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* Center for Molecular Medicine and Therapeutics, British Columbia Research Institute for Children’s and Women’s Health, and Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada; and
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
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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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SDF-1-mediated activation of CXCR4 results in increased
phosphorylation of focal adhesion components, activation and
phosphorylation of phosphatidylinositol-3-kinase (PI3K), and increased
activity of the NF-
B transcription factor (8). PI3K
activation generates the membrane bound second messengers,
phosphatidylinositol-3,4-bisphosphate (PI(3, 4)P2) and
phosphatidylinositol-3,4,5-trisphosphate (PI(3, 4, 5)P3), that recruit
and activate cytosolic proteins containing pleckstrin homology (PH)
domains, such as members of the family of serine/threonine kinases that
includes protein kinase B (PKB). Studies using either PI3K inhibitors
or activated and dominant negative mutants of PI3K have indicated that
the PI3K pathway is critical to SDF-1-induced chemotaxis
(9, 10, 11). Furthermore, SDF-1 is able to induce sustained
signaling and to promote prolonged activation of downstream effectors
such as PKB and mitogen-activated protein kinase (MAPK; Ref.
11).
Given the importance of SDF-1-induced PI3K activation, it is noteworthy that the tumor suppressor gene, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), has been shown to down-regulate this signaling pathway (12). In addition to sharing a catalytic signature motif with dual-specificity phosphatases, PTEN is capable of dephosphorylating the 3' position of PI(3, 4, 5)P3 and PI(3, 4)P2. This lipid phosphatase activity appears to account for the tumor suppressor effect of PTEN (13). For example, by modulating phosphatidylinositol levels, PTEN can negatively regulate PKB-dependent cell survival signals (14). Also, mice heterozygous for Pten spontaneously develop malignancies and show resistance to proapoptotic stimuli (15, 16, 17, 18). In addition to cancer susceptibility, Pten+/- mice develop a nonmalignant lymphoproliferative disorder that is accompanied by autoimmune features (16, 19) similar to that of mice expressing a constitutively active form of PI3K (20). This suggests that hyperresponsiveness of the PI3K signaling pathway to external stimuli might contribute to the lymphoproliferative disorder in Pten+/- mice. In support of this hypothesis, expression of active PKB in a T cell transgenic model system disturbed both T and B cell homeostasis and resulted in an inflammatory/autoimmune phenotype that closely resembled that seen in the Pten+/- mice (21). T cell-specific loss of Pten leads to activation of PKB and secondarily to defects in T cell homeostasis (22), suggesting that Pten, through its ability to regulate the PI3K/PKB pathway, is important in controlling lymphoid activation and development.
Several lines of evidence have suggested a role for PTEN in the control of cell movement. For example, while PTEN overexpression inhibited fibroblast motility and directional movement through effects on focal adhesion kinase (FAK; Refs. 23, 24, 25), murine embryonic fibroblasts lacking Pten exhibited increased cell motility, an effect attributed to Rac1 and Cdc42 dysregulation (26). Consistent with the importance of PI3K and lipid second messengers in directed migration, increased chemotaxis was seen in both thymic and splenic hematopoietic cells obtained from mice lacking Src homology 2 domain containing inositol phosphatase (SHIP), a lipid phosphatase that specifically targets the 5' position of PI(3, 4, 5)P3 (27).
We hypothesized that Pten levels might regulate chemokine-dependent cell migration. To examine this possibility, we studied the migratory response of Pten+/- B cells to SDF-1 in vitro. Purified B cells from Pten heterozygous mice demonstrated an augmented sensitivity to this chemokine that was accompanied by an increase in SDF-1-dependent chemotaxis. Thus, PTEN protein levels may be an important factor in the regulation of chemokine-dependent events in lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SDF-1 (residues 1–67) was a generous gift from I. Clark-Lewis (University of British Columbia, Vancouver, Canada). Abs with the following specificities were used: PTEN (6H2.1; A. G. Scientific, San Diego, CA), phospho-specific MAPK, MAPK, phospho-PKB (Ser473), and PKB (from Cell Signaling Technology, Ontario, Canada).
Cell preparations and migration assays
Spleens were isolated from 8-wk-old Pten+/- mice and sex-matched littermate controls. All animal work was approved by the Canadian Council of Animal Care. To isolate B cells, single-cell suspensions of splenocytes were first incubated with RBC lysis buffer and then washed twice in cold HBSS. T cells were depleted from the cell suspension by two separate incubations with monoclonal anti-Thy-1.2 (HO13.4), anti-CD4 (2B6), and anti-CD8 (3.155) Abs together with low endotoxin rabbit complement (Cedarlane, Hornby, Canada) for 45 min at 37°C. The remaining B cells were then isolated by Percoll density centrifugation as previously described (28). This cell population consisted of 80–94% CD19+ cells as determined by flow cytometry. For chemotaxis experiments, purified B lymphocytes were resuspended in RPMI (without serum) at 1 x 107 cells/ml. Migration assays were conducted using Transwell polycarbonate membranes (Corning-Costar, Cambridge, MA) as previously described (29).
For assessment of chemotaxis in the presence of PI3K inhibitors, 100 nM wortmannin was added to both the upper and lower chambers and the migration response compared with that seen without the inhibitor. For assessment of chemokinesis vs chemotaxis, migration was tested in both the presence and absence of an SDF-1 gradient. To create a situation with no SDF-1 gradient, cells placed in the upper chamber were resuspended in the same SDF-1 solution used for the lower chamber and then migration was allowed to proceed for 3 h.
Immunoblotting and densitometry
Primary B lymphocytes were resuspended in RPMI (without serum) at 1 x 108 cells/ml and stimulated with 50 µg/ml SDF-1. Immunoblotting was conducted as described (29) with 25–50 µg of total protein loaded per lane. Densitometry was conducted on between two to five immunoblots derived from independent experiments. The values obtained for the phospho-specific blots were expressed as a percentage of the density of the protein loading control. Protein loading controls were done in parallel on each of the samples using Abs specific for the nonphosphorylated forms of the proteins. For each experiment, values were normalized and results represent relative values of phosphorylation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the effect of Pten heterozygosity on cell
migration, we examined the response of primary B cells to SDF-1. In
addition to showing a characteristic bell-shaped response to SDF-1
stimulation (Fig. 1
a), it was
apparent that an increased number of
Pten+/- cells had migrated to the lower
chamber, at concentrations ranging between 500 and 2000 ng/ml, as
compared with control cells. The peak chemotactic response occurred at
1500 ng/ml SDF-1 for the Pten+/- B cells,
whereas the wild-type cells responded maximally at doses between 2000
and 3000 ng/ml (Fig. 1a). These results suggested that
Pten+/- B cells had an altered response
threshold to SDF-1.
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and Fig. 1
b). At this
concentration, Pten+/- B cells showed a
2.5-fold (p < 0.01) greater chemotactic
response. Pten+/- B cells also displayed
elevated chemokinesis compared with controls, showing a statistically
significant (2-fold) increase in their ability to move to the lower
chamber in the absence of SDF-1. To demonstrate that the increased
migration of Pten+/- cells at 1500 ng/ml
of SDF-1 was due to chemotaxis, as opposed to chemokinesis, migration
in the presence or absence of an SDF-1 gradient was assessed. The
contribution of chemokinesis, as assessed by migration in the absence
of an SDF-1 gradient, was small as compared with the maximal migratory
response (Fig. 1c). However,
Pten+/- B cells did show a small but
statistically significant increase in chemokinetic movement
(p < 0.03) as well as the previously observed
increase in migration due to chemotaxis. Taken together, these results
suggest that Pten+/- leads to an
alteration in the migratory response of B cells to SDF-1.
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Pten heterozygosity could lead to
differences in lymphocyte migration owing to a reduction of levels of
Pten, an important negative regulator of PI3K-mediated signal
transduction pathways. To confirm that
Pten+/- mice did in fact show a reduced
amount of Pten protein in primary B lymphocytes in vivo, immunoblot
analyses on purified populations of primary lymphocytes were conducted
(Fig. 2 and data not shown). Lymphocytes
from Pten+/- mice revealed an 50%
reduction in Pten as compared with lymphocytes from wild-type
littermate controls. Protein levels of Pten have been shown to be
regulated by phosphorylation and protein degradation (30, 31) as well as by promoter methylation (32). Thus,
it was important to confirm that Pten levels did not change throughout
the time course of SDF-1 stimulation. For all time points, Pten levels
in Pten+/- cells remained at about half
those of wild-type controls (data not shown).
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To investigate a potential mechanism by which Pten
heterozygosity leads to enhanced chemotaxis in B lymphocytes, we
examined two signaling events lying downstream of SDF-1, PKB, and MAPK
phosphorylation. Chemotaxis was abrogated when 1500 ng/ml SDF-1 was
added to B cells in the presence of 100 nM wortmannin, indicating that
the chemotactic response of both Pten+/-
and control B cells was PI3K-dependent (Fig. 1b).
Hypothesizing that differences in the activation of the PI3K pathway
might account for the differential response of the
Pten+/- and control B lymphocytes to
SDF-1, we examined PKB phosphorylation, an event reflective of PI3K
activation. It has been demonstrated that PKB phosphorylation at
Ser473 correlates with the kinase activity in
vitro (14, 33, 34). We observed an increased level
phosphorylation of PKB on Ser473 upon stimulation
of Pten+/- B cells with 50 µg/ml SDF-1,
compared with controls (Fig. 3a). This SDF-1 concentration
was selected so that differences in intracellular signaling events
occurring within minutes of SDF-1 stimulation could be readily
evaluated. An alteration in the kinetics of PKB phosphorylation was
also evident in Pten+/- B cells, which
showed a prolonged phosphorylation response to SDF-1 as compared with
wild-type cells. To quantitate the differential response of
Pten+/- and control cells to SDF-1,
densitometric analysis of multiple independent experiments was
conducted (Fig. 3b).
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a. Densitometric analyses
from multiple experiments revealed that
Pten+/- cells did not exhibit any
significant differences in phosphorylation of MAPK as compared with
wild-type cells (Fig. 4b). These results support the idea
that the differential response of Pten+/-
B lymphocytes to SDF-1 is linked to dysregulation of PI3K-dependent
events, including PKB phosphorylation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SDF-1 is known to activate multiple signal transduction pathways
including receptor-associated trimeric G proteins, phospholipase C
and protein kinase C, PI3K and PKB, small G proteins, and specific
protein tyrosine kinase pathways (8, 9, 10, 35).
SDF-1-induced chemotaxis is a complex and coordinated phenomenon that
involves polarized localization and activation of signaling molecules
such as PKB (36). These events in turn lead to increased
intracellular Ca2+ levels, cytoskeletal
reorganization, and ultimately, the chemotactic response of the cell.
Tyrosine phosphorylation of multiple focal adhesion proteins such as
RAFTK/Pyk2, p130Cas, Paxillin, FAK, CrkL, and Crk is also seen
following SDF-1 stimulation (37). Human PTEN has been
implicated in the dephosphorylation of some of these intermediates,
such as FAK and p130Cas, suggesting a role for the protein tyrosine
phosphatase domain of PTEN in the regulation of integrin-mediated cell
adhesion (24, 25). In addition to the activation of
protein kinase signaling pathways, SDF-1 also causes an accumulation of
PI(3, 4, 5)P3 in the membrane and SDF-1-induced chemotaxis is fully
dependent on PI3K activation (38). Because Pten has been
implicated as an important negative regulator of the PI3K pathway, the
link between SDF-1 and Pten could be through dysregulation of signaling
pathways dependent on PI(3, 4, 5)P3 and PI(3, 4)P2 in
Pten+/- cells.
Using specific inhibitors, it has been shown that the PI3K pathway, but not the MAPK pathway, is required for both SDF-1-induced phosphorylation of focal adhesion proteins and SDF-1-induced migration (37). Also, consistent with the importance of PI(3, 4, 5)P3 levels in chemokine dependent migration, lymphocytes lacking the 5' inositol phosphatase Src homology 2 containing inositol phosphatase showed increased responsiveness to SDF-1 and other chemokines (27). These results provided a further rationale for the hypothesis that Pten is capable of regulating SDF-1-induced events.
The chemotaxis of Pten+/- B lymphocytes that we observed indicated that reduced Pten levels were associated with an increased responsiveness to SDF-1, implicating Pten in the control of lymphocyte migration to this chemokine. In Pten+/- B cells, Pten levels were about half those of wild-type controls. We hypothesized that the reduced levels of Pten were accompanied by a decrease in Pten activity. Other studies support the hypothesis that Pten heterozygosity leads to haploinsufficiency (39).
We found that phosphorylation of MAPK remained unchanged. Although there is some debate as to whether Pten is able to regulate MAPK phosphorylation (40, 41, 42, 43, 44), we observed no reproducible difference in MAPK phosphorylation in Pten+/- B lymphocytes exposed to SDF-1. Taken together, our results suggest that the differential response of Pten+/- B lymphocytes to SDF-1 is linked to dysregulation of PI3K dependent events, including PKB phosphorylation.
The PKB PH domain rapidly and transiently translocates to the plasma membrane upon chemoattractant stimulation and is found at the leading edge in migrating cells (36). It has been proposed that activation of PI3K at the leading edge leads to the formation of PI(3, 4, 5)P3- and PI(3, 4)P2-enriched lipid domains that function as docking sites for diverse PH domain containing proteins. This results in a clustering of these signaling proteins, which leads to the formation of a new pseudopod and directed cellular movement (45, 46). In addition to this pivotal role at the leading edge of cells, PKB has also been implicated in the regulation of chemotaxis through its ability to cause the phosphorylation of G protein-coupled receptors (47). Although the increased phosphorylation of PKB in Pten+/- cells suggests a plausible mechanism for the enhanced response of these cells to SDF-1, it is possible that other PH domain containing proteins are also involved.
Pten heterozygosity does not appear to initially compromise normal lymphoid development, as reflected in the normal ratios of CD4+, CD8+, and B220+ lymphocytes in the lymph nodes of young Pten+/- mice (16). However, over time a progressive lymphoproliferative disorder develops in these mice (16). If Pten+/- lymphoid cells were abnormally sensitive to chemokines in vivo, it is plausible that the aberrant recruitment and accumulation of lymphoid cells would contribute to the lymphoproliferative disorder of Pten+/- mice.
It has been well-established that activation of PKB-dependent signaling pathways promote cell growth and survival (48). A complete lack of functional Pten, due to biallelic gene mutations that are frequently seen in tumors and tumor cell lines, and the resulting constitutive activation of PKB is thought to be a major contributing factor in tumorigenesis. In contrast, our observations suggest that even a reduction in Pten protein levels accompanying the loss of a single Pten allele might be able to confer a selective advantage on cells during multistep tumorigenesis. In keeping with the importance of this, chemokines have been implicated in tumor cell growth, angiogenesis, and metastasis (49); and recently, the SDF-1:CXCR4 axis has been shown to be involved in breast cancer metastasis (50). This latter study and others have found that CXCR4 is overexpressed in specific cancer cell lines and tumor samples as compared with normal tissues (50, 51, 52). Also, SDF-1 expression is high in tissues that are often targets for metastatic cells such as lung, liver, and lymph nodes (50). Pten+/- mice have been shown to develop malignant lymphomas that produce metastatic infiltrates of solid organs including the lung and the liver (16, 18, 19). It has also been shown that neutralization of CXCR4 in vivo inhibits tumor cell metastasis, suggesting that SDF-1 acts as an important attractive force in metastasis (50). Thus, our finding that Pten+/- cells have enhanced responsiveness to SDF-1 suggests a mechanism by which loss of a single Pten allele would be under selective pressure. For example, Pten+/- cells might have an increased ability for directed migration toward favorable microenvironments, thus promoting tumor progression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frank R. Jirik, Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive Northwest, Calgary, Alberta, Canada T2N 4N1. E-mail address: jirik{at}ucalgary.ca
3 Abbreviations used in this paper: SDF-1, stromal cell derived factor-1; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; MAPK, mitogen-activated protein kinase; PI(3,4)P2, phosphatidylinositol-3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol-3,4,5-triphosphate; PH, pleckstrin homology; PTEN, phosphatase and tensin homolog deleted on chromosome 10; FAK, focal adhesion kinase.
Received for publication September 25, 2001. Accepted for publication April 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chemokine, stromal cell-derived factor-1, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J. Biol. Chem. 273:23169.-induced lymphocyte polarization and chemotaxis. J. Immunol. 163:4001.B activation, and Bcl-xL levels in vivo. J. Exp. Med. 191:1721. triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB J. 13:1699. stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C. Blood 95:2505.This article has been cited by other articles:
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  D. J. J. de Gorter, R. M. Reijmers, E. A. Beuling, H. P. H. Naber, A. Kuil, M. J. Kersten, S. T. Pals, and M. Spaargaren The small GTPase Ral mediates SDF-1-induced migration of B cells and multiple myeloma cells Blood, April 1, 2008; 111(7): 3364 - 3372. [Abstract] [Full Text] [PDF]
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 S. Basu, N. T. Ray, S. J. Atkinson, and H. E. Broxmeyer Protein Phosphatase 2A Plays an Important Role in Stromal Cell-Derived Factor-1/CXC Chemokine Ligand 12-Mediated Migration and Adhesion of CD34+ Cells J. Immunol., September 1, 2007; 179(5): 3075 - 3085. [Abstract] [Full Text] [PDF]
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P. Gao, R. L. Wange, N. Zhang, J. J. Oppenheim, and O. M. Z. Howard Negative regulation of CXCR4-mediated chemotaxis by the lipid phosphatase activity of tumor suppressor PTEN Blood, October 15, 2005; 106(8): 2619 - 2626. [Abstract] [Full Text] [PDF]
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Y. Le, M. Honczarenko, A. M. Glodek, D. K. Ho, and L. E. Silberstein CXC Chemokine Ligand 12-Induced Focal Adhesion Kinase Activation and Segregation into Membrane Domains Is Modulated by Regulator of G Protein Signaling 1 in Pro-B Cells J. Immunol., March 1, 2005; 174(5): 2582 - 2590. [Abstract] [Full Text] [PDF]
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A. V. Gulino, D. Moratto, S. Sozzani, P. Cavadini, K. Otero, L. Tassone, L. Imberti, S. Pirovano, L. D. Notarangelo, R. Soresina, et al. Altered leukocyte response to CXCL12 in patients with warts hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome Blood, July 15, 2004; 104(2): 444 - 452. [Abstract] [Full Text] [PDF]
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D. G. Cronshaw, C. Owen, Z. Brown, and S. G. Ward Activation of Phosphoinositide 3-Kinases by the CCR4 Ligand Macrophage-Derived Chemokine Is a Dispensable Signal for T Lymphocyte Chemotaxis J. Immunol., June 15, 2004; 172(12): 7761 - 7770. [Abstract] [Full Text] [PDF]
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J. L. Moody, L. Xu, C. D. Helgason, and F. R. Jirik Anemia, thrombocytopenia, leukocytosis, extramedullary hematopoiesis, and impaired progenitor function in Pten+/-SHIP-/- mice: a novel model of myelodysplasia Blood, June 15, 2004; 103(12): 4503 - 4510. [Abstract] [Full Text] [PDF]
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J. M. Dyson, A. D. Munday, A. M. Kong, R. D. Huysmans, M. Matzaris, M. J. Layton, H. H. Nandurkar, M. C. Berndt, and C. A. Mitchell SHIP-2 forms a tetrameric complex with filamin, actin, and GPIb-IX-V: localization of SHIP-2 to the activated platelet actin cytoskeleton Blood, August 1, 2003; 102(3): 940 - 948. [Abstract] [Full Text] [PDF]
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