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CHEMOKINES
From the Division of Experimental Medicine and
Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard
Medical School, Boston, MA.
Stromal cell-derived factor-1 (SDF-1), the ligand for the CXCR4
receptor, is a highly efficacious chemoattractant for CD34+
hematopoietic progenitor cells. However, the SDF-1/CXCR4 signaling pathways that regulate hematopoiesis are still not well defined. This
study reports that SDF-1 Blood cells are generated from hematopoietic stem
cells within the bone marrow microenvironment. During fetal
development, stem cells migrate to the bone marrow from the fetal
liver.1 In bone marrow transplantation, hematopoietic stem
cells home to the extravascular compartment of the bone marrow and
durably repopulate transplanted recipients with both myeloid and
lymphoid blood cells.2,3 Chemoattractants appear to play
essential roles in directing the migration of hematopoietic stem cells
to the bone marrow, although how they act on a molecular level is not
yet fully understood. Stromal cell-derived factor-1 (SDF-1), also
called pre-B-cell growth-stimulating factor, is an Although our understanding of the functional role of SDF-1 More recently, it has been proposed that Janus kinase (JAK)/signal
transduction and activation of transcription (STAT) pathways may also
be involved in CXCR4 receptor signaling.24 In a model T-cell line, MOLT4, SDF-1 Reagents and antibodies
Cell culture and ligand stimulation of cells
Before stimulation, the CTS cells were starved in serum-free RPMI-1640
medium for 4 hours. During the last hour of starvation, 0.1 nM 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 next stimulated in vitro with 20 nM SDF-1 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. Following 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 protein A-Sepharose (10% suspension). Nonspecific bound proteins were removed by washing the Sepharose beads 2 times with HNTG buffer (50 mM HEPES, pH 7.0; 150 mM NaCl; 10% glycerol; 0.1% Triton X-100; 1 mM PMSF; 10 µg/mL each of aprotinin, leupeptin and pepstatin; and 10 mM sodium orthovanadate) and one 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% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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 or at 4°C overnight. Immunoreactive bands were visualized by using horseradish peroxidase-conjugated secondary antibody and the enhanced chemiluminescent system (Amersham Pharmacia Biotech).Assays of PI3-kinase activity Unstimulated or SDF-1 -stimulated cells were lysed in
ice-cold lysis buffer containing 137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 1 mM sodium orthovanadate, 10% glycerol, 1%
NP-40, and 1 mM PMSF. Immunoprecipitation was performed by using
antiphosphotyrosine antibody (PY20) or anti-p85 antibody.
Immunoprecipitates were washed 3 times with lysis buffer; 3 times with
buffer containing 0.1 M Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium
orthovanadate; and 2 times with Tris, NaCl, EDTA (TNE) buffer
containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.1 mM
sodium orthovanadate. Samples were resuspended in 20 µL TNE buffer,
20 µL phosphoinositol (10 µg; Avanti Polar Lipids, Alabaster, AL),
and 10 µL adenosine triphosphate (ATP) mix (1 mM HEPES, 10 µM ATP, 1 µM MgCl2, 10 µM
32P-ATP), then incubated at 37°C for 10 minutes. The
reaction was stopped by adding 40 µL 3 M HCl and 160 µL
chloroform:methanol (1:1 vol/vol). Lipids were separated on
oxalate-impregnated silica thin layer chromatography (TLC) plates using
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 healthy consenting donors and depleted of adherent cells, as previously described.28Isolation of human bone marrow CD34+ progenitor cells CD34+ cells were isolated from the bone marrow of healthy donors by using the Direct CD34+ Progenitor Cell Isolation Kit (Miltenyi Biotec GMbH, Germany), according to the manufacturer's instructions. The purity of the CD34+ cells selected by this method was found to be more than 95% by flow-activated cell sorter analysis. After isolation, cells were washed 2 times with migration medium and then resuspended in the same medium (see below).Chemotaxis assays Chemotaxis assays were performed in triplicate using 5 µm-pore filters (Transwell, 24-well cell clusters; Costar, Boston, MA) as described previously.23 Briefly, the filters were rinsed with migration medium (RPMI-1640 with 0.5% bovine serum albumin [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. CTS cells
(2 × 105) or CD34+ cells
(1.5 × 105), 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 nM 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, then
quantitated for viable cells using the trypan blue exclusion method. To
assess the effects of AG490, cells were preincubated with various
concentrations of this inhibitor for 2 hours, and then the chemotaxic
assays were done as described above. Cell migration is shown as the
percentage of cell input.
Statistical analysis The results are expressed as the means ± SD of data obtained from 3 or more experiments performed in triplicate. Statistical significance was determined using the Student t test.
SDF-1
stimulation, CTS cells were serum-starved and stimulated with 20 nM of
SDF-1 for the indicated times (Figure
1). Cell lysates were immunoprecipitated
with a specific anti-JAK2 (Figure 1A), anti-JAK1 (Figure 1B), anti-JAK3
(Figure 1C), or anti-TYK2 (Figure 1D) antibody, and the
immunoprecipitates were then analyzed by Western blotting with
antiphosphotyrosine antibody. As shown in Figure 1A, SDF-1
stimulation induced the rapid and transient tyrosine phosphorylation of
JAK2. Maximum phosphorylation of JAK2 was detected as early as 1 minute
after the addition of 20 nM SDF-1 . Thereafter, the tyrosine
phosphorylation declined gradually. JAK1 (Figure 1B, upper panel) and
TYK2 (Figure 1D, upper panel) were also tyrosine-phosphorylated in
response to SDF-1 stimulation. The time course of phosphorylation of
JAK1 and TYK2 was similar to that of JAK2 (data not shown). However,
the tyrosine phosphorylation of JAK3 was not altered by SDF-1
stimulation (Figure 1C). Loading of equal amounts of protein in all
lanes was confirmed by stripping and reprobing with the same antibodies
used for the immunoprecipitations (Figure 1A-D, lower panels).
Activation of the JAK tyrosine kinases enables these kinases to mediate
the tyrosine phosphorylation of specific STAT
proteins.29,30 Therefore, in further analysis, we examined
the effects of SDF-1
JAK2 is involved in the
SDF-1 stimulation activates the
PI3-kinase pathway in hematopoietic cells. The PI3-kinase pathway appears to be required for SDF-1 -induced migration in CTS cells and
CD34+ bone marrow progenitor cells.23 It has
been reported that activation of JAK2 kinase is required for
SDF-1 -induced migration in T cells.24 Thus, we sought
to determine the functional role of JAK2 in the activation of
PI3-kinase and their potential interrelationship.
These experiments were designed to examine the effects of AG490, a
specific JAK2 inhibitor,31 on the tyrosine phosphorylation of the p85 subunit of PI3-kinase. Serum-starved CTS cells at
10 × 106/mL were preincubated with 100 µM AG490 or its
diluent, dimethyl sulfoxide (DMSO), for 2 hours at 37°C. The cells
were then stimulated with 20 nM SDF-1
To examine the possible physical association between JAK2 and the
p85 subunit of PI3-kinase, cells were pretreated with AG490 or its
diluent, DMSO, followed by SDF-1
Inhibition of JAK2 with AG490 ablates
the SDF-1 stimulates the
tyrosine phosphorylation of several focal adhesion proteins, including CrkII, CrkL, p130Cas, paxillin, RAFTK, and FAK.18,22,23
These focal adhesion proteins are believed to play a critical role in the formation of focal adhesions and in the regulation of cell adhesion
and migration. Here, we observed that JAK2 is required for the
SDF-1 -induced activation of the PI3-kinase pathway. These observations led us to study whether JAK2 is also required for the
SDF-1 -induced tyrosine phosphorylation of these focal
adhesion proteins.
Serum-starved CTS cells were first treated with AG490 or its diluent,
DMSO, as described above. The cells were then stimulated with 20 nM
SDF-1
Inhibition of JAK2 kinase decreases
SDF-1 -induced migration of hematopoietic
progenitor cells, either CTS cells (Figure
6A) or CD34+ human bone
marrow cells (Figure 6B), after their pretreatment with different
concentrations of AG490. Cell migration in response to SDF-1 was
examined using a Transwell migration assay as described in "Materials
and methods." We observed that treatment with AG490 significantly
inhibited the SDF-1 -induced migration of CTS cells in a
dose-dependent manner. The AG490 treatment had no significant effect on
cell migration in the absence of SDF-1 or with SDF-1 in both the
upper and lower chambers (Figure 6A). Similar results were
found with CD34+ primary bone marrow progenitor cells
(Figure 6B). These results suggest that activation of JAK2 is required
for the SDF-1 -induced migration of hematopoietic
progenitor cells.
SDF-1 Although the JAK/STAT signaling pathway is conventionally considered to
be a common feature of all members of the cytokine receptor
superfamily,29,30 it also has been reported to participate in the signaling of the G-protein coupled receptor.34,35
It has been demonstrated that Next, we sought to identify the possible downstream components of the
JAK kinase pathway that are activated on CXCR4 receptor signaling. To
date, besides the SDF-1 receptor reported recently,24 it
is known that JAK2 is also associated with the erythropoietin (Epo)
receptor,38 thrombopoietin (Tpo)
receptor,39,40 GM-CSF receptor,41 IL-3
receptor,42 granulocyte CSF-R (G-CSF-R),43 and cytokine receptors containing gp130 in their receptor chain complex
(interleukin-6 receptor, ciliary neurotrophic factor receptor, leukemia
inhibitory factor receptor, and oncostatin M
receptor).44 Because JAK2 participates in the
signaling of many hematopoietic growth factor receptors, we
specifically focused on this kinase in our analyses. The major function
of the JAK kinases is generally considered to be the activation of the
STAT transcription factors. However, this is clearly not the only role
that JAK kinases play in signaling. For example, JAK kinases are
directly implicated in the activation of tyrosine kinases
RAFTK,45-47 FAK,48 and PI3-kinase,49,50 and in the stimulation of the Ras-MAPK
pathway.51,52 We examined the functional role of JAK2 in
the activation of PI3-kinase, which has been shown to be required for
CXCR4 receptor-mediated signaling and cell migration.23
The increase in PI3-kinase activity is much less impressive after
immunoprecipitation with anti-p85 (2.4 times, compared with
unstimulated control) than after immunoprecipitation with PY20 (4.5 times, compared with unstimulated control). This finding suggests that
p85 is either tyrosine-phosphorylated or associates with
phosphotyrosine-containing molecules after stimulation with SDF-1 Focal adhesions are cytoskeletal structures that form the adherent
contacts of cells with the extracellular matrix. The formation of such
focal adhesions is important for cell adhesion and migration. A number
of proteins, including tyrosine kinases (FAK and RAFTK), adapter
proteins (p130Cas, Crk, and CrkL), and cytoskeletal proteins (paxillin), are involved in the formation of such focal adhesions. The
tyrosine phosphorylation of focal adhesion proteins is believed to be
associated with the cell migration induced by cytokines or chemokines.
We have previously shown that SDF-1
We are grateful to Janet Delahanty for editing this manuscript and to Daniel Kelley for assistance in preparing the figures. Xue-Feng Zhang is a Bertlesmann Cancer research fellow.
Submitted June 28, 2000; accepted January 23, 2001.
Supported in part by grants HL 53745-02, HL 55187-01, HL 51456-02, and HL 55445-01 from the National Institutes of Health.
The first two authors contributed equally to this work.
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.
Reprints: Jerome E. Groopman, Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115; e-mail: jgroopma{at}caregroup.harvard.edu.
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