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The Journal of Immunology, 2001, 166: 3067-3074.
Copyright © 2001 by The American Association of Immunologists

Stromal Cell-Derived Factor 1{alpha}-Induced Chemotaxis in T Cells Is Mediated by Nitric Oxide Signaling Pathways1

Rama P. Cherla and Ramesh K. Ganju2

Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stromal cell-derived factor 1{alpha} (SDF1{alpha}) 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{alpha}-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-{kappa}B in SDF1{alpha}-induced T cell migration. SDF1{alpha} 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{alpha}-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-{kappa}B, which is linked to NO signaling pathways, also significantly blocked the SDF1{alpha}-induced chemotactic response. However, these compounds did not have a significant effect on SDF1{alpha}-induced mitogen-activated protein kinase activity. In addition, the MAP/Erk kinase kinase inhibitor PD98059 did not abrogate SDF1{alpha}-induced chemotaxis. AKT, which has been shown to mediate NO production, was also phosphorylated upon SDF1{alpha} stimulation. These studies suggest that NO-related signaling pathways may mediate SDF1{alpha}-induced chemotaxis, but not mitogen-activated protein kinase activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemokines, which are a group of low molecular weight proteins, mediate several cellular functions. They play an important role in the regulation of hematopoiesis, leukocyte maturation, angiogenesis, trafficking, and homing of T and B lymphocytes and in the development of lymphoid tissue (1, 2, 3, 4, 5, 6). Based on the arrangement of their first two of four-conserved cysteine residues, the chemokine superfamily has been divided into four subfamilies: {alpha} (C-X-C), {beta} (C-C), {gamma} (C), and {delta} (C-X-X-X-C) (1, 2, 6).

Stromal cell-derived factor 1{alpha} (SDF1{alpha})3 is a member of the CXC or {alpha}-chemokine subfamily and is the only known ligand for the chemokine receptor CXCR4 (7, 8, 9, 10). SDF1{alpha} 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{alpha} and the CXCR4 receptor also regulate embryonic development (16, 17, 18, 19). Furthermore, knockout mice lacking CXCR4 or SDF1{alpha} 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{alpha} 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-{beta} (23, 24, 25). Furthermore, we also showed that mitogen-activated protein (MAP) kinase and NF-{kappa}B are activated upon SDF1{alpha} stimulation (23). Phosphatidylinositol 3-kinase (PI3K), which has been shown to regulate chemotaxis (26), is also involved in SDF1{alpha}-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-{kappa}B are key components of the SDF1{alpha}-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-{kappa}B is a major transcription factor that regulates transcription of genes encoding many inflammatory cytokines and cell adhesion molecules (41). NF-{kappa}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-{kappa}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{alpha} treatment activates NOS in Jurkat T cells. Furthermore, NO donors enhance SDF1{alpha}-induced chemotaxis, whereas NO inhibitors potently abrogate this chemotaxis. SDF1{alpha} 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and materials

The NO donors S-nitroso-N-acetyl penicillamine (SNAP), sodium nitroprusside (SNP), and 4-phenyl-3-furoxancarbonitrite (PFC); the protease inhibitor N{alpha}-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-{kappa}B inhibitors N{alpha}-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 {alpha}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-{kappa}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{alpha}. 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-{kappa}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{alpha} (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 [{gamma}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SDF1{alpha} enhances NOS activity

To elucidate the effect of SDF1{alpha} 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{alpha} 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. 1Go, SDF1{alpha} treatment resulted in an increase in total NOS activity. Maximum activity was obtained around the 3-h time period.



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  		FIGURE 1. Activation of NOS upon SDF1{alpha} stimulation of Jurkat cells. Cell lysates were obtained from untreated cells or cells stimulated with SDF1{alpha} (100 ng/ml) for the indicated times. The samples were assayed for NOS activity by measuring the release of [3H]L-citrulline as described in Materials and Methods. The activity of the unstimulated samples was subtracted from the total activity obtained from the SDF1{alpha}-induced samples. The results presented in the graph are representative of three experiments.

 
NO donors enhance SDF1{alpha}-mediated chemotaxis

To explore the role of NO in SDF1{alpha}-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. 2Go, NO donors significantly enhanced chemotaxis in a concentration-dependent manner. There was about a 60% increase in migration with SNAP at 1000 nM (Fig. 2GoA), a 75% increase with SNP at 0.1 µM (Fig. 2GoB), and a 70% increase with PFC at 0.1 µM (Fig. 2GoC) 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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  		FIGURE 2. NO donors enhance the SDF1{alpha}-induced chemotaxis of Jurkat cells. Cells were preincubated with different concentrations of the NO donors SNAP (A), SNP (B), or PFC (C) or the appropriate solvent as a control for 30–60 min and then subjected to chemotactic assay in the presence of SDF1{alpha} (50 ng/ml) as described in Materials and Methods. *, Results that are significantly different from the control: A, p < 0.05; B, p < 0.01; and C, p < 0.001.

 
NO inhibitors attenuate SDF1{alpha}-induced chemotaxis

The role of NO in SDF1{alpha}-induced chemotaxis was further confirmed by using various inhibitors. As shown in Fig. 3Go, 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{alpha}-induced chemotaxis in a dose-dependent manner. L-NMMA pretreatment resulted in a 60–70% inhibition at 0.1–1.0 mM (Fig. 3GoA), whereas L-NAME at 2 mM inhibited by 40% the chemotactic response induced by SDF1{alpha} (Fig. 3GoB). Maximum inhibition (90%) was achieved with TLCK, at 100 µM (Fig. 3GoC). These NO donor and inhibitor studies revealed that NO is an important mediator of chemotactic responses induced by the {alpha} chemokine SDF1{alpha} 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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  		FIGURE 3. NO inhibitors abrogate SDF1{alpha}-induced migration. Jurkat cells were pretreated with various NO inhibitors such as L-NMMA (A), L-NAME (B), or TLCK (C) for 60 min. Next, the cells were subjected to chemotactic assay in the presence of SDF1{alpha} (50 ng/ml) as described in Materials and Methods. *, Results that are significantly different from the control: A, p < 0.005; B, p < 0.05; and C, p < 0.005.

 
NF-{kappa}B inhibitors inhibit SDF1{alpha}-induced chemotaxis

We have recently shown that SDF1{alpha} activates NF-{kappa}B (23). Furthermore, NOS expression has been shown to be regulated by NF-{kappa}B. To determine the role of NF-{kappa}B in SDF1{alpha}-induced chemotaxis, we used the NF-{kappa}B chemical inhibitor TPCK and the peptide inhibitor SN50. These inhibitors markedly attenuated SDF1{alpha}-induced chemotactic effects in a concentration-dependent manner (Fig. 4Go). About 80% inhibition was achieved with 100 µM TPCK as compared with the methanol control (Fig. 4GoA). Similarly, 90% inhibition was obtained with the SN50 peptide (100 µg/ml). A mutant peptide, SN50M, which does not affect NF-{kappa}B activity under similar conditions, had no significant effects on SDF1{alpha}-induced chemotaxis (Fig. 4GoB). SN50 is a cell permeable peptide inhibitor containing the nuclear localization sequence of the NF-{kappa}B component p50, which inhibits translocation of NF-{kappa}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-{kappa}B may regulate SDF1{alpha}-induced chemotaxis.



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  		FIGURE 4. NF-{kappa}B inhibitors block migration induced by SDF1{alpha}. The NF-{kappa}B chemical inhibitor TPCK (A) or peptide inhibitor SN50 (B) was incubated with cells for 30–60 min. An appropriate solvent was used as the control for TPCK and SN50M (a mutated peptide) was used as the control for SN50. Cells were subjected to chemotactic assay for 3 h in the presence of SDF1{alpha} (50 ng/ml) as described in Materials and Methods. *, Results that are significantly different from the control: A, p < 0.005 and B, p < 0.005.

 
MAP kinase inhibitor has no effect on SDF1{alpha}-induced chemotaxis

In previous studies, we have shown that besides chemotactic activity, SDF1{alpha} 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{alpha}-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{alpha} was then determined. As shown in Fig. 5Go, PD98059 at various concentrations did not inhibit SDF1{alpha}-induced migration. These studies suggest that the MAP kinase signaling pathway is not involved in the chemotaxis induced by SDF1{alpha} in T cells.



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  		FIGURE 5. MEK kinase inhibitor PD98059 does not inhibit SDF1{alpha}-induced chemotaxis. Jurkat cells were pretreated with various concentrations of the MEK kinase inhibitor PD98059, or DMSO as a solvent control, for 60 min. Cells were subjected to chemotactic assay in the presence of SDF1{alpha} (50 ng/ml).

 
Effect of various compounds on the SDF1{alpha}-induced chemotaxis of activated PBLs

We also confirmed the effect of various compounds on the SDF1{alpha}-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. 6GoA, 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{alpha}-induced chemotaxis (Fig. 6Go, B–D). TLCK inhibited chemotaxis by 90–95% at a 10 µM concentration (Fig. 6GoB). Similarly, the NF-{kappa}B inhibitor TPCK (10 µM; Fig. 6GoC) or SN50 (100 µg/ml; Fig. 6GoD) inhibited ~90–95% of the PBL chemotaxis induced by SDF1{alpha} as compared with the control-treated samples. However, the MEK kinase inhibitor PD98059 (Fig. 6GoE) did not have any effect on SDF1{alpha}-induced chemotaxis at a 10 or 50 µM concentration. These studies further confirmed that NO-linked signaling pathways participate in SDF1{alpha}-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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  		FIGURE 6. Effect of various compounds on SDF1{alpha}-induced chemotaxis in PBLs. Activated PBLs were pretreated with various compounds such as SNP (A), TLCK (B), TPCK (C), SN50 (D), or PD98059 (E). Cells pretreated for 30–60 min with the appropriate solvents or peptide (SN50M) were used as controls. The cells were subjected to chemotaxis in the presence of SDF1{alpha} (50 ng/ml) for 1.5–3 h. *, Results that are significantly different from the control: A, p < 0.01 and B–D, p < 0.005.

 
SDF1{alpha} 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. 7Go, stimulation with SDF1{alpha} increased the phosphorylation of AKT in Jurkat cells (Fig. 7GoA) and PBLs (Fig. 7GoB) 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. 7Go, bottom panels). These results suggest that AKT may regulate SDF1{alpha}-induced NO production and chemotaxis.



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  		FIGURE 7. SDF1{alpha} induces phosphorylation of AKT in Jurkat cells and PBLs. Cell lysates (50 µg) obtained from Jurkat cells (A) or PBLs (B), unstimulated (0) or stimulated for varying time periods with 100 ng/ml SDF1{alpha}, were run on SDS-PAGE and immunoblotted with anti-phospho-AKT Ab (upper panels). The blots were stripped and reblotted with AKT Ab (bottom panels).

 
NO inhibitors have no significant effect on SDF1{alpha}-induced MAP kinase activity

In this study, we also determined the effects of the NO inhibitor TLCK, the NF-{kappa}B inhibitor SN50, and the MEK kinase inhibitor PD98059 on the SDF1{alpha}-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. 8Go, Aa (upper panel) and 8Ab, TLCK pretreatment had no significant effect (only a 10–15% inhibition) on SDF1{alpha}-induced Erk-1/2 phosphorylation. Equivalent amounts of Erk-1/2 protein were present in each lane (Fig. 8GoAa, lower panel). Similar results were obtained with MAP kinase activity (data not shown). Likewise, preincubation with SN50 did not inhibit SDF1{alpha}-induced Erk-1/2 phosphorylation (Fig. 8Go, 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. 8GoBa, lower panel). However, with the MEK kinase inhibitor PD98059, 60–70% inhibition of SDF1{alpha}-induced Erk-1/2 phosphorylation (Fig. 8Go, 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. 8GoCa, lower panel).



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  		FIGURE 8. TLCK or SN50 does not significantly inhibit SDF1{alpha}-induced MAP kinase activity. Jurkat cells were pretreated with the NO inhibitor TLCK (Aa and Ab), NF-{kappa}B inhibitor SN50 (Ba and Bb), or the MEK kinase inhibitor PD98059 (Ca and Cb) for 60 min and then stimulated with SDF-1{alpha} (100 ng/ml) for the indicated time periods. Cell lysates (50 µg), unstimulated (0) or stimulated with SDF1{alpha} for various time periods, were separated on an 8% SDS-PAGE gel, transferred onto nitrocellulose membranes, and probed with phosphospecific anti-p44/42 MAP kinase (P-Erk-1/2) Ab (Aa–Ca, upper panels). The same blots were stripped and reprobed with anti-p44/42 MAP kinase (Erk-1/2) (Aa–Ca, bottom panels). The OD values obtained after densitometric scanning of the P-Erk-1/2 bands are presented as bar graphs (Ab–Cb). The above results are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The {alpha} chemokine SDF1{alpha} 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{alpha} (23, 24, 25). These studies have shown activation of PI3 kinase and focal adhesion components such as RAFTK/Pyk2, paxillin, and Crk in SDF1{alpha}-induced signaling and in chemotaxis responses of pre-B and T cells (23, 24, 25, 27). SDF1{alpha} 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{alpha}-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{alpha}-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{alpha}-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{alpha} stimulation (23, 24, 25). NO also induces the tyrosine nitration of the cytoskeletal protein p130cas, which is phosphorylated by SDF1{alpha} (68).

We have recently shown that SDF1{alpha} induced NF-{kappa}B activation in CXCR4 transfectants (23). In this study, we have demonstrated that NF-{kappa}B inhibitors abrogate SDF1{alpha}-induced chemotactic effects. NO-related compounds have been shown to activate NF-{kappa}B activity (42). Moreover, the transcriptional activity of NF-{kappa}B has been shown to regulate iNOS gene expression (68, 69). Furthermore, NF-{kappa}B has been shown to play an important role in cell migration when using various inhibitors that block NF-{kappa}B activation (70, 71, 72).

PI3 kinase has been shown to regulate SDF1{alpha}-induced migration, which in turn has been shown to activate AKT (protein kinase B). In the present study, we have shown that SDF1{alpha} 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-{kappa}B activation (73, 74) induced by TNF and platelet-derived growth factor. AKT was also shown to associate with I{kappa}B kinase and to phosphorylate I{kappa}B kinase at threonine 23.

We have observed that the MAP kinase inhibitor PD98059 had no effect on SDF1{alpha}-induced chemotaxis of T cells, whereas at similar concentrations it inhibited SDF1{alpha}-induced MAP kinase activation by 60–70%. This is consistent with recent results showing the SDF1{alpha}-induced chemotactic activity of stem cells (75). These results suggest that the MAP kinase pathway is not involved in the chemotaxis induced by SDF1{alpha} 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{alpha} (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-{kappa}B may mediate SDF1{alpha}-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 {alpha} chemokine SDF1{alpha} and its receptor CXCR4 to regulate T cell migration, an important event in both physiological and pathological processes.


   Acknowledgments
 
We are grateful to Dr. Jerome E. Groopman for his advice and support for our research project. We thank Janet Delahanty for editing this manuscript, Daniel Kelley for preparation of the figures, and Simone Jadusingh for facilitating receipt of the reagents for the experiments.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant CA76950 (to R.K.G). Back

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. Back

3 Abbreviations used in this paper: SDF1{alpha}, stromal cell-derived factor 1{alpha}; 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{alpha}-tosyl-lysine-chloromethylketone; L-NAME, NG-nitro-L-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine monoacetate; TPCK, N{alpha}-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. Back

Received for publication August 1, 2000. Accepted for publication December 20, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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