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Department of Pharmacy and Pharmacology, Bath University, Claverton Down, Bath, Avon, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokines, CC or ß-chemokines, C or
-chemokines, and the CX3C or 
-chemokines
(1, 3, 6).
Stromal cell-derived factor
(SDF-1)4 was first
described as a factor that is produced by bone marrow stromal cells and
shown to induce proliferation of B cell progenitors and regulate B cell
maturation (7). Two isoforms, SDF-1 and SDF-1ß, have
been identified that are encoded by a single gene and arise from
alternative splicing (8). SDF-1 is widely expressed and
is a highly efficacious chemoattractant for monocytes, T lymphocytes,
and CD34+ human progenitor cells
(7, 8, 9, 10, 11). SDF-1 is the biological ligand for the
chemokine receptor CXCR4, a seven-transmembrane G protein-coupled
receptor (12, 13, 14, 15, 16). CXCR4 is expressed on PBL, monocytes,
thymocytes, pre-B cells, as well as dendritic and endothelial cells
(17, 18, 19, 20, 21). Moreover, CXCR4 is the coreceptor for the
binding of T-tropic HIV strains (5, 12, 14, 15).
Accordingly, SDF-1 and its various analogues inhibit CXCR4-mediated
HIV-1 infection in vitro (15, 22, 23). Consistent with the
effects of SDF-1 on pre-B cell proliferation, knockout mice lacking
SDF-1 show abnormalities in B cell lymphopoiesis, bone marrow
myelopoiesis, and cerebellar neuron migration, and also have nonfatal
ventricular septal defects (24). Similar defects have been
reported in CXCR4-/- mice, which also exhibit
defective vascularization of the gastrointestinal tract
(25, 26, 27).
While our understanding of the biological role of SDF-1 has increased
substantially in recent years, relatively little is known about the
signaling pathways that may mediate these effects. SDF-1 has been shown
to elicit elevation of
[Ca2+]]i in a number of
settings (10, 15) and has also been reported to stimulate
phosphorylation of both MEK-1 and ERK1/2 in several cell models
(28, 29, 30, 31). SDF-1 stimulation also enhanced tyrosine
phosphorylation of focal adhesion complex components (including Pyk-2,
paxillin, and Crk), increased NF-
B activity, and induced PI 3-kinase
activity associated with antiphosphotyrosine immunoprecipitates
(29, 31). Thus, SDF-1 can couple to distinct signaling
pathways that may mediate cell growth, migration, and transcriptional
activation.
The prototypical class 1A PI 3-kinase consists of
an 85-kDa regulatory subunit (responsible for protein-protein
interactions via Src homology 2 domain interaction with phosphotyrosine
residues), and a catalytic 110-kDa subunit (32). A
distinct lipid kinase termed PI 3-kinase- is activated by G
protein-coupled receptors, and this is the only characterized member of
the class 1B G protein-coupled PI 3-kinase
family, consisting of a unique 101-kDa regulatory subunit and a
distinct 110-kDa catalytic subunit termed p110
(32, 33, 34). Nevertheless, there is some evidence that G
protein-coupled receptors such as fMLP receptors are also able to
activate the p85/p110 PI 3-kinase (35, 36). In this
respect, the p85/p110 heterodimer has been demonstrated to be
synergistically activated by the ß subunits of G proteins and by
phosphotyrosyl peptides (36). The class I PI 3-kinases can
potentially generate three lipid products, namely
phosphatidylinositol-(3)-monophosphate
(PtdIns(3)P),
phosphatidylinositol-(3, 4)-bisphosphate
(PtdIns(3, 4)P2),
and phosphatidylinositol-(3, 4, 5)-trisphosphate
(PtdIns(3, 4, 5)P3), which are
collectively known as D-3 phosphoinositide lipids (reviewed in Refs.
37, 38). In addition, both the p85/p110 heterodimer and
PI 3-kinase- exhibit dual specificity as both a lipid kinase and a
serine protein kinase (39, 40). At present, both
PtdIns(3, 4)P2 and
PtdIns(3, 4, 5)P3 can be regarded as
signaling molecules, whereas PtdIns(3)P is thought to
regulate membrane trafficking (37, 38). PI 3-kinase(s) is
now regarded as an important intracellular signal that is upstream of a
variety of responses including insulin-stimulated glucose uptake
(41), membrane ruffling (42), and superoxide
production (43). Moreover, activation of a number of
downstream signaling proteins is known to be regulated by PI 3-kinase
and its lipid products including protein kinase B (PKB), p70S6 kinase,
and Rac (44, 45, 46).
Given the functional role of SDF-1 in chemotaxis (7, 8, 9, 10, 11), it is interesting to note that the PI 3-kinase inhibitor wortmannin inhibits SDF-stimulated chemotaxis of CXCR4-expressing pre-B cells (31) as well as chemotaxis of several other cell types in response to other CXC chemokines (e.g., IL-8) (47) or CC chemokines (e.g., RANTES and monocyte-chemoattractant protein-1) (48, 49). In this study, therefore, we have investigated the possible involvement of PI 3-kinase(s) in SDF-1 signal transduction and chemotaxis in T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rSDF-1 was purchased from PeproTech (Rocky Hill, NJ ).
SDF-1 peptide analogues were a kind gift of Ian Clark-Lewis (University
of British Colombia). The anti-CXCR4 mAb 12G5 (50) was
obtained from the National Institute of Health AIDS Research and
Reference Reagent Program. The goat anti-PI 3-kinase- polyclonal
Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All
cell culture reagents and pertussis toxin were purchased from Life
Technologies (Paisley, U.K.). Wortmannin and standard
phosphatidylinositol lipids were purchased from Sigma (Poole, Dorset,
U.K.). [32P]Orthophosphate (8500–9120 Ci/mmol)
was from DuPont-NEN (Boston, MA). All other reagents were purchased
from Sigma.
Cell culture
The human leukemic T cell line Jurkat expressing CXCR4 was cultured in humidified incubators at 37°C, 5% (v/v) CO2 in RPMI 1640 medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 µg/ml amphotericin B. CHO cells transfected with B7.1 cDNA (CHO-B7.1+) were established and maintained as previously described (51).
T cell purification and T lymphoblast preparation
Heparinized blood samples were separated on a Histopaque (1.077) density gradient. PBMC were removed from the gradient, and purified T cells were obtained by negative selection, as described (51). Alternatively, PBMCs (106 cells/ml) were stimulated with staphylococcal enterotoxin B (1 µg/ml) for 72 h. The cells were washed and growth maintained by supplementing every 2 days with 0.1 nM IL-2. After 10 days, cells were deprived of IL-2 for at least 2 days and allowed to accumulate in the G0/G1 stage of the cell cycle (52).
Flow cytometry
Peripheral blood-derived T lymphocytes, IL-2-maintained T
lymphoblasts, or Jurkat cells (2 x 105)
were stained with 10 µg/ml anti-CXCR4 Ab 12G5 or IgG isotype
control (IgG2a) for 45 min at 4°C, washed, and incubated for a
further 45 min at 4°C with 10 µg/ml anti-IgG FITC secondary Ab.
Cells were washed and subsequently analyzed using a Becton Dickinson
(San Jose, CA) FACS Vantage; excitation 
488 nm, emission
530 nm.
D-3 phosphoinositide lipid labeling, extraction, and HPLC separation
A total of 1 x 108 cells were labeled with 1 mCi [32P]orthophosphate (8500–9120 Ci/mmol; DuPont-NEN), as described (53). 32P-labeled Jurkat cells were aliquoted at 107/120 µl and stimulated as described in the figure legends, and the phospholipids were extracted with 700 µl chloroform:methanol:H2O (32.6%:65.3%:2.1% v/v/v, respectively) (53). The samples were deacylated and analyzed by anion-exchange HPLC analysis using a Partisphere SAX column (Whatman, Maidstone, Kent, U.K.) (53). The eluate was fed into a Canberra Packard A-500 Flo-One on-line radiodetector, and the results were analyzed by the Flo-One data program (Radiomatic). Eluted peaks were compared with retention times for standards prepared from 3H-labeled phosphoinositide lipids (Amersham-Pharmacia Biotech, Amersham, Bucks, U.K.) and 32P-labeled D-3 phosphoinositides described elsewhere (54).
Cell lysis and in vitro PI 3-kinase assays
A total of 1 x 107 cells/ml were
equilibrated for 10 min at 37°C and then stimulated in RPMI 1640
medium, as described in the figure legends. Reactions were terminated
by pelleting cells in a microfuge for 10 s, followed by aspiration
of the supernatant and addition of 0.5 ml ice-cold lysis buffer (1%
(v/v) Nonidet P-40, 100 mM NaCl, 20 mM Tris (pH 7.4), 10 mM
iodoacetamide, 10 mM NaF, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 10 µg/ml ß-glycerophosphate, and 1 mM sodium
orthovanadate). Lysates were rotated at 4°C for 15 min, followed by
centrifugation at 14,000 rpm. The supernatants were precleared, and
immunoprecipitation was performed using anti-PI 3-kinase- mAb (1
µg/ml). Immunoprecipitates were washed and subjected to in vitro
lipid kinase assays using a lipid mixture of 100 µl of 0.1 mg/ml
PtdIns and 0.1 mg/ml phosphatidylserine dispersed by sonication in 25
mM HEPES, pH 7.4, and 1 mM EDTA (49). The reaction was
initiated by the addition of 10 µCi of
[-32P]ATP (3000Ci/mmol; DuPont-NEN) and 100
µM ATP to the immunoprecipitates suspended in 80 µl of kinase
buffer (5 mM MgCl2, 0.25 mM EDTA, 20 mM HEPES, pH
7.4). The reaction was terminated after 15 min, and phospholipids were
then separated by TLC (49). The TLC plates were stained
with iodine to confirm even extraction of substrate lipid between
individual samples, and 32P-labeled
PtdIns(3)P was visualized by autoradiography
(49).
Immunoblotting
Aliquots of cell lysate supernatant were boiled in Laemmli buffer and electrophoresed through 7.5% (v/v) acrylamide gels by SDS-PAGE, and the proteins were transferred by electroblotting onto nitrocellulose (Schleicher & Schuell, Keene, NH), as described previously (49). The blots were probed with a phosphospecific PKB Ab (0.5 µg/ml), which only has affinity for the active Ser473-phosphorylated forms of PKB (New England Biolabs, Knowl Piece, Herts, U.K.), and proteins were visualized using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ) with a goat anti-rabbit Ig (0.1 µg/ml) conjugated with HRP as a secondary Ab. Where appropriate, blots were completely stripped of Abs by incubation at 55°C for 60 min with stripping solution (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 100 mM 2-ME). After extensive washing, blots were reblocked, and total levels of PKB were detected by reprobing with 0.5 µg/ml anti-PKB Ab (New England Biolabs). Alternatively, cell lysates were separately probed with phosphoprotein-specific Abs detecting ERK1/2 (p44/p42) when phosphorylated at Thr202 and Tyr204, followed by stripping and reprobing with Abs to detect total levels of ERK1/2 (New England Biolabs).
Determination of [Ca2+]i
Jurkat cells and T lymphoblasts were suspended at
107 cells/ml in RPMI 1640 medium supplemented
with 10% FCS and incubated for 30 min at 37°C with 2.5 µM fura-2
acetoxymethyl ester, as described previously (55). The
fluorescence of a 2-ml cellular suspension was monitored with a Photon
Technology International Delta Scan Fluorometer (dual excitation
340 and 380 nm, single emission 510 nm) at 37°C. Cytosolic free
calcium concentration
([Ca2+]i) was determined
by fluorescence using Photon Technology International software program
(South Brunswick, NJ).
Chemotaxis assays
Chemotaxis was examined using a 96-well chemotaxis chamber
(Neuro Probe, Cabin John, MD). The wells of the 96-well plate were
filled with 380 µl of chemoattractant diluted in RPMI 1640 containing
0.1% BSA and covered with an adhesive polyvinylpyrrolidine-free
polycarbonate membrane (8 µM pore size). A total of 2 x
105 Jurkat cells or peripheral blood-derived T
lymphocytes were added to each upper well in a volume of 200 µl, and
the chamber was incubated at 37°C for 2 h. The cell suspension
was subsequently aspirated off, and 200 µl of Versene (Life
Technologies) was added to each well. After 20-min incubation at 4°C,
the 96-well plate and membrane were centrifuged at 1500 rpm for 10 min,
the supernatant was removed, and the cells were resuspended in 100 µl
of RPMI containing 0.1% BSA. Cell migration was assessed by adding 20
µl of Cell Titer 96 AQueous solution (Promega,
Southampton, U.K.) to each well. After a 2-h incubation at 37°C, the
plate was read at 490 nm, subtracting the readings at a reference
650 nm to reduce the background contributed by nonspecific
absorbance.
Actin polymerization
Purified T lymphocytes (2 x 106/0.5
ml) in RPMI 1640 were incubated at 37°C between 15 s and 30 min
in the presence of SDF-1, and the cells were then fixed by the addition
of 0.5 ml of 7.4% formaldehyde in PBS. After washing and
permeabilization in 0.1% Triton solution in PBS for 10 min, the cells
were incubated with 100 µl of 0.3 µM FITC-phalloidin at 4°C for
30 min. The cells were then washed twice in PBS and resuspended in 500
µl of 1% paraformaldehyde/PBS solution. Data were analyzed on a
Becton Dickinson FACS Vantage, excitation 488 nm, emission
530 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The leukemic T cell Jurkat has been used previously to investigate
biochemical responses to CXCR4 (28) and expresses high
levels of this receptor (Fig. 1
A). Therefore, we have used
32P-labeled Jurkat cells to investigate the
effect of SDF-1 stimulation on the activation of PI 3-kinase, as
assessed by the accumulation of one of its products, namely
PtdIns(3, 4, 5)P3. Accordingly,
treatment of Jurkat cells with SDF-1 resulted in a significant
concentration-dependent accumulation of
PtdIns(3, 4, 5)P3 above resting levels
(Fig. 2A). The maximum levels
of PtdIns(3, 4, 5)P3 accumulation
following stimulation with SDF-1 were approximately one-half that
observed in response to a maximal type stimulus for p85/p110 PI
3-kinase activation in Jurkat cells resulting from ligation of CD28
(54, 55). The SDF-1-induced increase in
PtdIns(3, 4, 5)P3 exhibited bell-shaped
characteristics, with the maximum response observed in the presence of
10 nM SDF-1 (Fig. 2A). Furthermore, the SDF-1-stimulated
formation of PtdIns(3, 4, 5)P3 was
extremely rapid and transient, because it was detectable 15
s after stimulation and had returned toward basal levels 2–5 min after
SDF-1 treatment (Fig. 2B).
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). However, when these partial agonist
peptides were added in combination with SDF-1, the resulting
PtdIns(3, 4, 5)P3 accumulation was
reduced compared with that induced by SDF-1 alone (Fig. 3). The peptide
antagonist SDF-1 1–9 [P2G] dimer and the anti-CXCR4 Ab 12G5 had
no effect on basal levels of
PtdIns(3, 4, 5)P3, but did markedly
inhibit accumulation of this lipid in response to 10 nM SDF-1
(Fig. 3).
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, A and B).
However, despite the low level of expression of CXCR4 on T
lymphoblasts, SDF-1 could still elicit elevation of
[Ca2+]]i in these cells
(Fig. 4A), thus both
confirming previous observations (14) and demonstrating
that CXCR4 stimulation can lead to biochemical events in this model.
Pretreatment with the Gi protein inhibitor
pertussis toxin completely inhibited the elevation of
[Ca2+]i in response to
SDF-1 (Fig. 4A). In marked contrast, although CXCR4 was
expressed at much greater levels on Jurkat cells, 100 nM SDF-1 did not
stimulate any detectable changes in
[Ca2+]]i in Jurkat cells
(data not shown). This suggests that the CXCR4 receptor is
differentially coupled to biochemical signaling pathways in different T
cell models, at least with respect to calcium mobilization. However,
this differential signaling does not appear to extend to coupling to PI
3-kinase because accumulation of
PtdIns(3, 4, 5)P3 in response to SDF-1
was also observed in T lymphoblasts (Fig. 4B), the kinetics
of which were similar to that observed for
PtdIns(3, 4, 5)P3 accumulation in Jurkat
cells after SDF-1 treatment (Fig. 2B). Studies with PI
3-kinase inhibitors in other systems have demonstrated a requirement
for D-3 phosphoinositide lipids for optimal receptor-stimulated calcium
mobilization (57). However, pretreatment with the PI
3-kinase inhibitor wortmannin (58) had no effect on
SDF-1-stimulated elevation of
[Ca2+]i in T lymphoblasts
(Fig. 4A).
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To further characterize the SDF-1-stimulated PI 3-kinase activity,
we used the PI 3-kinase inhibitor wortmannin and the
Gi protein inhibitor pertussis toxin.
Pretreatment for 10 min with wortmannin abrogated SDF-1-induced
PtdIns(3, 4, 5)P3 accumulation in both
Jurkat cells and T lymphoblasts (Fig. 5,
A and B). Similarly, pretreatment of
Jurkat cells for 16 h with 100 ng/ml pertussis toxin
completely abrogated the SDF-1-induced increase in
PtdIns(3, 4, 5)P3 (Fig. 5A),
suggesting that the accumulations of
PtdIns(3, 4, 5)P3 following SDF-1
treatment appear to involve a Gi protein-mediated
mechanism. To verify this, we determined whether immunoprecipitates of
PI 3-kinase- derived from SDF-1-stimulated Jurkat cells exhibited
enhanced in vitro lipid kinase activity vs that present in
immunoprecipitates derived from unstimulated cells. Accordingly, SDF-1
stimulated an increase in the in vitro activity of PI 3-kinase- that
was extremely rapid and transient, because it was detectable 30 s
after stimulation and had returned toward basal levels 5–10 min after
SDF-1 treatment (Fig. 5C).
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Having established that SDF-1 could strongly stimulate the
activation of PI 3-kinase, we next examined the outcome of SDF-1
treatment on the activity of PKB, a known downstream effector of the PI
3-kinase-dependent signaling cascade (44). Hence, cell
lysates derived from resting and SDF-1-stimulated cells were
immunoblotted using a phosphospecific Ab that recognizes only the
Ser473-phosphorylated, active form of PKB.
Indeed, SDF-1 was shown to activate PKB within 30 s above the
basal levels of PKB activity that were detectable under these
conditions. Pretreatment of Jurkat cells for 16 h with 100 ng/ml
pertussis toxin completely abrogated the SDF-1-stimulated PKB
phosphorylation (Fig. 6). Similarly,
5-min pretreatment with the PI 3-kinase inhibitor wortmannin also
inhibited SDF-1-stimulated PKB phosphorylation (Fig. 6). Basal levels
of PKB activity were unaffected by either pertussis toxin or wortmannin
(Fig. 6).
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Because chemokine receptor stimulation can have biological effects
in the absence of measurable calcium mobilization (48), we
investigated whether SDF-1 could stimulate chemotaxis of Jurkat cells
and, if so, whether PI 3-kinase activation was involved. Indeed, SDF-1
stimulated the chemotaxis of Jurkat cells and freshly isolated
peripheral blood-derived T lymphocytes in a bell-shaped,
concentration-dependent manner that is characteristic of
chemokine-dependent chemotaxis (Fig. 7,
A and B) (48). The involvement of PI
3-kinase in this SDF-1-stimulated functional response was assessed by
the use of the PI 3-kinase inhibitors wortmannin and LY294002. Jurkat
cell and peripheral blood-derived T lymphocyte chemotaxis in response
to SDF was inhibited by pretreatment of the cells with wortmannin (Fig. 7, A and B) and LY294002 (Fig. 7C).
The IC50 values for wortmannin and LY294002
inhibition of SDF-1-stimulated chemotaxis were 7 ± 4 nM and
1 ± 0.2 µM (n = 4). Pertussis toxin also
inhibited the SDF-1-stimulated chemotaxis of Jurkat cells and
peripheral blood-derived lymphocytes (Fig. 7, A and
B).
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Rearrangement of the actin cytoskeleton is an early cellular
response during chemotactic responses (59). Given the
strong activation of a pertussis toxin-sensitive PI 3-kinase by SDF-1
and its apparent involvement in chemotaxis, we therefore investigated
the effect of PI 3-kinase inhibitors on SDF-1-stimulated actin
polymerization. We were unable to detect any SDF-1-stimulated changes
in actin polymerization above the high basal levels observed in Jurkat
cells (data not shown), even though SDF-1 stimulates chemotaxis of
Jurkat cells. The reasons for this are unclear, but it is likely that
levels of polymerized actin were so high as to prevent detection of any
further effect of SDF-1 using the assay employed. We therefore used
normal peripheral blood-derived T cells that exhibited much lower
levels of basal actin polymerization, and SDF-1 induced a marked
concentration-dependent increase in actin polymerization in these cells
(Fig. 8A), confirming previous
observations (9). Moreover, the increase in filamentous
actin was transient, occurring within 15 s and returning to basal
levels within 30 min (Fig. 8B). Pretreatment with either
wortmannin or LY294002 partially inhibited actin polymerization by
50 ± 3% and 58 ± 6%, respectively (Fig. 8C).
In contrast, pertussis toxin pretreatment completely abrogated actin
polymerization in response to SDF-1 treatment of the cells (Fig. 8C).
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Having established that SDF-1 could strongly stimulate a pertussis
toxin-sensitive PI 3-kinase, we next examined the outcome of PI
3-kinase inhibitors on SDF-1-stimulated ERK1/2 MAP kinase activation
because the pertussis toxin-sensitive PI 3-kinase- has been
demonstrated to mediate Gß-dependent regulation of the MAP kinase
signaling pathway in other systems (60, 61). Hence, cell
lysates derived from control unstimulated or SDF-1-stimulated Jurkat
cells were immunoblotted using a phosphospecific Ab to the
phosphorylated active forms of ERK1/2. Indeed, SDF-1 was shown to
activate ERK1/2 within 30 s (Fig. 9A). Pretreatment of Jurkat
cells for 10 min with wortmannin inhibited the SDF-1-stimulated ERK
phosphorylation in a concentration-dependent manner (Fig. 9, B and D). The activation of ERK1/2 in response to
SDF-1 was also inhibited by 16-h pretreatment with pertussis toxin
(Fig. 9C). Blots were routinely stripped and reprobed with
anti-ERK1/2 Ab to verify equal loading and efficiency of protein
transfer (Fig. 9A–D).
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Given that PI 3-kinase activation is required for ERK1/2
activation as well as chemotaxis in response to SDF-1, we investigated
whether the chemotactic response of peripheral blood-derived T
lymphocytes was also dependent on ERK1/2 activation using the MEK
inhibitor PD098059 (62). Indeed, peripheral blood-derived
T cell chemotaxis, in response to a concentration of SDF-1 sufficient
to elicit optimal chemotaxis (10 nM), was attenuated by pretreatment of
the cells with PD098059 (Fig. 10).
Although PD098059 inhibition of SDF-1-stimulated chemotaxis was
concentration dependent, the highest concentration (10 µM) afforded
only partial inhibition to 58 ± 7% of control migration
(n = 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The elevation of PtdIns(3, 4, 5)P3
observed in response to SDF-1 may be the result of activation of more
than one PI 3-kinase (e.g., the p85/p110 PI 3-kinase and PI
3-kinase-). However, the accumulation of
PtdIns(3, 4, 5)P3 in Jurkat cells
stimulated by SDF-1 could be completely inhibited by pretreatment with
pertussis toxin, strongly indicating that D-3 phosphoinositide lipid
accumulation occurs via a Gi protein-coupled PI
3-kinase. To date, the only characterized Gi
protein-coupled PI 3-kinase is the class 1B PI
3-kinase- (32). Previous studies using different cell
models have reported that SDF-1 stimulation induces a lipid kinase
activity to coassociate with antiphosphotyrosine immunoprecipitates,
implying the activation of the class 1A p85/p110
heterodimer, although this was not formally demonstrated
(31). Other studies have reported an increase in PI
3-kinase activity associated with antiphosphotyrosine
immunoprecipitates after activation of G protein-coupled receptors
(35, 48, 49). Certainly, SDF-1 can induce the protein
tyrosine phosphorylation of a number of substrates
(28, 29, 30, 31), while other G protein-coupled receptors have
also been shown to stimulate protein tyrosine kinases after appropriate
stimulation with bombesin and vasopressin (63) or
monocyte-chemoattractant protein-1 (49). Because
synergistic activation of the p85/p110 PI 3-kinase by
tyrosine-phosphorylated peptides and ß subunits of GTP-binding
proteins has been reported (36, 64), it is possible that
the p85/p110 heterodimer may contribute to the accumulation of
PtdIns(3, 4, 5)P3 observed after
stimulation with SDF-1. However, it seems unlikely that the p85/p110
heterodimeric PI 3-kinase makes any contribution to SDF-1-stimulated
PtdIns(3, 4, 5)P3 accumulation, because
this response is completely abrogated by pertussis toxin pretreatment,
while the protein tyrosine kinase inhibitor herbimycin A had no effect
on the PtdIns(3, 4, 5)P3 accumulation
(our unpublished observations). Hence, the different levels of
PtdIns(3, 4, 5)P3 accumulation
stimulated by SDF-1 and CD28 may be simply explained by the fact that
they stimulate different subclasses of PI 3-kinase, namely PI
3-kinase- and p85/p110, respectively.
Studies with PI 3-kinase inhibitors in other systems have demonstrated
a requirement for D-3 phosphoinositides in the activation of
phospholipase C- and hence for optimal calcium mobilization in
response to ligation of the B cell Ag receptor (57, 65).
Such a mechanism is thought to involve direct interaction of D-3
phosphoinositide lipids with the tandem Src homology 2 domains and/or
the amino-terminal PH domains of phospholipase C-. In addition, the
D-3 phosphoinositides can interact with the PH domains of the Tec
family of protein tyrosine kinases, thereby influencing their membrane
targeting and activation, which in turn influences phospholipase C-
activation. However, CXCR4 appears to be coupled to the pertussis
toxin-sensitive phospholipase Cß and it is unlikely that
phospholipase C is activated by CXCR4. The G protein-coupled ß
isoforms of phospholipase C also contain a PH domain that can
potentially interact with the D-3 phosphoinositides formed in response
to SDF-1 and hence facilitate optimal calcium mobilization. However,
this seems an unlikely event given that we were unable to detect any
inhibitory effect of the PI 3-kinase inhibitor wortmannin on elevation
of [Ca2+]i in T
lymphoblasts in response to SDF-1 stimulation.
The SDF-1-induced activation of a wortmannin-sensitive PI 3-kinase
appears to be an important signal required for SDF-stimulated
biochemical events such as ERK1/2 and PKB phosphorylation. Although
SDF-1 can couple to these distinct signaling pathways that can mediate
cell survival, growth, migration, and transcriptional activation, it is
unable to support IL-2 production and T cell proliferation either alone
or in combination with anti-CD3 or anti-CD28 Abs (unpublished
observations). One possibility is that SDF-1 may regulate the threshold
for T cell activation. Indeed, some studies have demonstrated that
SDF-1 can exert inhibitory effects on critical components of the TCR
signaling cascade such as reduced tyrosine phosphorylation of ZAP-70,
SLP-76, and linker for activation of T cells (66), while
PI 3-kinase has been proposed to play a negative role in TCR function
(67). Our observation that SDF-1 activates PKB also fits
well with the previous demonstration that PKB is a downstream effector
of PI 3-kinase- (68). Indeed, several other G
protein-coupled receptors, including those activated by the chemokines
RANTES and IL-8, have been shown to activate PKB in a PI
3-kinase-dependent manner, although the functional significance of
these observations has not been determined (69, 70). PKB
is a key mediator of growth factor-induced cell survival and protection
against c-Myc-induced cell death (71, 72, 73). However, we
have demonstrated that pretreatment of Jurkat cells with SDF-1 is not
sufficient to protect against Fas-induced Jurkat cell death
(unpublished observations). Nevertheless, activation of PKB by SDF-1 is
hard to reconcile with evidence implicating SDF-1 and CXCR4 with the
promotion of cell death in various systems (74, 75, 76).
Several studies have recently reported that SDF-1 stimulates
phosphorylation of MEK-1 and ERK1/2 in leukemic T cell lines, T cell
clones, and a pre-B cell lymphoma cell line (28, 29, 30, 31). Our
data indicate that PI 3-kinase inhibitors prevent SDF-1-stimulated
activation of ERK1/2, implying an upstream requirement for PI 3-kinase
activation. These observations correlate well with observations that PI
3-kinase- has been demonstrated to mediate Gß-dependent
regulation of both the ERK1/2 MAP kinase and PKB signaling pathway in
other systems (60, 61, 68). Moreover, the MEK inhibitor
PD098059 partially inhibits SDF-1-stimulated chemotaxis, suggesting
that ERK1/2 activation may be involved at least in part, as a
downstream effector of a PI 3-kinase-regulated signaling cascade that
culminates in a chemotactic response. This would correlate with
previous observations indicating a role for MAP kinases in amoeboid
chemotaxis in response to cAMP and fibroblast chemotaxis in response to
fibronectin (77, 78, 79). However, it should be emphasized
that chemotaxis of neutrophils in response to the related chemokine
IL-8 or fMLP has been reported to be independent of ERK1/2 (47, 80, 81). Hence, our observation that ERK1/2 activation is
involved at least partially in SDF-1-stimulated chemotaxis in T cells
may reflect differences between cell types and/or chemoattractants with
respect to the biochemical pathways that facilitate chemotactic
responses.
The role of SDF-1-stimulated biochemical signals in T cell activation
remains unclear, but it seems that SDF-1-stimulated activation of
Gi protein-coupled PI 3-kinase plays a pivotal
role in chemotaxis, given that PI 3-kinase inhibitors prevent
chemotaxis of Jurkat cells and peripheral blood-derived T lymphocytes.
This correlates well with previous studies that have indicated that PI
3-kinase and its metabolic products play an important role in signaling
pathways mediating chemotaxis (47, 48, 49, 80, 81). Moreover,
PI 3-kinase- has been shown to play an important role in regulating
reorganization of the actin cytoskeleton (82), a process
thought to be a prerequisite for cell movement (59).
However, it is interesting to note that SDF-1-stimulated actin
polymerization is only partially inhibited by PI 3-kinase inhibitors.
This may indicate that while chemotaxis is fully dependent on PI
3-kinase activation, actin polymerization is subject to distinct
biochemical regulation involving additional PI 3-kinase-independent
pathways. In this respect, it is interesting to note that SDF-1 induces
CXCR4 coupling to both Gi (pertussis
toxin-sensitive) (9, 19) and Gq
(pertussis toxin-insensitive) (9, 19, 20, 83) family
members of G proteins, so it is possible that CXCR4 uses more than one
G protein subunit and may initiate signaling pathways that are
independent of PI 3-kinase(s). Another possibility is that there may be
more than one receptor for SDF-1 and these receptors may be
differentially coupled to signaling pathways and functional events.
Indeed, while SDF-/- and
CXCR4-/- mice have similar phenotypes relating
to B cell development, they may not be identical in other respects
(24, 25, 26, 27) and a splice variant of CXCR4 has indeed recently
been identified (84). It is also interesting to note that
the optimal changes in actin polymerization were observed 30 s to
1 min after SDF-1 stimulation, but chemotaxis was not measured until
2 h after stimulation. Therefore, a final possibility to explain
the different sensitivities of SDF-1-stimulated chemotaxis and actin
polymerization to PI 3-kinase inhibitors is that while the observed
changes in polymerized actin may well be a representation of initiation
of motile response, these changes may have little to do with the
subcellular contractile machinary for sensing chemotactic gradients and
facilitating ordered and coordinated cell migration.
This investigation has demonstrated that SDF-1 activates a pertussis
toxin-sensitive PI 3-kinase that appears necessary for the activation
of PKB and ERK in response to SDF-1. Although the role of PKB in SDF-1
functional responses is not understood, it seems that PI
3-kinase-dependent ERK activation is required for SDF-1-stimulated
chemotaxis. Interestingly, other studies have reported that G
protein-coupled receptors can activate multiple PI 3-kinase effectors
such as Rac and PKB (82, 85). However, while cytoskeletal
reorganization and lamellipodium formation are PI 3-kinase-mediated
events, they occur independently of PKB (82, 85). Use of
PI 3-kinase inhibitors such as wortmannin and LY294002 does not
distinguish between the lipid or protein kinase activities of PI
3-kinases. Given that PKB activation is dependent on D-3
phosphoinositide products of PI 3-kinase-, while MAP kinase
activation is mediated by the protein kinase activity of PI
3-kinase- in other systems (68), it will be an
important aim of future studies to ascertain whether it is the lipid or
protein kinase activity of PI 3-kinase responsible for mediating
functional effects of SDF-1.
|
Footnotes |
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2 Current address: Novartis Horsham Research Centre, Wimblehurst Road, Horsham West Sussex, U.K.
3 Address correspondence and reprint requests to Dr. Stephen G. Ward, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, Avon, BA2 7AY, U.K. E-mail address:
4 Abbreviations used in this paper: SDF-1, stromal cell-derived factor-1; [Ca2+]i, intracellular calcium concentration; ERK, extracellular signal-related kinase; MAP, mitogen-activated protein; MEK, MAP/ERK kinase; PH, pleckstrin homology; PI 3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; PtdIns, phosphatidylinositol; PtdIns(3)P, phosphatidylinositol-(3)-monophosphate; PtdIns(3,4)P2, phosphatidylinositol-(3,4)-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol-(3,4,5)-trisphosphate.
Received for publication July 19, 1999. Accepted for publication September 20, 1999.
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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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M. Inngjerdingen, K. M. Torgersen, and A. A. Maghazachi Lck is required for stromal cell-derived factor 1alpha (CXCL12)-induced lymphoid cell chemotaxis Blood, May 29, 2002; 99(12): 4318 - 4325. [Abstract] [Full Text] [PDF]
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M. Ticchioni, C. Charvet, N. Noraz, L. Lamy, M. Steinberg, A. Bernard, and M. Deckert Signaling through ZAP-70 is required for CXCL12-mediated T-cell transendothelial migration Blood, May 1, 2002; 99(9): 3111 - 3118. [Abstract] [Full Text] [PDF]
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M. Nishita, H. Aizawa, and K. Mizuno Stromal Cell-Derived Factor 1{alpha} Activates LIM Kinase 1 and Induces Cofilin Phosphorylation for T-Cell Chemotaxis Mol. Cell. Biol., February 1, 2002; 22(3): 774 - 783. [Abstract] [Full Text] [PDF]
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H. Geminder, O. Sagi-Assif, L. Goldberg, T. Meshel, G. Rechavi, I. P. Witz, and A. Ben-Baruch A Possible Role for CXCR4 and Its Ligand, the CXC Chemokine Stromal Cell-Derived Factor-1, in the Development of Bone Marrow Metastases in Neuroblastoma J. Immunol., October 15, 2001; 167(8): 4747 - 4757. [Abstract] [Full Text] [PDF]
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B.-S. Youn, Y. J. Kim, C. Mantel, K.-Y. Yu, and H. E. Broxmeyer Blocking of c-FLIPL-independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction Blood, August 15, 2001; 98(4): 925 - 933. [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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S. Montaner, A. Sodhi, S. Pece, E. A. Mesri, and J. S. Gutkind The Kaposi's Sarcoma-associated Herpesvirus G Protein-coupled Receptor Promotes Endothelial Cell Survival through the Activation of Akt/Protein Kinase B Cancer Res., March 1, 2001; 61(6): 2641 - 2648. [Abstract] [Full Text]
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R. P. Cherla and R. K. Ganju Stromal Cell-Derived Factor 1{{alpha}}-Induced Chemotaxis in T Cells Is Mediated by Nitric Oxide Signaling Pathways J. Immunol., March 1, 2001; 166(5): 3067 - 3074. [Abstract] [Full Text] [PDF]
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R. D. Chernock, R. P. Cherla, and R. K. Ganju SHP2 and cbl participate in {alpha}-chemokine receptor CXCR4-mediated signaling pathways Blood, February 1, 2001; 97(3): 608 - 615. [Abstract] [Full Text] [PDF]
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F. Sanz-Rodriguez, A. Hidalgo, and J. Teixido Chemokine stromal cell-derived factor-1{alpha} modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1 Blood, January 15, 2001; 97(2): 346 - 351. [Abstract] [Full Text] [PDF]
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E. Haddad, J. L. Zugaza, F. Louache, N. Debili, C. Crouin, K. Schwarz, A. Fischer, W. Vainchenker, and J. Bertoglio The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis Blood, January 1, 2001; 97(1): 33 - 38. [Abstract] [Full Text] [PDF]
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 V. Grabovsky, S. Feigelson, C. Chen, D. A. Bleijs, A. Peled, G. Cinamon, F. Baleux, F. Arenzana-Seisdedos, T. Lapidot, Y. van Kooyk, et al. Subsecond Induction of {alpha}4 Integrin Clustering by Immobilized Chemokines Stimulates Leukocyte Tethering and Rolling on Endothelial Vascular Cell Adhesion Molecule 1 under Flow Conditions J. Exp. Med., August 14, 2000; 192(4): 495 - 506. [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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P. H. Naccache, S. Levasseur, G. Lachance, S. Chakravarti, S. G. Bourgoin, and S. R. McColl Stimulation of Human Neutrophils by Chemotactic Factors Is Associated with the Activation of Phosphatidylinositol 3-Kinase gamma J. Biol. Chem., July 28, 2000; 275(31): 23636 - 23641. [Abstract] [Full Text] [PDF]
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V. Kansra, C. Groves, J. C. Gutierrez-Ramos, and R. D. Polakiewicz Phosphatidylinositol 3-Kinase-dependent Extracellular Calcium Influx Is Essential for CX3CR1-mediated Activation of the Mitogen-activated Protein Kinase Cascade J. Biol. Chem., August 17, 2001; 276(34): 31831 - 31838. [Abstract] [Full Text] [PDF]
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R. E. Gerszten, E. B. Friedrich, T. Matsui, R. R. Hung, L. Li, T. Force, and A. Rosenzweig Role of Phosphoinositide 3-Kinase in Monocyte Recruitment under Flow Conditions J. Biol. Chem., July 13, 2001; 276(29): 26846 - 26851. [Abstract] [Full Text] [PDF]
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)
wortmannin (100 nM for 10 min, •), or pertussis toxin (100 ng/ml for
16 h, 
) at 37°C and then incubated with increasing
concentrations of SDF-1 (1–100 nM) in a 96-well chemotaxis chamber at
37°C for 2 h. C, A total of 2 x
105 Jurkat (
