or mouse IgG were purchased from Organon
Teknika Corp (Westchester, PA). Wortmannin, a PI-3 kinase inhibitor;
GF109203X, a PKC inhibitor; and PD98059, a MEK kinase inhibitor, were
obtained from Calbiochem (La Jolla, CA). Electrophoresis reagents and
nitrocellulose membrane were obtained from Bio-Rad Laboratories
(Hercules, CA). Protein A-Sepharose CL-4B and Glutathione Sepharose 4B
were obtained from Amersham Pharmacia (Piscataway, NJ). The protease
inhibitors leupeptin, aprotinin, and alpha 1 antitrypsin and all other
reagents were obtained from Sigma Chemical Co (St Louis, MO).
Cells and cell culture
The CTS cell line was a gift from Dr Takeyuki Sato (Chiba
University, Chiba, Japan). The CTS hematopoietic cell line was
established from a patient with acute myeloblastic leukemia (AML) and
was grown at 37°C in 5% CO2 in RPMI-1640 medium
containing 10% FCS, 50 µg/mL penicillin, and 50 µg/mL
streptomycin, as described previously.30
Ligand stimulation of cells
CTS cells were starved in serum-free RPMI-1640 medium for 5 hours.
During the last hour of starvation, 0.1 nmol/L of sodium vanadate was
added. After starvation, cells were washed twice with serum-free
RPMI-1640 medium and then resuspended at
15 × 106/mL. Cells were then stimulated in vitro
with 20 nmol/L SDF-1 for different time periods at 37°C. After
stimulation, cell lysates were prepared by lysis in lysis buffer (50 mmol/L HEPES, pH 7.0, 150 mmol/L NaCl, 10% glycerol, 1% Triton-X 100, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10 mmol/L sodium
pyrophosphate, 100 mmol/L NaF, 10 mmol/L dithiothreitol, 1 mmol/L PMSF,
10 µg/mL of aprotinin, leupeptin and pepstatin, 10 mmol/L sodium
orthovanadate). Total cell lysates (TCL) were clarified by
centrifugation at 10 000g for 10 minutes. Protein
concentrations were determined by protein assay (Bio-Rad Laboratories).
To assess the effects of the PI-3 kinase inhibitor wortmannin, the PKC
inhibitor GF109203X, or the MEK inhibitor PD98059 for ERK1/2, cells
were preincubated with each of these compounds for 45 minutes before
SDF-1 stimulation as described above.
Immunoprecipitation and Western blot analysis
For the immunoprecipitation studies, identical amounts of protein
from each sample were clarified by incubation with protein A-Sepharose
for 1 hour at 4°C. After the removal of protein A-Sepharose by
brief centrifugation, the solution was incubated with different primary
antibodies as detailed below for each experiment for 4 hours or
overnight at 4°C. Immunoprecipitations of the antibody-antigen complexes were performed by incubation for 2 hours at 4°C with 75 µL of protein A-Sepharose (10% suspension). Nonspecific bound proteins were removed by washing the Sepharose beads 2 times with HNTG
buffer (50 mmol/L HEPES, pH 7.0, 150 mmol/L NaCl, 10% glycerol, 0.1%
Triton-X 100, 1 mmol/L PMSF, 10 µg/mL aprotinin, leupeptin, and
pepstatin, 10 mmol/L sodium orthovanadate) and 1 time with phosphate-buffered saline (PBS). Bound proteins were solubilized in 40 µL of 2 × Laemmli sample buffer and further analyzed by immunoblotting. Samples were separated on 8% to 12% SDS-PAGE and then
transferred to nitrocellulose membranes. The membranes were blocked
with 5% nonfat milk protein and probed with primary antibody for 2 hours at room temperature (RT) or 4°C overnight. Immunoreactive bands were visualized using horseradish peroxidase (HRP)-conjugated secondary antibody and the enhanced chemiluminescent (ECL) system (Amersham Pharmacia). Immunoreactive bands were quantitated by scanning
the blot under a Model GS-700 Imaging Densitometer (Bio-Rad). Control
lanes were assigned a value of 1, and the quantitations of the
immunoreactive bands were expressed as multiples of the control, based
on the densitometry values. Each experiment was repeated at least 3 times and the presented blots are representative of these experiments.
Assays of phosphoinositide-3 kinase activity
SDF-1-stimulated or -unstimulated cells were lysed in ice-cold
lysis buffer containing 137 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.4), 1 mmol/L MgCl2, 1 mmol/L sodium orthovanadate, 10% glycerol, 1% NP-40, and 1 mmol/L PMSF. Immunoprecipitation was performed using
antiphosphotyrosine mAb (PY20) (for phosphotyrosine-associated PI-3-kinase activity). Immunoprecipitates were washed 3 times with
lysis buffer, 3 times with buffer containing 0.1 mol/L Tris-HCl (pH
7.4), 5 mmol/L LiCl, and 0.1 mmol/L sodium orthovanadate, 2 times with
TNE buffer containing 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, and 0.1 mmol/L sodium orthovanadate. Samples were
resuspended in 20 µL TNE buffer, 20 µL phosphoinositol (10 µg;
Avanti Polar Lipids, Alabaster, AL), and 10 µL ATP mix (1 mmol/L
HEPES, 10 µmol/L ATP, 1 µmol/L MgCl2, 10 µmol/L
32P-ATP), then incubated at 37°C for 10 minutes. The
reaction was stopped by adding 40 µL of 3 mol/L HCl and 160 µL
chloroform:methanol (1:1 vol/vol). Lipids were separated on
oxalate-impregnated silica thin-layer chromatography (TLC) plates with
a solvent system of chloroform:methanol:water:ammonium hydroxide (28%)
(35:35:3.5:7). TLC plates were dried and subjected to autoradiography
at  80°C.
Preparation of human bone marrow cells
Light-density bone marrow mononuclear cells were obtained from
normal consenting donors and depleted of adherent cells as previously
described.33
Cell separation or isolation by immunomagnetic beads
Nonadherent light-density bone marrow cells were resuspended in
ice-cold PBS with 1% FCS and 0.5% BSA (isolation medium). CD34+ cells were selected by the Dynal CD34 Progenitor Cell
Selection System (DynalbeadsTM M-450 CD34 and
DETACHaBEADTM CD34, Dynal Inc, Lake Success, NY)
according to the manufacturer's instructions. The beads binding on the
cells were detached and the cells were washed with and resuspended in a
medium for the migration assays (see below). The purity of the
CD34+ cells selected by this method was found to be more
than 95%.
Chemotaxis assays
Chemotaxis assays were performed in triplicate using 5-µm pore
filters (Transwell, 24-well cell clusters; Costar, Boston, MA) as
described previously.11 Briefly, the filters were rinsed with migration medium (RPMI-1640 with 0.5% BSA for the CTS cells; complete -medium with 0.5% BSA for the CD34+ bone
marrow cells) and the supernatant was aspirated immediately before
loading the cells. Either 2 × 105 CTS cells or
1.5 × 105 CD34+ cells suspended in 100 µL migration medium were loaded into each Transwell filter. Filters
were then carefully transferred to another well containing 650 µL
migration medium with 20 nmol/L SDF-1 (R&D Systems). The plates were
incubated at 37°C in 5% CO2 for 3.5 hours. Next, the
upper chambers were carefully removed and the cells in the bottom
chambers were collected. The cells were washed and resuspended in
proper volume and quantitated for viable cells using the trypan blue
exclusion method. To assess the effects of wortmannin, GF109203X, or
PD98059, the cells were pre-incubated with various concentrations of
these inhibitors for 45 minutes, and then the chemotaxic assays were
performed, as described above.
Statistical analysis
The results are expressed as the mean ± SD of data obtained from
3 or more experiments performed in duplicate or triplicate. Statistical
significance was determined using the Student t test.
| |
Results |
Stromal cell-derived factor-1 stimulation induces activation of
phosphoinositide-3 kinase and the tyrosine phosphorylation of PLC-
in CTS cells
Cytokine or chemokine-induced cell migration is a complex process,
mediated by multiple signaling mechanisms. PI-3 kinase, PKC, or MAPK
pathways have been reported to be involved in cytokine or
chemokine-induced migration in various cell types.34-39 We
assessed whether these signaling pathways were involved in
SDF-1-induced migration in hematopoietic progenitor cells.
Using CXCR4 receptor-transfected L1.2 mouse pre-B cells, we previously
showed that SDF-1 stimulation via the CXCR4 receptor selectively
activated p44/42 ERK kinase, but not p38 or JNK kinase.36 In CTS cells, we previously observed a similar activation of p44/42 ERK
by SDF-1.18 Here, we examined the effects of SDF-1 on PI-3 kinase activity and on the tyrosine phosphorylation of PLC- in
CTS cells.
SDF-1 stimulation of CTS cells induced tyrosine phosphorylation of
the p85 PI-3 kinase subunit as detected by immunoblotting of
anti-phosphotyrosine (PY) immunoprecipitates with anti-p85 anti-body
(Figure 1A). To determine whether SDF-1
stimulated PI-3 kinase activity, cell lysates from SDF-1 stimulated
or unstimulated CTS cells were immunoprecipitated with anti-PY
antibody. The tyrosine phosphorylation-associated PI-3 kinase activity
was measured by an in vitro PI-3 kinase assay. As shown in Figure 1B,
PI-3 kinase activity was enhanced by SDF-1 stimulation in a time
course that paralleled p85 tyrosine phosphorylation.
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| Fig 1.
SDF-1 stimulation results in increased tyrosine
phosphorylation and activation of PI-3 kinase in CTS cells. (A)
Total cell lysates, prepared from SDF-1-stimulated CTS cells for
the indicated times, were immunoprecipitated with anti-PY antibody
(PY20). The immune complexes were subjected to immunoblot analysis with
anti-p85 antibody. The changes in the immunoblotted bands of p85 are
indicated (by fold increase) based on the densitometry values. (B)
Total cell lysates were immunoprecipitated with anti-PY antibody
(PY20). PI-3 kinase activity was measured by a PI-3 kinase assay as
described in "Materials and methods."
|
|
PLC- has been shown to play an important role in G-protein coupled
receptor signaling and is an upstream mediator for PKC activation.40,41 We thus investigated if PLC- was
tyrosine-phosphorylated after SDF-1 stimulation of CTS cells. Cell
lysates from SDF-1 stimulated or unstimulated CTS cells were
immunoprecipitated with anti-PLC- antibody and subjected to serial
immunoblotting with anti-PY antibody and anti-PLC- antibody. We
observed that PLC- was significantly tyrosine-phosphorylated on
SDF-1 stimulation (Figure 2, upper
panel). The tyrosine phosphorylation of PLC- was rapid and
transient, reached a maximum at 1 minute, and returned to a basal level
at 5 to 10 minutes after stimulation. Equal amounts of PLC- were
present in each lane (Figure 2, lower panel). The tyrosine
phosphorylation of PLC- in response to SDF-1 stimulation suggested that the PKC pathway may be involved in CXCR4 receptor signaling.
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| Fig 2.
SDF-1 stimulation induces tyrosine phosphorylation of
PLC- in CTS cells. CTS cells were serum-starved and stimulated
with SDF-1 for the indicated times. Total cell lysates were
immunoprecipitated with anti-PLC- antibody, and subjected to serial
immunoblotting with anti-PY antibody (4G10) (upper panel) and
anti-PLC- antibody (lower panel). The increase in tyrosine
phosphorylation of PLC- is indicated (by fold increase) based on the
densitometry values.
|
|
Stromal cell-derived factor-1 stimulates tyrosine phosphorylation
of focal adhesion kinase and its association with p130 Cas and paxillin
To determine whether FAK is tyrosine-phosphorylated after SDF-1
stimulation, CTS cells were serum-starved and stimulated with SDF-1
for the indicated times (Figure 3). Cell
lysates were immunoprecipitated with a specific anti-FAK antibody, and
the immunoprecipitates were then analyzed by Western blotting with anti-PY antibody. As shown in Figure 3, SDF-1 stimulation induced a
rapid and transient tyrosine phosphorylation of FAK. The maximum tyrosine phosphorylation of FAK was detected as early as 1 minute after
the addition of 20 nmol/L of SDF-1. Thereafter, tyrosine phosphorylation of FAK declined gradually to almost baseline levels after 5 minutes (Figure 3, upper panel). We verified that similar amounts of FAK were recovered from the lysates of cells untreated or
treated with SDF-1 for various times (Figure 3, lower panel).
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| Fig 3.
SDF-1 treatment induced the tyrosine phosphorylation
of FAK in CTS cells. Total cell lysates from CTS cells unstimulated
or stimulated with SDF-1 for the indicated times were
immunoprecipitated with anti-FAK antibody. The immune complexes were
subjected to immunoblotting analysis with anti-PY antibody (4G10)
(upper panel) and anti-FAK antibody (lower panel). The increase in
tyrosine phosphorylation of FAK is indicated (by fold increase) based
on the densitometry values.
|
|
Tyrosine-phosphorylated FAK can form a complex with other focal
adhesion proteins. The adaptor protein, p130 Cas, has been shown to
associate with FAK and mediate cell migration.26 The cytoskeletal protein, paxillin, is also a substrate of
FAK.42 Our previous study showed that, in CTS cells, both
p130 Cas and paxillin were tyrosine phosphorylated on SDF-1
stimulation.18 Therefore, we now investigated if FAK
associated with either p130 Cas or paxillin. Serum-starved CTS cells
were stimulated with SDF-1 for various times. Cell lysates from
unstimulated or SDF-1 stimulated CTS cells were immunoprecipitated
with anti-FAK polyclonal antibody. The immunoprecipitates were resolved
on SDS-PAGE gels, followed by immunoblotting with anti-p130 Cas (Figure
4A, upper panel) or anti-paxillin antibody
(Figure 4B, upper panel). SDF-1 stimulation enhanced the association
of FAK both with p130 Cas (Figure 4A, upper panel) and with paxillin
(Figure 4B, upper panel). Similar amounts of FAK were recovered from
the lysates of cells untreated or treated with SDF-1 (Figure 4A and
B, lower panels).
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| Fig 4.
SDF-1 stimulation enhanced the association of FAK with
p130 Cas or paxillin. CTS cells unstimulated or stimulated with
SDF-1 for 1 minute were lysed in a lysis buffer and immunoprecipitated
with anti-FAK antibody. (A) The immune complexes were resolved on 8%
SDS-PAGE and immunoblotted with anti-p130 Cas (upper panel), anti-FAK
(lower panel). (B) The immunocomplexes were resolved on 8% SDS-PAGE
and immunoblotted with anti-paxillin antibody (upper panel) or anti-FAK
antibody (lower panel). TCL represents total cell lysates. The changes
in the association of FAK with p130 Cas (A) or with paxillin (B) are
indicated (by fold increase) based on the densitometry values.
|
|
These results indicate that SDF-1 stimulation induces the tyrosine
phosphorylation of FAK and its association with p130 Cas and paxillin
in hematopoietic progenitor cells.
Stromal cell-derived factor-1 induces the tyrosine
phosphorylation of Crk and Crk-L and enhances their association with
p130 Cas
Crk and Crk-L are structurally similar, but encoded by separate
genes.43 Both Crk and Crk-L are composed of 1 SH2 and 2 SH3
domains. Crk and Crk-L share overall 60% amino acid similarity and
their SH2 and SH3 domains are highly conserved.43 Crk and Crk-L have been shown to function as adaptor proteins, linking different proteins in signaling.44 Thus, we sought to
determine whether the adaptor protein Crk or Crk-L is tyrosine
phosphorylated in CTS cells on SDF-1 stimulation. Cell lysates from
unstimulated or SDF-1 stimulated CTS cells were immunoprecipitated
with anti-Crk or anti-Crk-L, followed by immunoblotting with anti-PY
antibody. As shown in Figure 5E and F,
SDF-1 stimulation resulted in the tyrosine phosphorylation of both
Crk (Figure 5E) and Crk-L (Figure 5F). Moreover, we observed that
several proteins which coimmunoprecipitated with Crk or Crk-L were also
tyrosine phosphorylated. We verified that similar amounts of Crk
(Figure 5E, lower panel) or Crk-L (Figure 5F, lower panel) were
recovered from the lysates of cells untreated or treated with
SDF-1.
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| Fig 5.
Effects of wortmannin, GF109203X, or PD98059 on the
SDF-1-induced tyrosine phosphorylation of various adhesion proteins
in CTS cells. Serum-starved CTS cells were incubated without or
with 100 nmol/L wortmannin, 3 µmol/L GF109203X, or 20 µmol/L
PD98059 (PD) for 45 minutes, then stimulated with SDF-1 (20 nmol/L)
or left untreated for 1 minute at 37°C. Total cell lysates were
immunoprecipitated with antibodies for FAK (A), RAFTK (B), p130 Cas
(C), paxillin (D), Crk (E), or Crk-L (F), respectively. The
immunoprecipitates were subjected to immunoblot analysis with anti-PY
antibody (4G10) (upper panels), followed by reprobing with the same
antibody used for each of the specific immunoprecipitations (lower
panels). Lanes 1 to 8 represent the control, untreated cells with
SDF-1 stimulation (SDF-1), wortmannin-treated cells without
(Wort.) or with SDF-1 (Wort.+SDF-1) stimulation,
GF109203X-treated cells without (GF) or with SDF-1 (GF+SDF-1)
stimulation, PD98059-treated cells without (PD) or with SDF-1
(PD+SDF-1) stimulation, respectively. The increase in tyrosine
phosphorylation of each adhesion protein is indicated (by fold
increase) based on the densitometry values.
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|
Both Crk and Crk-L have been reported to associate with p130
Cas.25,45,46 Thus, we investigated if the
tyrosine-phosphorylated proteins that coimmunoprecipitated with Crk or
Crk-L included p130 Cas. Cell lysates from unstimulated or
SDF-1-stimulated CTS cells were immunoprecipitated with anti-Crk
(Figure 6A) or anti-Crk-L (Figure 6B), and
then immunoblotted with p130 Cas. We observed a constitutive
association between p130 Cas and Crk-L but not between p130 Cas and Crk
in the unstimulated CTS cells. However, SDF-1 stimulation enhanced
the association of both Crk and Crk-L with p130 Cas (Figure 6A and B,
upper panels). We verified that similar amounts of Crk (Figure 6A,
lower panel) or Crk-L (Figure 6B, lower panel) were recovered from the
lysates of the untreated or SDF-1 treated cells.
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| Fig 6.
Association of p130 Cas with Crk or Crk-L on stimulation
with SDF-1 in CTS cells. Total cell lysates from unstimulated or
SDF-1-stimulated CTS cells were immunoprecipitated with anti-Crk
antibody (A) or anti-Crk-L antibody (B). The immune complexes were
resolved on 10% SDS-PAGE and immunoblotted with anti-p130 Cas antibody
(A and B, upper panels). The immunoblots were stripped and reblotted
with anti-Crk (A, lower panel) or anti-Crk-L antibody (B, lower panel).
TCL represents total cell lysates. The changes in the association of
Crk (A) or Crk-L (B) with p130 are indicated (by fold increase) based
on the densitometry values.
|
|
These results indicate that both Crk and Crk-L are involved in CXCR4
receptor signaling in hematopoietic progenitor cells. SDF-1
stimulated the tyrosine phosphorylation of both Crk and Crk-L and
induced their association with p130 Cas.
Effects of signaling inhibitors on stromal cell-derived
factor-1-induced tyrosine phosphorylation of various adhesion
proteins in CTS cells
To assess the functional roles of PI-3 kinase, PKC, and p44/42 ERK,
before stimulation with SDF-1, CTS cells were pretreated with 100 nmol/L wortmannin, a PI-3 kinase inhibitor; 3 µmol/L GF109203X, a PKC
inhibitor; or 20 µmol/L PD98059, a MEK kinase inhibitor,
respectively. Changes in the tyrosine phosphorylation of focal adhesion
proteins on inhibitor treatment were investigated by
immunoprecipitation with specific antibodies for these proteins and by
immunoblotting with anti-PY antibody. As shown in Figure 5, the
SDF-1-induced tyrosine phosphorylation of FAK (A, upper panel),
RAFTK (B, upper panel), p130 Cas (C, upper panel), paxillin (D, upper
panel), Crk (E, upper panel), or Crk-L (F, upper panel) was
significantly reduced by pretreatment with wortmannin or GF109203X but
not PD98059. These results indicate that the tyrosine phosphorylation of these focal adhesion molecules was dependent on the activation of
PI-3 kinase or PKC but not on that of MEK/p44/42 ERK. When the protein
loading controls in each immunoblot were examined by reprobing with the
same antibody used for the immunoprecipitation, we observed that the
SDF-1-treated sample showed an apparent decrease in p130 Cas
protein level, along with an increase in the tyrosine phosphorylation
of this protein (Figure 5C, lower panel). However, similar amounts of
protein were recovered in each lane of the other immunoblots (Figure
5A, B, D, E, and F, lower panels).
Inhibition of phosphoinositide-3 kinase or protein kinase C, but
not MEK, inhibits stromal cell-derived
factor-1-induced migration of CTS cells or primary
bone marrow CD34+ progenitor
cells.
Activation of PI-3 kinase, PKC, and p44/42 ERK after SDF-1 treatment
indicated that CXCR4 signaling involves multiple pathways. To determine
the functional role of PI-3 kinase, PKC or ERK in SDF-1-induced
migration of hematopoietic progenitor cells, CTS cells were pretreated
with different concentrations of wortmannin (Figure
7A), GF109203X (Figure 7B), or PD98059
(Figure 7C). Cell migration in response to SDF-1 was examined by a
Transwell migration assay as described in "Materials and
methods." We observed that treatment with wortmannin (Figure 7A) or
GF109203X (Figure 7B) significantly inhibited SDF-1-induced
migration in a dose-dependent manner. However, treatment with PD98059
over a concentration range of 1 to 40 µmol/L did not alter cell
migration (Figure 7C). Because the specificity of wortmannin, at low
doses, was recently questioned,47 we also tested the
ability of LY294002,48 a competitive inhibitor of PI-3
kinase, to confirm this observed effect. Similar to the results with
wortmannin, LY294002 treatment inhibited cell migration in a
dose-dependent manner (data not shown).
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| Fig 7.
Wortmannin or GF109203X but not PD98059 inhibits
SDF-1--induced migration of CTS cells. CTS cells were treated
with either carrier (DMSO) alone, various concentrations of wortmannin
(A), GF109203X (B), or PD98059 (C) for 45 minutes. The cell migration
in response to SDF-1 (20 nmol/L) was measured in a chemotactic assay
as described in "Materials and methods." Cell migration is shown
as the percentage of cell input. The data shown represent the mean
± SD of 3 separate experiments.
|
|
We next examined the effects of wortmannin, GF109203X, or PD98059 on
the migration of primary bone marrow CD34+ cells in
response to SDF-1. CD34+ cells, isolated from normal
human bone marrow, were pretreated with each inhibitor and tested in a
Transwell migration assay. As shown in Figure
8, similar to that observed in CTS cells,
treatment with wortmannin or GF109203X, but not PD98059, inhibited the
SDF-1-induced migration of CD34+ progenitor cells in
response to SDF-1.
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| Fig 8.
Effects of the PI-3 kinase inhibitor, wortmannin, PKC
inhibitor, GF109203X or MEK kinase inhibitor, PD98059 on
SDF-1-induced migration of CD34+ marrow
cells. CD34+ cells were treated with either carrier
alone or various concentrations (shown in nanomolars) of wortmannin
(Wort.), GF109203X (GF, µM}, or PD98059 (PD, µM) for 45 minutes.
The cell migration in response to SDF-1 (20 nmol/L) or medium alone
(random) was measured in a chemotactic assay as described in
"Materials and methods." Cell migration was shown as the
percentage of cell input. The data shown represent the mean ± SD of 3 separate experiments. * and ** show statistical significance compared
with control (cells pretreated with carrier, DMSO) and are assessed as
P < .05 and P < .01, respectively.
|
|
| |
Discussion |
The molecular mechanisms that regulate hematopoietic progenitor cell
migration are not well characterized. On the basis of studies in mice
null for either the CXCR4 receptor or its cognate ligand SDF-1, it
appears that this chemokine is a major and essential physiological
regulator of effective hematopoiesis, because such mice have an absence
of both lymphoid and myeloid hematopoiesis.15,16 Moreover,
in vitro studies with human progenitors have demonstrated a
potent chemotactic effect of SDF-1.10-12 With this
background, we sought to characterize the signaling pathways triggered
after CXCR4 activation by SDF-1, first in a model
hematopoietic cell line, CTS, and then using primary bone marrow
CD34+ cells.
Our studies focused on components of the focal adhesion complex. Focal
adhesions are important structures that mediate cell adhesion and
migration. They consist of a constellation of signaling molecules and
cytoskeletal proteins and are believed to be essential in
migration.20,49,50 Our previous studies demonstrated that SDF-1 induced calcium flux in CTS cells, followed by the tyrosine phosphorylation of RAFTK, paxillin, and p130 Cas.18 Calcium flux is the signature initial change in G-protein coupled
receptors.5 RAFTK and the related FAK are well-recognized
platform kinases that have multiple binding motifs for a number of
different signaling molecules and act as putative bridges to transmit
signals to the cytoskeleton.51,52 RAFTK and FAK are highly
homologous, sharing an overall 45% amino acid sequence identity, with
60% identity in the catalytic domain. Several tyrosine residues appear
to be conserved between RAFTK and FAK, including an Src family tyrosine kinase SH2-binding site. Both RAFTK and FAK contain proline-rich motifs
capable of SH3 domain interaction.32,53 Given the high degree of structural and amino acid sequence similarity, RAFTK and FAK
may have some similar or even exchangeable functions. Recently, it has
been shown that RAFTK can phosphorylate FAK and functions as an
agonist-dependent regulator of FAK.54 FAK appears to play a
more important role than RAFTK in regulating cell
migration.55 Our studies demonstrated that both FAK and
RAFTK were phosphorylated on SDF-1 treatment of CTS cells, and this
phosphorylation was followed by their association with 2 other
important focal adhesion molecules, p130 Cas and paxillin (Dutt et
al18 and our unpublished data). These results indicated
that both RAFTK and FAK are involved in CXCR4 signaling induced by
SDF-1 in hematopoietic progenitor cells.
With the use of CXCR4-transfected L1.2 cells, our previous study
demonstrated that SDF-1 stimulation induced the tyrosine phosphorylation of Crk and its association with paxillin and
RAFTK.36 In the current study, we observed that not only
Crk but also Crk-L was tyrosine phosphorylated in CTS cells on SDF-1
stimulation. Crk-L has a high homology to Crk and contains a similar
domain structure, SH2-SH3-SH3.43,44 Like Crk, Crk-L has
been shown to be involved in focal adhesion formation by interacting
with several focal adhesion proteins, including p130 Cas, paxillin, Abl, and Bcr-Abl.44 Crk-L is most abundantly expressed in
hematopoietic cells,56 and regulates integrin-mediated cell
adhesion.57,58 Although Crk-L has similar in vitro binding
characteristics with Crk,43,44 there are a few differences
to be noted.45,59 One example of these differences is the
observation that Crk-L but not Crk constitutively associates with p130
Cas in Bcr-Abl transformed leukemia cells.45 Consistent
with this observation, we demonstrated that there was a constitutive
association of Crk-L, but not Crk, with p130 Cas in CTS cells. However,
SDF-1 stimulation significantly enhanced the association of both Crk
and Crk-L with p130 Cas. Our results indicated that both Crk and Crk-L,
through their interaction with p130 Cas, were involved in CXCR4 signaling.
p130 Cas was initially identified as a prominent
tyrosine-phosphorylated substrate of the oncoproteins v-src and
v-crk.60 p130 Cas contains an SH3 domain and numerous
potential SH2 domain docking sites, and also serves as a docking
protein for the recruitment of proteins involved in protein-tyrosine
kinase-mediated signaling pathways. p130 Cas localizes to focal
adhesions and interacts with other focal adhesion
components.61 It has been shown that the association of
p130 Cas with FAK26 or Crk25 is critical for
cell migration. This study, combined with our previous
studies,18,36 demonstrate that SDF-1 stimulates the
tyrosine phosphorylation of p130 Cas and enhances its association with
focal adhesion proteins, including RAFTK, FAK, Crk, and Crk-L. These
results indicate that p130 Cas may play an important role in CXCR4
signaling and cell migration. The apparent decrease in the protein
levels of p130 Cas after SDF-1 stimulation (Figure 5C, lower panel)
could be due to p130 Cas redistribution to actin-rich cytoskeletal
complexes in the Triton-insoluble fractions on its phosphorylation, as
reported previously.60,62,63 However, further study is
needed to elucidate the functional significance of p130 Cas
redistribution in CXCR4 signaling on SDF-1 stimulation.
Our data indicate that multiple adhesion proteins participated in
SDF-1 induced signaling in hematopoietic progenitor cells. The
changes in phosphorylation and interaction of these structural components may be important for the cell adhesion and migration mediated by SDF-1 in hematopoietic cells. Thus, we sought to define
which upstream signaling components may be critical for these
observed changes and the related cell migration induced by
SDF-1.
Accumulating data have implicated that multiple signaling mechanisms
exist to regulate cell migration. MAP kinase (p44/42 ERK),38,39 PI-3 kinase,34-36 and
PKC37 signaling pathways have been shown to regulate the
cell migration induced by chemokines or cytokines. Consistent with our
previous observation in CXCR4-transfected L1.2 cells,36 we
found that SDF-1 activated PI-3 kinase in CTS cells (Figure 1).
Additionally, we observed that SDF-1 significantly induced the
tyrosine phosphorylation of PLC- (Figure 2). Phosphorylated PLC-
hydrolyzes phosphatidylinositol diphosphate to the second-messenger molecules IP3 and DAG, which in turn activate
PKC.40,41 Our previous study demonstrated that SDF-1
also stimulated p44/42 ERK activity in CTS cells.18 To
investigate the functional roles of PI-3 kinase, PKC, and p44/42 ERK in
the SDF-1-induced phosphorylation of focal adhesion components and
cell migration, we used a series of compounds that have been used as
specific inhibitors for PI-3 kinase (wortmannin or LY294002), for PKC
(GF109203X), or for ERK (PD98059, MEK inhibitor). Our results
demonstrated that inhibition of PI-3 kinase or PKC significantly
decreased the tyrosine phosphorylation of various focal adhesion
proteins in response to SDF-1 stimulation, including FAK, RAFTK,
p130 Cas, paxillin, Crk, and Crk-L. Inhibition of PI-3 kinase or PKC
also decreased cell migration in response to SDF-1, both in CTS and
primary bone marrow CD34+ cells. These results
suggest that PI-3 kinase and PKC are both required for the
SDF-1-induced cell migration.
Although activation of the p44/42 ERK signaling pathway has been shown
to promote cell motility either by regulating gene expression38 or directly activating myosin light chain
kinase,39 this was not the case in SDF-1-induced cell
migration. Our studies using the MEK inhibitor PD98059 demonstrated
that inhibition of MEK kinase upstream of p44/42 ERK did not inhibit
SDF-1-induced migration in CTS cells or CD34+ bone
marrow progenitors. Furthermore, treatment with PD98059 could not
interfere with the SDF-1-induced tyrosine phosphorylation of
various focal adhesion proteins (Figure 5). These results imply that the ERK signaling pathway is not involved in the focal
adhesion formation and migration induced by SDF-1 in hematopoietic
progenitor cells.
In summary, our experiments, first done in the CTS cell line and then
confirmed in primary bone marrow CD34+ cells, indicate that
both PI-3 kinase and PKC are functionally important in inducing
SDF-1-mediated progenitor cell migration, whereas p44/42 ERK is
not. The CXCR4 receptor has also been characterized as the primary
coreceptor for certain strains of HIV, and binds the envelope
glycoproteins gp120/160 with high affinity. Furthermore, such binding
results in the activation of certain downstream signaling molecules,
including RAFTK/Pyk2.64,65 With better characterization of
these signal transduction pathways triggered by SDF-1 in hematopoietic progenitor cells, we now can study in the context of HIV infection whether such pathways are conserved or whether dysregulated
hematopoiesis might occur because of aberrant signaling on gp120/160
binding to CXCR4.
| |
Acknowledgments |
We thank our colleague William C. Hatch for his technical
assistance. We are grateful to Janet Delahanty for editing this manuscript and for preparation of the figures, as well as Daniel Kelley
for his assistance with the figures. Finally, we appreciate Delroy
Heath for facilitating our receipt of the needed reagents for the experiments.
| |
Footnotes |
Submitted July 1, 1999; accepted December 14, 1999.
Supported in part by NIH grants HL 53745-02, HL 55187-01, HL 51456-02, and HL 55445-01.
Reprints: Jerome E. Groopman, Division of Experimental
Medicine, Harvard Institutes of Medicine, Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, MA 02115; e-mail:
jgroopma{at}caregroup.harvard.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction