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-Induced Chemotaxis in T Cells Is Mediated by Nitric Oxide Signaling Pathways1
Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(SDF1) and its cognate
chemokine receptor CXCR4 act as potent chemoattractants and regulate
trafficking and homing of hematopoietic progenitor cells and
lymphocytes. However, the molecular mechanisms regulating
SDF1-driven cell migration are not well defined. In this study, we
have explored the roles of the second messenger NO and the
transcription factor NF-
B in SDF1-induced T cell migration.
SDF1 treatment of Jurkat T cells increased the activity of NO
synthase, which catalyzes the generation of NO. We observed that
pretreatment of Jurkat cells or activated PBLs with several NO donors
significantly enhanced the SDF1-induced migration, whereas various
inhibitors of NO synthase markedly abrogated the chemotactic response
in a concentration-dependent manner. Furthermore, we observed that
inhibitors of the transcription factor NF-B, which is linked to NO
signaling pathways, also significantly blocked the SDF1-induced
chemotactic response. However, these compounds did not have a
significant effect on SDF1-induced mitogen-activated protein kinase
activity. In addition, the MAP/Erk kinase kinase inhibitor PD98059 did
not abrogate SDF1-induced chemotaxis. AKT, which has been shown to
mediate NO production, was also phosphorylated upon SDF1
stimulation. These studies suggest that NO-related signaling pathways
may mediate SDF1-induced chemotaxis, but not mitogen-activated
protein kinase activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(C-X-C), 
(C-C), 
(C), and 
(C-X-X-X-C) (1, 2, 6).
Stromal cell-derived factor 1
(SDF1)3 is a member
of the CXC or -chemokine subfamily and is the only known ligand for
the chemokine receptor CXCR4 (7, 8, 9, 10). SDF1 and CXCR4
are constitutively expressed in a large number of tissues
(10, 11, 12, 13, 14). CXCR4 plays an important role in HIV infection
and pathogenesis (9, 10, 15). SDF1 and the CXCR4
receptor also regulate embryonic development (16, 17, 18, 19).
Furthermore, knockout mice lacking CXCR4 or SDF1 protein are
embryologically lethal. Knockout studies revealed that these two
proteins are mandatory for various developmental processes, including
chemotaxis or homing of myeloid stem cells from the fetal liver to the
bone marrow environment (17, 19).
SDF1 has been shown to act as a potent chemoattractant for PBLs,
monocytes, pre- and pro-B cells, and CD34+ human
progenitors (6, 11, 20, 21, 22). However, little is known
about the molecular mechanisms that mediate these functions. We and
others have recently shown that CXCR4 can mediate signaling through
various components of the focal adhesion complex such as the related
adhesion focal tyrosine kinase (RAFTK), also known as Pyk2 or CAK-
(23, 24, 25). Furthermore, we also showed that
mitogen-activated protein (MAP) kinase and NF-B are activated upon
SDF1 stimulation (23). Phosphatidylinositol 3-kinase
(PI3K), which has been shown to regulate chemotaxis (26),
is also involved in SDF1-induced and CXCR4-mediated chemotaxis and T
cell migration (23, 27). In our present study, we further
delineate the signaling pathways that regulate CXCR4-mediated
chemotaxis. We have observed that NO and NF-B are key components of
the SDF1-induced signaling cascade.
NO is a multifunctional signaling molecule that has been shown to regulate various cellular functions such as hemostasis, apoptosis, inflammation, vascular tone, and chemotaxis (28, 29, 30, 31, 32). NO is produced from L-arginine by at least three isoforms of the NO synthase (NOS) enzyme: type I (neuronal NOS), type II (inducible NOS), and type III (endothelial NOS (eNOS)) (28, 29, 30). All three isoforms have been shown to be expressed at very low levels in different types of T cells (33, 34, 35, 36). The activity of NOS can be regulated by its phosphorylation and by other posttranslational modifications. It has been shown that AKT/protein kinase B can phosphorylate eNOS on serine 1179, which leads to enzyme activation and NO production (37, 38, 39). NO modulates the functions of various proteins by nitrosylation and other posttranslational changes (40).
NF-B is a major transcription factor that regulates transcription of
genes encoding many inflammatory cytokines and cell adhesion molecules
(41). NF-B has been shown to influence NO production by
regulating expression of the inducible NO synthase gene
(42). Conversely, NO has also been shown to be capable of
modulating NF-B function in mononuclear leukocytes and endothelial
cells (43, 44).
Although NO can regulate migration in several cell types
(45, 46, 47, 48), its role in chemokine-mediated chemotaxis in T
cells is not known. We now report that SDF1 treatment activates NOS
in Jurkat T cells. Furthermore, NO donors enhance SDF1-induced
chemotaxis, whereas NO inhibitors potently abrogate this chemotaxis.
SDF1 stimulation also induces AKT phosphorylation. These studies
provide new information regarding signaling pathways that are involved
in the regulation of chemokine-induced chemotaxis in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The NO donors S-nitroso-N-acetyl
penicillamine (SNAP), sodium nitroprusside (SNP), and
4-phenyl-3-furoxancarbonitrite (PFC); the protease inhibitor
N-tosyl-lysine-chloromethylketone
(TLCK); the NOS inhibitors
NG-nitro-L-arginine
methyl ester (L-NAME) and
NG-monomethyl-L-arginine
monoacetate (L-NMMA); the NF-B inhibitors
N-tosyl-phenylalanine-chloromethylketone
(TPCK) chemical inhibitor and the cell permeable peptide inhibitor
(SN50); and the cell permeable inactive control peptide (SN50M) were
obtained from Calbiochem (San Diego, CA). Purified Abs to
phosphospecific p44/42 MAP kinase were obtained from New England
Biolabs (Beverly, MA). Protein Abs to p44/42 were obtained from Santa
Cruz Biotechnology (Santa Cruz, CA). The NOS Detect Assay kit was
obtained from Stratagene (La Jolla, CA). Phospho-AKT and AKT Abs were
obtained from New England Biolabs. Electrophoresis reagents and
nitrocellulose membrane were obtained from Bio-Rad (Hercules, CA). The
protease inhibitors leupeptin and 1 antitrypsin, and all other
reagents, were obtained from Sigma (St. Louis, MO).
Cell culture
The Jurkat T cell line was cultured at 37°C in 5% CO2 in RPMI 1640 with 10% FCS, 2 mM glutamine, 50 µg/ml penicillin, and 50 µg/ml streptomycin.
Stimulation of cells
Jurkat cells were washed twice with 1x HBSS (Mediatech
Laboratories, Cody, NY), then resuspended at 10 x
106 cells/ml in 1x HBSS and starved for 1 h
at 37°C in 5% CO2. The cells were then
preincubated with various NO donors, NOS inhibitors, or the NF-B
inhibitor along with the appropriate control solvents for 30–60 min at
37°C. Following this treatment, Jurkat cells were stimulated with 100
ng/ml SDF1. After stimulation, the cells were microfuged for 10
s and lysed with modified radioimmunoprecipitation assay (RIPA) buffer
(50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 150 mM NaCl, 200 mM PMSF,
10 µg/ml aprotinin, 1 µg/ml each of leupeptin and pepstatin, 2 mM
each of sodium vanadate and sodium fluoride, and 0.25 M sodium
pyrophosphate). Total cell lysates were clarified by centrifugation at
10,000 x g for 10 min. Protein amounts were determined
by a protein assay (Bio-Rad) to normalize the concentrations in all of
the samples.
Isolation of PBLs
PBLs were isolated as described previously (49). Briefly, PBLs were isolated from heparinized venous blood collected from healthy donors by Ficoll-Hypaque density gradient centrifugation at 3000 rpm for 25 min. The cells were suspended in RPMI 1640 containing 10% FCS, 2 mM glutamine, 50 µg/ml penicillin, and 50 µg/ml streptomycin. Monocytes were depleted by two rounds of adherence to plastic. Nonadherent cells were stimulated with PHA (5 µg/ml) for 3 days. Cells were removed to fresh medium supplemented with recombinant human IL-2 (Advanced Biotechnologies, Columbia, MD). Two-week-old cells were used for the chemotaxis assays. To determine whether any of the added agents were toxic, the viability of the cells following various treatments was monitored by trypan blue uptake. No toxicity was observed. Each chemotaxis assay was done in triplicate and each experiment was repeated twice.
Immunoprecipitation
Immunoprecipitation analysis was done as described elsewhere (50). Briefly, equivalent amounts of protein from each sample were precleared by incubating with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at 4°C. The supernatant from each sample was collected after brief centrifugation. Primary Ab was added for each experiment, and samples were incubated at 4°C for 4 h or overnight. The immune complexes were precipitated with 50 µl of protein A-Sepharose CL-4B (50% suspension). Nonspecific bound proteins were removed by washing the Sepharose beads three times with modified RIPA buffer and once with 1x PBS. The immune complexes were subjected to MAP kinase assay.
Western blotting
Western blot analyses were done as described previously (50). Briefly, equivalent amounts of protein from each sample were run on 8% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk and incubated with primary Ab for 2 h at room temperature or overnight at 4°C. The blots were washed and incubated with secondary Ab coupled to HRP for 2 h at room temperature or overnight at 4°C. The bands were visualized by using the ECL system (Amersham Pharmacia Biotech). The data shown are representative of findings from three experiments. The activity of the bands was quantified by densitometric analysis using a Bio-Rad Imager. The mean densities of the bands are represented as the OD in units per square millimeter.
Chemotaxis assays
Assays were done as previously described (23).
Briefly, Jurkat cells were washed twice and 10 x
106 cells/ml were suspended in medium containing
RPMI 1640 with 2.5% FCS. The chemotaxis assay was performed in 24-well
plates containing 5-µm porosity inserts (Costar, Kennebunk, ME).
Before performing the chemotaxis assays, the cells were treated with
different concentrations of NO donors, NOS inhibitors, NF-B
inhibitor, or the appropriate controls for 30–60 min. One hundred
microliters (1 x 106 cells) from each
sample were loaded onto the upper well. A total of 0.6 ml of medium
containing SDF1 (50 ng/ml) was added to the lower chamber. The
plates were incubated for 1.5–3 h at 37°C in 5%
CO2. After incubation, the porosity inserts were
removed carefully and the viable cells were counted using standard
procedures. The results are expressed as the number of cells migrated
to the bottom chamber. Each experiment was performed three or four
times in triplicate.
p44/42 MAP kinase assay
Cells were stimulated as described above. After normalizing the
protein concentration, the cell lysates were immunoprecipitated with
extracellular signal-regulated kinase (Erk)-1 and Erk-2 Abs (Santa Cruz
Biotechnology). The immune complexes were washed twice with RIPA buffer
and twice with kinase buffer (50 mM HEPES (pH 7.4), 10 mM
MgCl2, and 20 µM ATP). Finally, the immune
complexes were incubated in a total volume of 25 µl of kinase buffer
containing 7 µg of myelin basic protein (Upstate Biotechnology, Lake
Placid, NY) and 5 µCi of [-32P]ATP for 20
min at 30°C. The proteins were separated on 15% SDS-PAGE and bands
were detected by autoradiography.
NOS assay
This assay was performed by using the NOS Detect Assay kit (Stratagene). Briefly, the cells were washed once with 1x PBS containing 1 mM EDTA, suspended in 1x homogenization buffer, and then homogenized by repeated freezing and thawing in dry ice. The soluble fraction was separated by centrifugation at 10,000 x g for 5 min, normalized for protein concentration, and used for estimation of NOS activity according to the instructions of the manufacturer. Briefly, the cell lysate (5–10 µg protein) was added to the reaction mixture which contained the substrate [3H]L-arginine, buffer (Tris buffer (pH 7.4), 6 µM tetrahydrobiopterin, 2 µM flavin adenine dinucleotide, 2 µM flavin adenine mononucleotide), 6 mM calcium chloride, and 10 mM NADPH. The samples were then incubated for 30 min at room temperature. After termination of the reaction, the reaction product, citrulline, was separated from L-arginine using a resin that specifically binds to L-arginine, and the radioactivity was then counted. NOS activity is expressed as cpm/h/mg protein.
Statistical analysis
The results are expressed as the mean ± SD of data obtained from three or four experiments performed in duplicate or triplicate. The statistical significance was determined using the Student t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhances NOS activity
To elucidate the effect of SDF1 on NO production, Jurkat
T cells, which were uniformly positive for the CXCR4 receptor as
detected by FACS analysis (data not shown), were stimulated with
SDF1 for up to 6 h. NOS activity, which catalyzes the synthesis
of NO, was assessed by measuring the conversion of
[3H]L-arginine to
[3H]citrulline. As shown in Fig. 1
, SDF1 treatment resulted in an
increase in total NOS activity. Maximum activity was obtained around
the 3-h time period.
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-mediated chemotaxis
To explore the role of NO in SDF1-induced chemotaxis,
Jurkat T cells were pretreated with the different NO donors SNAP, SNP,
and PFC in a concentration-dependent manner and then a chemotaxis assay
was performed as described in Materials and Methods. As
shown in Fig. 2, NO donors significantly
enhanced chemotaxis in a concentration-dependent manner. There was
about a 60% increase in migration with SNAP at 1000 nM (Fig. 2A), a 75% increase with SNP at 0.1 µM (Fig. 2B), and a 70% increase with PFC at 0.1 µM (Fig. 2C) as compared with the solvent controls. SNP and PFC at
very high concentrations had no or a lesser effect. The effect of
various donors on the viability of cells was determined. No effect on
cell viability using various compounds was observed under these
conditions (data not shown).
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-induced chemotaxis
The role of NO in SDF1-induced chemotaxis was further
confirmed by using various inhibitors. As shown in Fig. 3, pretreatment of Jurkat cells with the
NOS inhibitors L-NMMA and L-NAME, and TLCK
which has been shown to inhibit NOS gene expression
(51, 52, 53), attenuated SDF1-induced chemotaxis in a
dose-dependent manner. L-NMMA pretreatment resulted in a
60–70% inhibition at 0.1–1.0 mM (Fig. 3A), whereas
L-NAME at 2 mM inhibited by 40% the chemotactic
response induced by SDF1 (Fig. 3B). Maximum inhibition
(90%) was achieved with TLCK, at 100 µM (Fig. 3C). These
NO donor and inhibitor studies revealed that NO is an important
mediator of chemotactic responses induced by the chemokine SDF1
in T cells. We also determined the effect of various inhibitors on cell
viability to rule out the possibility that the observed decrease in
chemotaxis was due to the decrease in cell viability. No effect on cell
viability using various inhibitors was observed under these conditions
(data not shown).
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B inhibitors inhibit SDF1-induced chemotaxis
We have recently shown that SDF1 activates NF-B
(23). Furthermore, NOS expression has been shown to be
regulated by NF-B. To determine the role of NF-B in
SDF1-induced chemotaxis, we used the NF-B chemical inhibitor TPCK
and the peptide inhibitor SN50. These inhibitors markedly attenuated
SDF1-induced chemotactic effects in a concentration-dependent manner
(Fig. 4). About 80% inhibition was
achieved with 100 µM TPCK as compared with the methanol control (Fig. 4A). Similarly, 90% inhibition was obtained with the SN50
peptide (100 µg/ml). A mutant peptide, SN50M, which does not affect
NF-B activity under similar conditions, had no significant effects
on SDF1-induced chemotaxis (Fig. 4B). SN50 is a cell
permeable peptide inhibitor containing the nuclear localization
sequence of the NF-B component p50, which inhibits translocation of
NF-B into the nucleus. SN50M is an inactive control peptide with
substitutions of two amino acids in the nuclear localization sequence
region (54). These results suggest that NF-B may
regulate SDF1-induced chemotaxis.
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-induced chemotaxis
In previous studies, we have shown that besides chemotactic
activity, SDF1 also induced MAP kinase activity in CXCR4-expressing
pre-B cells (23). In this study, we further assessed the
functional role of the MAP kinase pathway in SDF1-induced
chemotaxis. Jurkat cells were pretreated either with PD98059, which
inhibits MEK kinase upstream of p44/42 MAP kinase, or with control
solvent DMSO. Cell migration in response to SDF1 was then
determined. As shown in Fig. 5, PD98059
at various concentrations did not inhibit SDF1-induced migration.
These studies suggest that the MAP kinase signaling pathway is not
involved in the chemotaxis induced by SDF1 in T cells.
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-induced chemotaxis of
activated PBLs
We also confirmed the effect of various compounds on the
SDF1-induced chemotaxis of activated PBLs. These cells were found to
be 
60% positive for CXCR4 by FACS analysis (data not shown). As
shown in Fig. 6A, an
20–25% increase in chemotaxis was observed with the NO donor SNP
at 50–100 nM concentrations. However, NO and various other inhibitors
significantly blocked SDF1-induced chemotaxis (Fig. 6, B–D). TLCK inhibited chemotaxis by 90–95% at a 10 µM
concentration (Fig. 6B). Similarly, the NF-B inhibitor
TPCK (10 µM; Fig. 6C) or SN50 (100 µg/ml; Fig. 6D) inhibited 90–95% of the PBL chemotaxis induced by
SDF1 as compared with the control-treated samples. However, the MEK
kinase inhibitor PD98059 (Fig. 6E) did not have any effect
on SDF1-induced chemotaxis at a 10 or 50 µM concentration. These
studies further confirmed that NO-linked signaling pathways participate
in SDF1-induced chemotaxis. We also determined the effect of the
above-mentioned compounds on the viability of PBLs. No effect on cell
viability was observed under these conditions (data not shown).
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induces AKT phosphorylation
AKT has been shown to phosphorylate eNOS and mediate NO
production during vascular endothelial growth factor signaling
(37, 38, 39). As shown in Fig. 7, stimulation with SDF1 increased the
phosphorylation of AKT in Jurkat cells (Fig. 7A) and PBLs
(Fig. 7B) as observed using phospho-AKT (S473)-specific Ab.
Maximum phosphorylation was observed around 5–10 min of stimulation.
Equal amounts of AKT protein were present in each lane (Fig. 7, bottom panels). These results suggest that AKT may regulate
SDF1-induced NO production and chemotaxis.
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-induced MAP
kinase activity
In this study, we also determined the effects of the NO
inhibitor TLCK, the NF-B inhibitor SN50, and the MEK kinase
inhibitor PD98059 on the SDF1-induced phosphorylation of Erk-1/2
protein by using phosphospecific (Thr202 of p44
and Tyr204 of p42) mAb and also estimated MAP
kinase activity as described in Materials and Methods. As
shown in Fig. 8, Aa
(upper panel) and 8Ab, TLCK pretreatment had no
significant effect (only a 10–15% inhibition) on SDF1-induced
Erk-1/2 phosphorylation. Equivalent amounts of Erk-1/2 protein were
present in each lane (Fig. 8Aa, lower panel).
Similar results were obtained with MAP kinase activity (data not
shown). Likewise, preincubation with SN50 did not inhibit
SDF1-induced Erk-1/2 phosphorylation (Fig. 8, Ba,
upper panel, and 8Bb) and activity (data not
shown) as compared with the control inhibitor SN50M. Equivalent amounts
of Erk-1/2 protein were present in each lane (Fig. 8Ba,
lower panel). However, with the MEK kinase inhibitor
PD98059, 60–70% inhibition of SDF1-induced Erk-1/2 phosphorylation
(Fig. 8, Ca, upper panel, and 8Cb) and
activity was observed (data not shown). Equivalent amounts of Erk-1/2
protein were present in each sample (Fig. 8Ca, lower
panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chemokine SDF1 and its cognate receptor CXCR4
have recently sparked substantial interest because of their role in
embryonic development, immune regulation, and HIV pathogenesis
(9, 10, 16, 17, 18, 19). These molecules regulate chemotaxis and
cell viability, which are important for various cellular functions such
as wound repair, metastasis, inflammation, angiogenesis, and
development of lymphoid tissue (55, 56, 57). We and others
have recently studied signaling molecules activated by SDF1
(23, 24, 25). These studies have shown activation of PI3
kinase and focal adhesion components such as RAFTK/Pyk2, paxillin, and
Crk in SDF1-induced signaling and in chemotaxis responses of pre-B
and T cells (23, 24, 25, 27). SDF1 was also shown to
activate the JAK/STAT pathway (58). Since the coordinated
regulation of several signaling components is required for mediating
migration, we have further explored the signaling mechanisms regulating
SDF1-induced chemotaxis.
NO, generated by NOS, is known to be both an intercellular and
intracellular signaling messenger involved in multiple cellular
functions. Pretreatment of Jurkat cells or PBLs with NO donors markedly
enhanced SDF1-induced chemotaxis, whereas NO inhibitors
significantly abrogated this cell migration. However, NO donors SNP and
PFC at higher concentrations had no or a lesser effect, respectively,
on this migration. This could be due to feedback inhibition of NOS,
since various NO donors have been shown to attenuate NOS activity in a
concentration-dependent manner (59, 60). These results
demonstrated that NO-linked signaling pathways mediate SDF1-induced
chemotaxis. NO has been shown to have opposing and complex effects on
chemotactic responses exerted by various stimuli. For example, it has
been shown that NO enhances chemotactic responses in some cell types
(45, 46, 61, 62, 63) upon certain stimuli, while inhibiting
cell migration in others (47, 64). Furthermore, NO has
been shown to modulate T cell chemotaxis in Peyer’s patches and in the
nonlymphoid region of the intestine (65). NO can exert its
effects on cell motility through the modulation of cytoskeletal
proteins and via cell matrix interactions (66, 67). The
cytoskeletal proteins paxillin, Crk, and RAFTK/Pyk2 are activated upon
SDF1 stimulation (23, 24, 25). NO also induces the tyrosine
nitration of the cytoskeletal protein p130cas, which is phosphorylated
by SDF1 (68).
We have recently shown that SDF1 induced NF-B activation in
CXCR4 transfectants (23). In this study, we have
demonstrated that NF-B inhibitors abrogate SDF1-induced
chemotactic effects. NO-related compounds have been shown to activate
NF-B activity (42). Moreover, the transcriptional
activity of NF-B has been shown to regulate iNOS gene expression
(68, 69). Furthermore, NF-B has been shown to play an
important role in cell migration when using various inhibitors that
block NF-B activation (70, 71, 72).
PI3 kinase has been shown to regulate SDF1-induced migration,
which in turn has been shown to activate AKT (protein kinase B). In the
present study, we have shown that SDF1 enhances AKT phosphorylation.
Activated AKT has recently been shown to phosphorylate eNOS and enhance
its activity, leading to NO production (37, 38, 39). In
addition, AKT was demonstrated to mediate NF-B activation (73, 74) induced by TNF and platelet-derived growth factor. AKT was
also shown to associate with IB kinase and to phosphorylate IB
kinase at threonine 23.
We have observed that the MAP kinase inhibitor PD98059 had no
effect on SDF1-induced chemotaxis of T cells, whereas at similar
concentrations it inhibited SDF1-induced MAP kinase activation by
60–70%. This is consistent with recent results showing the
SDF1-induced chemotactic activity of stem cells (75).
These results suggest that the MAP kinase pathway is not involved in
the chemotaxis induced by SDF1 in T cells. However, in some cell
types and under certain stimuli, MAP kinase signaling has been shown to
regulate cell motility (76).
Our results also indicate that NO is an upstream activator of
chemotactic response, but has no significant effect on MAP kinase
stimulation in CXCR4-mediated signaling pathways. Furthermore, we
observed that MAP kinase does not regulate NOS activity as PD98059 (a
specific inhibitor of p44/42 MAP kinase) at a 50 µM concentration had
no effect on NOS activity induced by SDF1 (data not shown). However,
NO has been shown to have complex and opposing effects on the MAP
kinase activation induced by various stimuli. For example, it has been
shown that NO has partial or no effect on MAP kinase activation under
certain conditions (77, 78), whereas it mediates this
activation in several other cell types (79, 80, 81). In
addition, PD98059 has been shown to inhibit the expression of NOS
(82, 83).
Taken together, our results suggest that NO and NF-B may
mediate SDF1-induced chemotactic activity, but not MAP kinase
activation in T cells. These findings provide new information at the
molecular level on the signal transduction pathways used by the
chemokine SDF1 and its receptor CXCR4 to regulate T cell migration,
an important event in both physiological and pathological
processes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ramesh K. Ganju, Harvard Institutes of Medicine-Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Room 343, Boston, MA 02115.
3 Abbreviations used in this paper: SDF1, stromal cell-derived factor 1; NOS, NO synthase; RAFTK, related adhesion focal tyrosine kinase; SNAP, S-nitroso-N-acetyl penicillamine; SNP, sodium nitroprusside; PFC, 4-phenyl-3-furoxancarbonitrite; TLCK, N-tosyl-lysine-chloromethylketone; L-NAME, NG-nitro-L-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine monoacetate; TPCK, N-tosyl-phenylalanine-chloromethylketone; MAP, mitogen-activated protein; eNOS, endothelial NOS; RIPA, radioimmunoprecipitation assay; Erk, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; MEK, MAP/Erk kinase.
Received for publication August 1, 2000. Accepted for publication December 20, 2000.
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  X. Pi, Y. Wu, J. E. Ferguson III, A. L. Portbury, and C. Patterson SDF-1{alpha} stimulates JNK3 activity via eNOS-dependent nitrosylation of MKP7 to enhance endothelial migration PNAS, April 7, 2009; 106(14): 5675 - 5680. [Abstract] [Full Text] [PDF]
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 Z. Sameermahmood, M. Balasubramanyam, T. Saravanan, and M. Rema Curcumin Modulates SDF-1{alpha}/CXCR4-Induced Migration of Human Retinal Endothelial Cells (HRECs) Invest. Ophthalmol. Vis. Sci., August 1, 2008; 49(8): 3305 - 3311. [Abstract] [Full Text] [PDF]
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 N. A. Shahabi, K. McAllen, and B. M. Sharp Stromal cell-derived factor 1-{alpha} (SDF)-induced human T cell chemotaxis becomes phosphoinositide 3-kinase (PI3K)-independent: role of PKC-{theta} J. Leukoc. Biol., March 1, 2008; 83(3): 663 - 671. [Abstract] [Full Text] [PDF]
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 X. Cui, J. Chen, A. Zacharek, Y. Li, C. Roberts, A. Kapke, S. Savant-Bhonsale, and M. Chopp Nitric Oxide Donor Upregulation of Stromal Cell-Derived Factor-1/Chemokine (CXC Motif) Receptor 4 Enhances Bone Marrow Stromal Cell Migration into Ischemic Brain After Stroke Stem Cells, November 1, 2007; 25(11): 2777 - 2785. [Abstract] [Full Text] [PDF]
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A. Prasad, Z. Qamri, J. Wu, and R. K. Ganju Slit-2/Robo-1 modulates the CXCL12/CXCR4-induced chemotaxis of T cells J. Leukoc. Biol., September 1, 2007; 82(3): 465 - 476. [Abstract] [Full Text] [PDF]
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 K. N. Kremer, A. Kumar, and K. E. Hedin Haplotype-Independent Costimulation of IL-10 Secretion by SDF-1/CXCL12 Proceeds via AP-1 Binding to the Human IL-10 Promoter J. Immunol., February 1, 2007; 178(3): 1581 - 1588. [Abstract] [Full Text] [PDF]
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Y. Zhang, M. Wittner, H. Bouamar, P. Jarrier, W. Vainchenker, and F. Louache Identification of CXCR4 as a New Nitric Oxide-Regulated Gene in Human CD34+ Cells Stem Cells, January 1, 2007; 25(1): 211 - 219. [Abstract] [Full Text] [PDF]
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 S. Goda, H. Inoue, H. Umehara, M. Miyaji, Y. Nagano, N. Harakawa, H. Imai, P. Lee, J. B. MaCarthy, T. Ikeo, et al. Matrix Metalloproteinase-1 Produced by Human CXCL12-Stimulated Natural Killer Cells Am. J. Pathol., August 1, 2006; 169(2): 445 - 458. [Abstract] [Full Text] [PDF]
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 S. Fukuda, H. E. Broxmeyer, and L. M. Pelus Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1{alpha}(CXCL12)/CXCR4 axis Blood, April 15, 2005; 105(8): 3117 - 3126. [Abstract] [Full Text] [PDF]
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 S.-B. Peng, V. Peek, Y. Zhai, D. C. Paul, Q. Lou, X. Xia, T. Eessalu, W. Kohn, and S. Tang Akt Activation, but not Extracellular Signal-Regulated Kinase Activation, Is Required for SDF-1{alpha}/CXCR4-Mediated Migration of Epitheloid Carcinoma Cells Mol. Cancer Res., April 1, 2005; 3(4): 227 - 236. [Abstract] [Full Text] [PDF]
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B.-C. Lee, T.-H. Lee, S. Avraham, and H. K. Avraham Involvement of the Chemokine Receptor CXCR4 and Its Ligand Stromal Cell-Derived Factor 1{alpha} in Breast Cancer Cell Migration Through Human Brain Microvascular Endothelial Cells Mol. Cancer Res., June 1, 2004; 2(6): 327 - 338. [Abstract] [Full Text] [PDF]
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A. Prasad, A. Z. Fernandis, Y. Rao, and R. K. Ganju Slit Protein-mediated Inhibition of CXCR4-induced Chemotactic and Chemoinvasive Signaling Pathways in Breast Cancer Cells J. Biol. Chem., March 5, 2004; 279(10): 9115 - 9124. [Abstract] [Full Text] [PDF]
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A. Z. Fernandis, R. P. Cherla, and R. K. Ganju Differential Regulation of CXCR4-mediated T-cell Chemotaxis and Mitogen-activated Protein Kinase Activation by the Membrane Tyrosine Phosphatase, CD45 J. Biol. Chem., March 7, 2003; 278(11): 9536 - 9543. [Abstract] [Full Text] [PDF]
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E. Y. Choe, E. S. Schoenberger, J. E. Groopman, and I.-W. Park HIV Nef Inhibits T Cell Migration J. Biol. Chem., November 22, 2002; 277(48): 46079 - 46084. [Abstract] [Full Text] [PDF]
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M. L. Giustizieri, C. Albanesi, C. Scarponi, O. De Pita, and G. Girolomoni Nitric Oxide Donors Suppress Chemokine Production by Keratinocytes in Vitro and in Vivo Am. J. Pathol., October 1, 2002; 161(4): 1409 - 1418. [Abstract] [Full Text] [PDF]
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 H.-S. Choi, P. R. Rai, H. W. Chu, C. Cool, and E. D. Chan Analysis of Nitric Oxide Synthase and Nitrotyrosine Expression in Human Pulmonary Tuberculosis Am. J. Respir. Crit. Care Med., July 15, 2002; 166(2): 178 - 186. [Abstract] [Full Text] [PDF]
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A. Z. Fernandis, R. P. Cherla, R. D. Chernock, and R. K. Ganju CXCR4/CCR5 Down-modulation and Chemotaxis Are Regulated by the Proteasome Pathway J. Biol. Chem., May 10, 2002; 277(20): 18111 - 18117. [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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N. C. Ottoson, J. T. Pribila, A. S. H. Chan, and Y. Shimizu Cutting Edge: T Cell Migration Regulated by CXCR4 Chemokine Receptor Signaling to ZAP-70 Tyrosine Kinase J. Immunol., August 15, 2001; 167(4): 1857 - 1861. [Abstract] [Full Text] [PDF]
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