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The Journal of Immunology, 2007, 179, 3075 -3085
Copyright © 2007 by The American Association of Immunologists, Inc.
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Protein Phosphatase 2A Plays an Important Role in Stromal Cell-Derived Factor-1/CXC Chemokine Ligand 12-Mediated Migration and Adhesion of CD34+ Cells1

Sunanda Basu2,*,{dagger},§, Nicole T. Ray{ddagger}, Simon J. Atkinson{ddagger} and Hal E. Broxmeyer*,{dagger},{ddagger},§

* Department of Microbiology and Immunology, {dagger} Walther Oncology Center, and {ddagger} Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202; and § Walther Cancer Institute, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Migration of hemopoietic stem and progenitor cells (HSPC) is required for homing to bone marrow following transplantation. Therefore, it is critical to understand signals underlying directional movement of HSPC. Stromal cell-derived factor-1 (SDF-1)/CXCL12 is a potent chemoattractant for HSPC. In this study, we demonstrate that the serine-threonine protein phosphatase (PP)2A plays an important role in regulation of optimal level and duration of Akt/protein kinase B activation (a molecule important for efficient chemotaxis), in response to SDF-1. Inhibition of PP2A, using various pharmacological inhibitors of PP2A including okadaic acid (OA) as well as using genetic approaches including dominant-negative PP2A-catalytic subunit (PP2A-C) or PP2A-C small interfering RNA, in primary CD34+ cord blood (CB) cells led to reduced chemotaxis. This was associated with impairment in polarization and slower speed of movement in response to SDF-1. Concomitantly, SDF-1-induced Akt phosphorylation was robust and prolonged. Following SDF-1 stimulation, Akt and PP2A-C translocate to plasma membrane with enhanced association of PP2A-C with Akt observed at the plasma membrane. Inhibition of PI3K by low-dose LY294002 partially recovered chemotactic activity of cells pretreated with OA. In addition to chemotaxis, adhesion of CD34+ cells to fibronectin was impaired by OA pretreatment. Our study demonstrates PP2A plays an important role in chemotaxis and adhesion of CD34+ CB cells in response to SDF-1. CD34+ CB cells pretreated with OA showed impaired ability to repopulate NOD-SCID mice in vivo, suggesting physiological relevance of these observations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although cord blood (CB)3 contains significant numbers of hemopoietic stem and progenitor cells (HSPC), its applicability in HSPC transplantation remains largely for use in children (1). Enhanced homing of CB HSPC has the potential to increase applicability of CB transplantation in adults (2). Stromal cell-derived factor-1 (SDF-1)/CXCL12 is an important player in HSPC homing and engraftment (3, 4, 5). SDF-1 binds to CXCR4, stimulating a series of intracellular events downstream of this G protein-coupled receptor (GPCR) (6, 7, 8). Major processes regulated by the SDF-1-CXCR4 axis in vivo are highlighted in SDF-1–/– and CXCR4–/– mice (9, 10, 11). The importance of SDF-1 in homing and engraftment of HSPC is demonstrated by immunoimaging experiments that suggest that specific microvascular domains in bone marrow express high levels of E-selectin and SDF-1; in vivo colocalization studies confirmed that HSPC were restricted to these vessel beds (12). Because chemotaxis is indispensable to homing, understanding molecular mechanisms underlying SDF-1-directed chemotaxis of HSPC is crucial for designing more efficient HSPC transplantation protocols.

Initiation of chemotaxis requires binding of a chemoattractant to its receptor, which results in activation of the PI3K pathway. Activation of PI3K leads to generation of phosphatidylinositol (3, 4) biphosphate and phosphatidylinositol (3, 4, 5) triphosphate that recruit various pleckstrin homology (PH) domain-containing proteins, including Akt (13, 14, 15). Recruitment of PH domain-containing proteins, which bind phosphoinositides on the membrane, and their regulation, is a key event, downstream of G protein activation, leading to pseudopodia production (16). Although activation of Akt is critical for efficient cell movement, it is transient and highly regulated (14).

A balance of kinases and phosphatases is necessary for cells to maintain appropriate transducing signal levels (17). In Dictyostelium, in addition to PI3K, phosphatase and tensin homologue (PTEN) determines level and duration of phosphatidylinositol (3, 4, 5) triphosphate production (18), which in turn regulates duration of association of PH domains with the membrane. PTEN, therefore, indirectly regulates activity of various PH domain-containing proteins, including Akt, whose activity is tightly regulated during chemotaxis (14). Although PI3K and its downstream target, Akt, have been shown to play an important role in chemotaxis of mammalian leukocytes (19, 20, 21), the complete regulation of this pathway is not yet clearly understood. For example, there are conflicting reports on the role of PTEN in chemotaxis of mammalian leukocytes (22, 23). However, in addition to PTEN, Akt activation is also regulated by protein phosphatase (PP)2A (24). PP2A is a multimeric serine-threonine phosphatase that is highly conserved in eukaryotes (25). It is a holoenzyme consisting of a heterotrimer of 65-kDa structural subunit A, 55- to 130-kDa regulatory subunits B, and 36-kDa catalytic subunit C. A major opponent of phosphoinositol-dependent kinase 1 in regulation of Akt/protein kinase B (PKB) is PP2A (26). Akt/PKB is inactivated by PP2A in vitro (24) and is stimulated in cells upon treatment with okadaic acid (OA), an inhibitor of serine-threonine phosphatases (27).

In this study, we investigated a role for PP2A in SDF-1-mediated responses of primary human CD34+ CB cells, a population enriched for HSPC. Because limited numbers of cells are obtained from each CB collection and CD34+ cells are present at low frequency in CB, it is difficult to get enough of these rare cells to do in-depth molecular analysis of intracellular signaling with them. Most such studies have been done with established cell lines, casting doubt as to the relevance of such information in terms of primary cell function. Thus, it is extremely important that these studies be done in primary cells. For these reasons, we chose to evaluate an intracellular signaling role for PP2A in SDF-1-mediated chemotaxis and adhesion in primary CD34+ CB cells. To compensate for low numbers of primary CD34+ cells, we expanded these cells ex vivo, with maintenance of cellular function, and used them for intracellular biochemical analysis. Our study elucidates a role for a previously unrecognized intracellular signaling molecule that mediates SDF-1-dependent cell migration and adhesion in primary CD34+ CB cells. Inhibition of PP2A in primary CD34+ cells isolated from umbilical CB led to prolonged SDF-1-induced Akt phosphorylation; this was associated with impaired chemotaxis and SDF-1-stimulated adhesion of CD34+ cells to fibronectin, suggesting PP2A plays an important role in SDF-1-mediated functional responses of CD34+ CB cells.


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

The following Abs were used for cell staining: FITC-conjugated CD34 (Miltenyi Biotec), PE-conjugated CXCR4, and allophycocyanin-conjugated CD38 (BD Pharmingen). For confocal microscopy, anti-PP2A-catalytic subunit (PP2A-C) (Upstate Biotechnology/Chemicon International) and anti-CXCR4 Ab (Santa Cruz Biotechnology) were used. Phalloidin rhodamine was purchased from Invitrogen Life Technologies. Abs used for Western blot were anti-phospho-ERK1/2 mouse mAb, ERK1/ERK2 rabbit polyclonal Ab, anti-phospho-Akt (Ser473 and Thr308) rabbit Ab and anti-Akt rabbit polyclonal Ab, anti-phospho-glycogen synthase kinase (GSK)3 Ab (Cell Signaling Technology), and anti-GSK3 (Santa Cruz Biotechnology). Secondary Abs were peroxidase-labeled anti-mouse IgG and anti-rabbit IgG HRP-linked Abs (Amersham Biosciences). OA was purchased from Upstate Biotechnology, and GSK3 inhibitor IX, inhibitor-2, and Fostriecein were purchased from Calbiochem. BSA was purchased from Sigma-Aldrich. Human rSDF-1 was purchased from BioVision.

Cells

Human CB was obtained after informed consent with institutional review board approval, and CD34+ cells were isolated, as described previously (28).

Expansion and pretreatment of CD34+ CB cells

Enriched CD34+ cells were expanded using 100 ng/ml recombinant human thrombopoietin, 100 ng/ml human rFlt-3 ligand, and 50 ng/ml recombinant human stem cell factor, as described previously (28). Freshly isolated or cytokine-expanded CD34+ cells were pretreated with OA or medium alone (IMDM plus 2% BSA) for 40 min, washed, and used. In experiments evaluating the effect of PI3K inhibitor LY294002 on inhibition of chemotaxis by OA, cells were pretreated with the inhibitor(s) and then used directly in chemotaxis assay.

PP2A activity

PP2A activity of OA-pretreated or untreated CD34+ cell lysates was measured using specific phosphopeptide and buffering conditions, as per manufacturer’s instructions (Promega).

Transfection of CD34+ cells with small interfering RNA (siRNA) and plasmid

We used siRNA SMARTpool PP2A-C, {alpha} isoform from Upstate Biotechnology/Chemicon International for silencing {alpha} PP2A-C subunit in freshly isolated CD34+ CB cells. Transfection was done by electroporation using the Nucleofection System (Amaxa), according to the manufacturer’s instructions. Cells were transfected with 2 µg of oligonucleotides with control siRNA (Qiagen) or PP2A-C siRNA under identical conditions and cultured for 40 h. Control siRNA was labeled with fluorescein and used to monitor transfection efficiency. In cells transfected with control siRNA, >90% of cells were found to be fluorescein positive by flow cytometer, 4 h posttransfection. For the first 20 h, cells were kept in mixture of cytokines described above. For the next 20 h, cytokine concentrations were halved to slow cell proliferation. After 40 h, cells were analyzed for chemotaxis. Protein expression was studied using Western blot. Plasmids encoding wild-type (WT) PP2A-C or dominant-negative mutant of PP2A-C (DNPP2A-C; provided by Dr. B. A. Hemmings, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) or constitutively active Akt (CAAkt) (29) and corresponding vector alone (pcDNA) (a gift from Dr. H. Nakshatri, Indiana University School of Medicine, Indianapolis, IN) were cotransfected with plasmid-encoding GFP (pmaxGFP; Amaxa) in pooled freshly isolated CD34+ cells using Amaxa Nucleofection kit. A total of 2–3 µg of each plasmid (pcDNA and pcDNACAAkt) and 0.75 µg of GFP encoding plasmid was used per transfection. Following transfection, cells were cultured for 20 h in medium containing above mixture of cytokines. After 20 h, cells were sorted on the basis of GFP expression; some of the sorted cells from each transfection were used for chemotaxis assay, and the rest of the cells were lysed and analyzed by Western blot.

Migration assay

Chemotaxis assays were performed using 24-well chemotaxis chambers, pore size 5.0 µM (Corning Costar). CD34+ cells in 100 µl of chemotaxis medium (IMDM plus 1% BSA) were added to the membrane. Chemotaxis medium alone (600 µl) or containing 200 ng/ml SDF-1 was added to bottom well. After 4 h at 37°C in 5% CO2, cells migrated to the lower chamber and input cells were counted for 30 s using FACScan under identical flow conditions. In a few experiments, SDF-1 was added to both the upper and lower well, and the movement of cells was assessed, as described above.

Time-lapse video microscopy and motility analysis using Dunn chamber

Motility of CD34+ cells was recorded using Dunn chamber (30). Glass coverslips were coated with Retronectin (human fibronectin fragment CH296; Takara Shuzo) and blocked using 2% BSA. CD34+ cells were allowed to adhere for 15 min at 37°C. Coverslips were mounted on the Dunn chamber with a gradient of 0–200 ng/ml SDF-1 between the inner and outer wells of the chamber. Cell positions were tracked using particle-tracking capabilities in Metamorph 6.1 software (Universal Imaging). Length and breadth of cells were measured using Photoshop (Adobe Systems). Ratio of length to breadth expresses extent of polarization of cells in response to SDF-1.

Progenitor cell colony assay

Methylcellulose progenitor assay was performed, as described (31).

Adhesion assays

Adhesion assay was performed, as described (28).

Confocal microscopy

Effect of SDF-1 stimulation on subcellular localization of Akt/pAkt (Thr308) was studied using Zeiss LSM510 confocal microscope using x63 objective lens and 5x or 2x digital zoom. Cytokine-expanded CD34+ cells were stimulated with SDF-1 (200 ng/ml) for various lengths of time, permeabilized with formalin, and fixed using methanol. The cells were then stained with either Akt or pAkt (Thr308) Ab (Cell Signaling Technology), washed, and stained with anti-rabbit Alexa 488 Ab (Molecular Probes). Then cells were stained with PP2A-C Ab (Upstate Biotechnology), washed, and stained with anti-rabbit Alexa 647 Ab. Finally, cells were stained with 4',6-diamidino-2-phenylindole.

Western blot analysis and coimmunoprecipitation

Whole cell protein lysates were prepared, and Western blot was done, as described (28). For coimmunoprecipitation, cells were treated, washed with PBS, and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, and 2 mM EDTA) containing protease inhibitor mixture (Roche), sodium fluoride, and sodium vanadate. Immunoprecipitation was done using anti-PP2A-C or anti-Akt Abs using catch and release kit (Chemicon International). Proteins in the immunocomplex were resolved on 8% SDS-PAGE.

Mice

NOD-SCID mice were bred and maintained in ventilated sterile microisolator cages. Experiments were approved by Animal Ethics Committee of Indiana University. Eight-week-old mice were given a sublethal dose of 300 cGy from a cobalt source 4 h before transplantation. Freshly isolated CD34+ CB cells were preincubated with OA or medium alone for 40 min and washed twice with PBS. Cells were then injected i.v. Mice were sacrificed after 30–35 days (32). Bone marrow cells were flushed from femurs, and percentages of human cells were determined by staining cells with anti-human CD45-allophycocyanin Ab (BD Pharmingen). Various blood cell lineages were determined with anti-human CD34-FITC, CD38-FITC, CD33-PE, and CD19-FITC Abs (BD Pharmingen).

Statistical analysis

Data are represented as mean ± SD, unless indicated otherwise. Student’s two-tailed t test was used for statistical analysis. Level of significance is indicated by p value.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Due to limited numbers of CD34+ cells present in CB collections for intracellular biochemical analysis of signal transduction events, we expanded these cells ex vivo using thrombopoietin, Flt-3 ligand, and stem cell factor. After 4 days of expansion, cells still remain essentially CD34+ (28). To verify intact cellular responses, effects on chemotaxis and adhesion were performed with freshly isolated and cytokine-expanded CD34+ cells.

Effects of OA on CD34+ CB cells

OA is a cell-permeable molecule that inhibits PP2A in vitro at 100-fold lower concentrations than PP1 (33). Binding of OA to PP2A-C is tight and withstands a lysis process (34). OA has a 50% inhibitory concentration of 1 nM in cell-free systems; however, much higher concentrations are required to observe its effects using intact cells. This is due to relatively inefficient transport of this molecule into the cell and higher concentrations of PP2A in some cells (26, 33, 35). Concentrations of OA up to 1 µM preferentially inhibit PP2A enzymatic activity in cells, without detectable intracellular inhibitory effects on PP1, PP2B, or PP2C (26). As seen in Fig. 1A, PP2A activity in the soluble fraction of lysates from CD34+ cells pretreated with various concentrations of OA was reduced by 60–70% with 100 nM OA, with further reductions at 1 µM. Viability of CD34+ cells was unaffected by OA pretreatment when cells were cultured in medium containing 20% FCS without or with SDF-1 (52.0 ± 3.2% and 73.6 ± 2.0% for medium vs 51 ± 3% and 61.5 ± 2% for OA, without and with SDF-1, respectively). Similarly, after 3 days in culture in 2% BSA, no significant difference in survival of CD34+ cells, cultured in the absence or presence of SDF-1, was observed between OA-pretreated and control CD34+ cells (Fig. 1B, i and ii).


Figure 1
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  		FIGURE 1. OA decreases PP2A activity without affecting viability of CD34+ cells. A, Effect of OA on PP2A activity in freshly isolated CD34+ cells. Results from a representative experiment in which PP2A-C enzymatic activity in CD34+ cells pooled from three CB collections was determined, as described in Materials and Methods. B, Viability of freshly isolated CD34+ cells pretreated either with medium (i) or OA (ii) 1 µM alone cultured in IMDM containing 2% BSA alone ({square}) or along with SDF-1 (200 ng/ml) ({blacksquare}). Viable cell counts were enumerated using trypan blue. Independent wells were used for each day cell count. Data are from one of two independent experiments.

 
Effect of PP2A inhibition on chemotaxis of CD34+ cells toward SDF-1

Pretreatment with OA inhibited chemotactic response of both freshly isolated (Fig. 2Ai) and cytokine-expanded (Fig. 2Aii) CD34+ cells to SDF-1. The effect was dose dependent: chemotactic activity was reduced by 50% with 100 nM OA, and reduced up to 90% with 1 µM OA (Fig. 2B). OA pretreatment affected SDF-1-directed chemotaxis without effects on chemokinesis of the CD34+ cells. In the absence of a net SDF-1 gradient, motility of the cells pretreated with OA was comparable to background chemotaxis. As additional controls for effects of PP2A, we evaluated fostriecin and inhibitor-2, other serine-threonine phosphatase inhibitors, on chemotaxis of CD34+ cells. Fostriecin, an antitumor antibiotic, is a highly selective inhibitor of PP2A and inhibits PP2A at 10,000–40,000 times lower concentrations than that required for inhibition of PP1 in vitro (36, 37). Fostriecin, when used at 5 µM, inhibited chemotaxis of CD34+ cells toward SDF-1 to a level comparable to that observed with 100 nM OA. In contrast, inhibitor-2, a selective inhibitor against PP1 in vitro and in vivo (38, 39), did not affect chemotaxis of CD34+ cells at concentrations up to 2 µM (Fig. 2B). These findings suggested that PP2A plays a role in SDF-1-directed chemotaxis of CD34+ CB cells.


Figure 2
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  		FIGURE 2. Inhibition of PP2A impairs chemotaxis of CD34+ cells toward SDF-1. A, Chemotaxis of freshly isolated (i) or cytokine-expanded (ii) CD34+ cells pretreated with medium alone or OA toward SDF-1 (200 ng/ml). Data represent mean ± SD of three independent experiments. B, Effect of pretreatment with various serine-threonine phosphatase inhibitors on chemotaxis of freshly isolated CD34+ cells toward SDF-1. Data represent mean ± SD of three independent experiments. C, Effect of DNPP2A-C (H118N) on chemotaxis of CD34+ cells. CD34+ cells pooled after purification from six CB collections were transfected with either DNPP2A-C (H118N) or WT PP2A-C, and the chemotaxis of the transfected cells toward 200 ng/ml SDF-1 was evaluated 20 h after transfection. Data represent mean ± SD of one of the two experiments performed. D, Effect of PP2A-C siRNA on PP2A-C protein level (i) and chemotaxis of CD34+ cells (ii). CD34+ cells pooled after purification from three CB collections were transfected with either control siRNA or siRNA for PP2A-C subunit. Western blot analyses of lysates prepared from a part of the cultures were blotted with PP2A-C, PP1, and actin Abs to evaluate effect and specificity of si-RNA transfection. Chemotaxis of remaining cells was studied using transwell assay. Data represent mean ± SD of triplicates. E, Effect of OA pretreatment on composition of colony-forming cells (CFCs) in the migrated cell population (i) and on CFCs in the input cell population (ii). Results shown are from one of the two experiments performed.

 
To extend our above noted findings that used pharmacological inhibitors to more specific inhibition of PP2A, we transfected freshly isolated CD34+ CB cells with WT PP2A-C or a DNPP2A-C (H118N) (40). Cells transfected with DNPP2A-C (H118N) demonstrated 70% decrease in migration to SDF-1 compared with cells transfected with WT PP2A-C (Fig. 2C). We also evaluated CD34+ CB cells transfected with control siRNA or PP2A-catalyic subunit (PP2A-C) siRNA. Forty hours after transfection, expression of PP2A-C was reduced by 40% (Fig. 2Di). This was accompanied by significantly reduced (35 ± 0.02%) chemotaxis of CD34+ CB cells toward SDF-1 (Fig. 2Dii). PP1 was unaffected by PP2A-C siRNA transfection (Fig. 2Di), demonstrating the specificity of PP2A-C siRNA. These findings confirm our observations using pharmacological inhibitors, and clearly establish that specific inhibition of PP2A impairs migration of CD34+ CB cells to SDF-1.

CD34+ cells are enriched, but not purified for HSPC. We, therefore, also assessed whether OA had any effects on SDF-1-induced migration of progenitor cells in CD34+ populations, as determined by colony assays for erythroid, multipotential, and granulocyte-macrophage progenitor cells. SDF-1-induced chemotaxis of CD34+ cells was significantly decreased (Fig. 2B) by OA pretreatment, but its inhibitory effects were similar for the different progenitor cells because it did not change the ratio of various progenitors in the migrated cell populations (Fig. 2Ei). As seen in Fig. 2Eii, OA did not affect the numbers or ratio of the input progenitor cell populations. Similarly, the proportion of phenotypically defined immature CD34+/CD38–/low and mature CD34+/CD38high progenitors that migrated in response to SDF-1 was unaffected by OA pretreatment (medium, 88.97 and 7.89% vs OA, 88.92 and 8.59%). Thus, the inhibitory effect of OA on SDF-1-induced chemotaxis is similar for primitive and more mature progenitors, by both phenotypic and functional analysis.

Effect of OA on adhesion of CD34+ cells to fibronectin

Homing requires integrin-mediated adhesion interactions of HSPC with endothelium. In addition, adhesion to stroma is required for proper engraftment and retention of HSPC in bone marrow (41). SDF-1-mediated adhesion of freshly isolated (Fig. 3i) and cytokine-expanded (Fig. 3ii) CD34+ cells to fibronectin was significantly reduced by pretreatment of cells with OA.


Figure 3
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  		FIGURE 3. Effect of OA on adhesion of CD34+ CB cells on fibronectin. Effect of OA (1 µM) pretreatment on SDF-1-stimulated adhesion of freshly isolated (i) and cytokine-expanded (ii) CD34+ cells to fibronectin. Results were normalized against adhesion of CD34+ cells pretreated with medium alone. Data represent mean ± SD of three independent experiments.

 
OA impairs ability of CD34+ cells to polarize

Chemoattractant regulation of actin polymerization is a major event in remodeling of cytoskeleton and is the driving force behind directed cell movement (42). SDF-1-induced F-actin polymerization in CD34+ cells was unaffected by OA pretreatment of fresh and expanded cells (data not shown). To understand why OA-pretreated CD34+ cells do not migrate efficiently toward SDF-1 despite being able to undergo SDF-1-induced F-actin polymerization as efficiently as untreated CD34+ cells, we evaluated the ability of cells to polarize in response to SDF-1. Motility experiments using Dunn chamber demonstrated that OA pretreatment impaired the ability of the cells to polarize in response to SDF-1 (Fig. 4, A and B). The speed of movement was also reduced when cells were pretreated with OA (Fig. 4C). To further confirm that inhibition of PP2A was responsible for reduced speed of movement of CD34+ cells toward SDF-1, CD34+ cells transfected with either WT PP2A-C or DNPP2A-C were evaluated for their chemotactic activity using Dunn chamber. Similar to OA-pretreated cells, CD34+ cells transfected with DNPP2A-C also exhibited reduced speed of movement compared with cells transfected with WT PP2A-C (Fig. 4D).


Figure 4
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  		FIGURE 4. OA pretreatment impairs SDF-1-induced polarization and speed of movement. Freshly isolated CD34+ cells pretreated with OA or medium alone or CD34+ cells transfected with WT PP2A-C or DNPP2A-C were loaded on Dunn’s chamber, and movement of cells was recorded by time-lapse video microscopy. Frames were taken at 10-s interval. A, Morphology of cells migrating in response to SDF-1 at indicated time. B, Polarization ability of cells was measured as change in shape of cell as determined by length to breadth ratio; 100 cells were examined for each treatment condition. Data are one representative of three experiments performed. C, The speed of movement of CD34+ cells (D) and CD34+ cells transfected with WT PP2A-C or DNPP2A-C (H118N) was analyzed using Metamorph software.

 
OA results in prolonged SDF-1-induced Akt phosphorylation

Because pretreatment of CD34+ cells with OA results in reduced chemotactic activity without influencing expression of CXCR4, we evaluated intracellular signaling events. SDF-1 induces Akt and ERK phosphorylation (7, 28). Both pathways have been implicated in chemotaxis (20, 28). Stimulation of CD34+ cells with SDF-1 enhanced phosphorylation of Akt at Ser473 and Thr308 (Fig. 5Ai). To measure the net change in phosphorylation of Akt in response to SDF-1 stimulation, we calculated the ratio of phosphorylated Akt to total Akt for each time point and then determined the fold change in phosphorylation following SDF-1 stimulation in relation to baseline values (Fig. 5A, ii and iii). Although SDF-1-induced phosphorylation of Akt at Ser473 appeared to be elevated more in OA-pretreated compared with control cells (Fig. 5A, i and ii), due to large intersample variability, the difference (both the fold change and kinetics) did not attain statistical significance. Akt phosphorylation at Thr308 was significantly higher and more prolonged in cells pretreated with OA (Fig. 5A, i and iii). At 10 min post-SDF-1 stimulation, pAkt (Thr308) returned to near basal level in control cells, whereas the level of pAkt (Thr308) was significantly elevated and nearly 1.5-fold higher than basal level in OA-pretreated cells. SDF-1 stimulation of OA-pretreated and untreated CD34+ cells led to enhanced ERK1/2 phosphorylation that peaked at 3 min post-SDF-1 stimulation and thereafter declined (Fig. 5Bi). OA pretreatment did not significantly alter the time-related enhancement of SDF-1-induced ERK1/2 phosphorylation (Fig. 5Bii), although ERK1/2 phosphorylation at baseline was more robust in OA-pretreated cells.


Figure 5
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  		FIGURE 5. Effect of OA pretreatment on SDF-1-induced activation of intracellular signaling. Western blot analysis of Ai phospho-Akt (Thr308 and Ser473) in lysates prepared from cytokine-expanded CD34+ cells. Cytokine-expanded CD34+ cells were harvested, washed, and kept in medium containing 2% BSA (growth factor starvation) for 30 min and then pretreated with OA (1.0 µM) or medium alone for 40 min, following which cells were stimulated with SDF-1 (200 ng/ml). Densitometric analysis of fold change in ratio of Aii, pAkt (Ser473) and Aiii, pAkt (Thr308) over baseline following SDF-1 stimulation is shown. Data represent mean ± SD of three independent experiments. *, p < 0.005 compared with control cells stimulated with SDF-1 for 10 min. Bi, Phospho-ERK1/2 in lysates prepared from cytokine-expanded CD34+ cells pretreated with OA (1.0 µM) or medium alone for 40 min and then stimulated with SDF-1 (200 ng/ml). Data are representative of one of three experiments performed. Bii, Densitometric analysis of relative increase in phospho-ERK1/2 over baseline following SDF-1 stimulation, represented as mean ± SD of three independent experiments. Ratio of phospho-ERK1/2 to total protein for each time point was calculated, and changes in phospho-ERK1/ERK2 level were expressed relative to baseline values.

 
Interaction of Akt with PP2A and β-arrestin-2 following SDF-1 stimulation

The importance of transient Akt activation for effective chemotaxis has been demonstrated in Dictyostelium (14). Because OA pretreatment of CD34+ cells resulted in impaired chemotaxis and prolonged Akt phosphorylation, we explored the effect of sustained activation of Akt on chemotaxis of CD34+ cells toward SDF-1. CD34+ cells were cotransfected with plasmids expressing CAAkt (or control vector), and GFP-CD34+ cells expressing CAAkt were identified by their GFP expression (Fig. 6Ai). Approximately 35–40% of cells were transfected. Twenty hours after transfection, GFP+ cells from both cultures (CD34+ cells transfected with control vector and CAAkt) were sorted and analyzed for chemotaxis. Overexpression of CAAkt in CD34+ cells (Fig. 6Aii) resulted in 49% inhibition of chemotaxis of CD34+ cells toward SDF-1 (Fig. 6Aiii). CAAkt expression did not affect chemokinesis or background movement of CD34+ cells (results not shown).


Figure 6
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  		FIGURE 6. Expression of CAAkt impairs chemotaxis to SDF-1, and association of Akt with PP2A is enhanced following SDF-1 stimulation. A, Expression of CAAkt impairs chemotaxis of CD34+ cells. Freshly isolated CD34+ cells pooled from six CB collections were cotransfected with plasmid-encoding GFP (pmaxGFP) and pcDNA (control vector) or pcDNACAAkt. Cells expressing GFP were sorted using FACSsort and analyzed for SDF-1-induced chemotaxis. Ai, Dot-blot representation of input and migrated CD34+ cells from a chemotaxis assay performed using GFP+ cells. Aii, Level of CAAkt expression, phospho-GSK, and total GSK in GFP+-sorted fraction of cells, as determined using Western blot. Aiii, Chemotactic activity of GFP+ fraction of CD34+ cells cotransfected with plasmids expressing GFP (pmaxGPF) and CAAkt or pcDNA (vector). Data represent mean ± SD of triplicates. B, Coimmunoprecipitation of Akt from pooled cytokine-expanded CD34+ cells (three to four CB collections). Analysis of Akt interaction with PP2A-C, GSK-3{alpha}, GSK-3β, and β-arrestin-2, as determined by immunoblotting with specific Abs. Shown is one representative of three experiments performed. C, Coimmunoprecipitation of PP2A-C in cytokine-expanded CD34+ cells from three to four CB collections. One representative experiment of three experiments is shown. D, Localization of Akt and PP2A-C in cytokine-expanded CD34+ CB cells before and after SDF-1 (200 ng/ml) stimulation. Cells were stained with 4',6'-diamidino-2-phenylindole (a–e), anti-PP2A-C, and anti-Akt, or anti-phospho-Akt (f) (Thr308) Abs. Images (a, b, d, e, and f) were taken using x63 optical magnification and 5x zoom. Image (c) was taken using x63 optical magnification and 2x zoom, and d shows the higher magnification image of the cell marked by square in c.

 
Serine-Threonine protein phosphatases negatively regulate Akt activity in various cellular systems (27, 43). Because OA pretreatment resulted in significantly greater and more prolonged Akt phosphorylation at Thr308 in response to SDF-1 stimulation, we considered it likely that inhibition of serine-threonine phosphatase by OA affected Akt dephosphorylation. To better understand regulation of Akt by PP2A, we performed coimmunoprecipitation experiments on cytokine-expanded CD34+ cells, because small numbers of CD34+ cells in a CB collection make it almost impossible to perform coimmunoprecipitation experiments using freshly isolated CD34+ cells. Coimmunoprecipitation experiments demonstrated that Akt and PP2A-C exist in a complex in CD34+ cells, and SDF-1 stimulation increases the amount of PP2A-C associated with Akt (Fig. 6, B and C). PP1 was not associated with Akt, in either unstimulated cells or in cells stimulated with SDF-1 (data not shown). In agreement with other reports (14, 15), Akt (Fig. 6D, a–e) and phosphorylated Akt (Thr308) (Fig. 6Df) translocated to the plasma membrane of CD34+ cells following SDF-1 stimulation. PP2A-C also translocated to the plasma membrane upon SDF-1 stimulation. These latter findings extend our coimmunoprecipitation observations and suggest that SDF-1-enhanced association of Akt with PP2A-C occurs primarily at the plasma membrane.

In addition to a classical role in desensitization of GPCR signaling, β-arrestin-2 also acts as a scaffolding protein, linking GPCR to a second wave of cell signaling via different signaling pathways (44, 45). β-arrestin-2 was found in the Akt-PP2A complex after, but not before, SDF-1 stimulation (Fig. 6B).

Inhibition of PI3K leads to partial recovery of chemotaxis in OA-pretreated CD34+ cells

Because enhanced and prolonged Akt phophorylation is at least in part responsible for inhibition of chemotaxis of CD34+ cells, we reasoned that inhibition of Akt phosphorylation in OA-pretreated cells may partially recover their chemotactic activity. Akt phosphorylation is positively regulated by PI3K and is negatively regulated by PP2A (24). To test whether reduction of Akt phosphorylation in OA-pretreated cells indeed recovers their chemotactic activity toward SDF-1, we evaluated effects of PI3K inhibition on SDF-1-directed chemotaxis. Pretreatment of CD34+ cells with LY294002, a specific inhibitor of PI3K (46), resulted in reduced chemotactic response of CD34+ CB cells toward SDF-1. However, it indeed led to partial recovery of chemotactic activity in OA-pretreated CD34+ CB cells (Fig. 7A). This effect was observed only when low-dose (2 µM) LY294002 was used. Pretreatment of CD34+ cells with low-dose LY294002 along with OA cells also led to a reduction in Akt phosphorylation at both Thr308 and Ser473 compared with cells pretreated with OA alone following SDF-1 stimulation (Fig. 7B). These findings demonstrate that PI3K is stimulated in these cells following SDF-1 stimulation and PP2A activity is required to regulate optimal levels and duration of Akt phosphorylation.


Figure 7
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  		FIGURE 7. Low-dose PI3K inhibitor partially recovers chemotactic activity of OA-pretreated cells. A, Chemotaxis of cytokine-expanded isolated CD34+ cells pretreated with LY294002 (2 µM) alone, OA (1 µM) alone, or with both LY294002 (2 µM) and OA (1 µM). Results represent mean ± SD of three independent experiments. B, Western blot analysis of lysates of CD34+ cells untreated or pretreated with OA or OA along with LY294002 and stimulated with SDF-1 (200 ng/ml) for 3 min from one of the experiments for which chemotaxis assay was performed. C, Phosphorylation of GSK3 in response to SDF-1 stimulation in untreated or OA-pretreated cells. One of three experiments performed is shown. D, Chemotaxis of CD34+ cells untreated or pretreated with GSK3 inhibitor IX was evaluated in in vitro Transwell assay. Result represents mean ± SD of three independent experiments.

 
Inhibition of GSK3 leads to reduced SDF-1-directed chemotactic activity

To determine how prolonged Akt phosphorylation might result in reduced chemotactic activity, we assessed the activation status of its substrates GSK3{alpha} and GSK3β. In addition to its important role in metabolism and proliferation (47), GSK3 plays an important role in cell migration (48). GSK3 activity is negatively regulated by phosphorylation at serine residues (49). PP2A has been reported to directly dephosphorylate GSK3β purified from rabbit skeletal muscle (50). Consistent with this report (50), we found that inhibition of PP2A by OA pretreatment led to increased level of GSK3 phosphorylation, particularly at 1 µM OA, and this was further enhanced upon SDF-1 stimulation (Fig. 7C). Because PP2A directly regulates GSK3 activity (51) and also dephosphorylates Akt (24, 50), we evaluated whether activated Akt in SDF-1-stimulated OA-pretreated cells played a role in GSK3 phosphorylation. Pretreatment of cells with 2 µM LY294002, even in the presence of 1 µM OA (Fig. 7B), resulted in decreased Akt phosphorylation with concomitant reduction in GSK3 phosphorylation at Ser21/9 sites in response to SDF-1. This demonstrates that inhibition of Akt phosphorylation leads to activation of GSK3 following SDF-1 stimulation, and is consistent with our observation that GSK3 associates with Akt in CD34+ cells upon SDF-1 stimulation (Fig. 7B). Moreover, in CD34+ cells transfected with CAAkt, although the direct interaction of CAAkt with GSK3 could not be evaluated due to limited numbers of CD34+ cells, GSK3 phosphorylation was enhanced in cells transfected with CAAkt. This further suggested that activated Akt associates with GSK3 and phosphorylates it (Fig. 6Aii). Unlike the interaction of GSK3 with Akt, which was only observed upon SDF-1 stimulation, interaction of GSK3 with PP2A-C was detected in both SDF-1-unstimulated and -stimulated CD34+ cells (Fig. 6C). Pretreatment of CD34+ CB cells with the specific GSK3 inhibitor, GSK3 inhibitor IX, significantly decreased SDF-1-directed chemotaxis (Fig. 7D) without affecting cell viability (data not shown), confirming that GSK3 plays a role in SDF-1-directed chemotaxis. Thus, Akt interacts with GSK3 in CD34+ cells following SDF-1 stimulation, and inhibition of GSK3 activity impairs chemotaxis of CD34+ cells.

OA pretreatment reduces engraftment of CD34+ cells in NOD-SCID mice

SDF-1 plays an important role in migration (homing) and retention of HSPC in bone marrow (3, 4, 12). NOD-SCID mice are a useful model to evaluate the engrafting properties and stem cell characteristics of human CD34+ cells (52). Consistent with our in vitro findings, inhibition of PP2A by OA significantly reduced engraftment of CD34+ cells in NOD-SCID mice (Fig. 8A, i and ii), without impairing the ability of transplanted cells to give rise to different cell lineages (Fig. 8B). This suggests an involvement of PP2A in the in vivo engraftment of human CD34+ CB cells in NOD-SCID mice.


Figure 8
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  		FIGURE 8. OA pretreatment impairs engraftment of CD34+ cells in NOD-SCID mice. A, Percentage of human leukocytes in the bone marrow of NOD-SCID mice 35 days after injection of untreated or OA (1.0 µM)-pretreated CD34+ cells. Two separate experiments are shown in which 1 x 105 (i) and 5 x 104 (ii) CD34+ CB cells were injected. B, The ability of injected CD34+ cells to give rise to different lineages was unaffected by OA pretreatment as determined by staining of bone marrow cells with Abs against various cell surface markers.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
SDF-1 is the most potent known chemoattractant for both human and mouse HSPC. Homing and engraftment of mouse and human HSPC are SDF-1 dependent (2, 3, 4, 5, 12). However, much remains to be determined regarding the intracellular signaling molecules responsible for directional movement of HSPC toward SDF-1. Our report not only directly implicates PP2A in this effect through both pharmacological inhibitors and more specific PP2A inhibition using genetic approaches, but also elucidates the intracellular molecules involved in this regulation. Most importantly, the cellular and intracellular studies were done with primary cells highly enriched for HSPC. Such intracellular signaling molecule interactions have rarely, if ever, been previously studied with these primary cells because of their rareness and limiting numbers. We have now shown that PP2A is an important player in SDF-1-mediated chemotaxis and adhesion of human HPSC in vitro. Inhibition of PP2A impairs ability of CD34+ cells to polarize in response to SDF-1. PP2A activity following SDF-1 stimulation is required for maintaining appropriate levels and duration of Akt phosphorylation for effective chemotaxis.

Inhibition of SDF-1-mediated chemotaxis of CD34+ cells was dependent on OA concentration. Concentrations of OA up to 1 µM can be used to selectively inhibit PP2A without detectable intracellular inhibitory effect on PP1, PP2B, or PP2C (26). In addition to OA, by using fostriecin, a more specific PP2A inhibitor, and DNPP2A-C as well as PP2A-C siRNA, we confirmed that inhibition of PP2A impairs chemotaxis of CD34+ cells toward SDF-1, and that this was specific for PP2A. Neither PP2A-C siRNA nor DNPP2A-C affected either CXCR4 or integrin molecule expression on CD34+ cells (data not shown).

Inhibition of SDF-1-mediated chemotaxis and adhesion by OA pretreatment was not due to differential changes in CXCR4 expression upon SDF-1 stimulation (data not shown). However, Akt phosphorylation at Thr308 was more robust and prolonged in OA-pretreated cells stimulated with SDF-1 and most likely a reason for impaired SDF-1-mediated chemotaxis upon OA pretreatment. Expression of CAAkt impairs chemotaxis in Dictyostelium (14); the same occurred when we expressed CAAkt (an Akt variant lacking its PH domain that becomes constitutively active on membrane targeting via the addition of the 14-aa src myristoylation signal (29, 53) in CD34+ cells). In addition to sustained Akt phosphorylation, the ability of CD34+ cells to polarize in respond to SDF-1 was impaired and the speed of cell movement was reduced upon OA pretreatment. OA pretreatment also impaired the ability of cells to adhere to fibronectin. Chemotaxis is a repetitive process wherein a cell develops a leading edge that protrudes and then attaches to the substratum, following which the cell retracts the tail, and this process continues until the cell stops migrating. Therefore, impairment in the ability of cell to adhere to fibronectin following OA pretreatment may in part contribute to inefficient migration of OA-pretreated CD34+ cells. No defect in F-actin polymerization was observed (data not shown). The phenotype of OA-pretreated cell is similar to that described for Dictyostelium in which myristylated Akt/PKB (constitutive high Akt/PKB activity) was overexpressed; in these cells, speed of movement and directional sense were found to be impaired (14). Although activation of Akt/PKB by chemoattractants plays an important role in controlling aspects of cell polarity and cell movement in Dictyostelium and mammalian cells (14, 15), it is dispensable for Rac/Cdc42 regulation of actin polymerization (13, 54, 55). ERK phosphorylation is associated with F-actin assembly (56), and in the present study SDF-1-induced ERK phosphorylation was not affected by OA pretreatment.

Chemotaxis is a dynamic cyclical process, and the orchestration of this complex process resides in many molecules that serve to distinguish the front from the rear of the cell and whose actions are carefully timed. Reciprocal localization of PI3K and various negative signals including PTEN (18, 22) spatially regulates production of phosphoinositol lipids, thereby regulating duration of activation of various PH domain-containing proteins, including Akt at the leading edge of a chemotaxing cell (14, 57). However, there is a lack of consensus in the literature regarding the role of PTEN in chemotaxis of mammalian leukocytes (22, 23, 58, 59). We chose to focus instead on PP2A. Our data demonstrate that in CD34+ cells, Akt phosphorylation is modulated following SDF-1 stimulation, and PP2A plays an important role in regulating both the levels and duration of Akt phosphorylation. Immunoprecipitation experiments demonstrated that there is some basal association of Akt with PP2A in CD34+ cells, but the amount of PP2A associated with Akt significantly increases following SDF-1 stimulation. Interaction of PP2A with Akt was not affected by OA pretreatment; however, interaction of GSK3 with Akt seemed to be reduced in OA-pretreated cells. The reason for the latter observation remains unclear. Increased translocation of Akt and PP2A to the plasma membrane was apparent after SDF-1 stimulation. The kinetics of Akt phosphorylation in CD34+ CB following stimulation with SDF-1 was slightly different from what has been described for other cells (14). This difference in kinetics of Akt translocation is most likely cell-type dependent. Optimal Akt phosphorylation in response to SDF-1 for effective chemotaxis is achieved by a balance of kinase, and phosphatase signal is illustrated by the fact that inhibition of PI3K, using low-dose LY294002, leads to partial recovery of chemotactic response in cells in which PP2A activity has been inhibited by OA, although by itself LY294002 inhibited chemotaxis of the cells. Thus, Akt phosphorylation following SDF-1 stimulation appears to be regulated by an optimal balance of PI3K and PP2A signals in CD34+ cells.

Interestingly, β-arrestin-2 was detected by coimmunoprecipitation in the Akt-PP2A complex following SDF-1 stimulation. In addition to its classical role in desensitization of GPCR signaling, β-arrestins can also act as a scaffolding protein (44, 45). A recent study suggested β-arrestin-2 is a positive mediator of dopaminergic synaptic transmission, and attributed this to its role as a signaling intermediate through a kinase/phosphatase scaffold (60). β-arrestin-2-deficient lymphocytes are defective in chemotaxis toward SDF-1 (61). It is therefore, possible that following SDF-1 stimulation, β-arrestin-2 acts as a scaffold to bring Akt and PP2A together. This interaction may be critical for effective SDF-1-directed chemotaxis of CD34+ CB cells.

Recently, GSK3 has been found to play a pivotal role in establishment of cell polarity by influencing microtubule stability. This was important for axon development and astrocyte migration (62, 63). However, global inhibition of GSK3 impairs polarity of cells (62). Cell migration is both positively and negatively affected by GSK3 (64, 65, 66). Cell migration may involve cyclic transient activation and inactivation of GSK3 as well as modulation of cellular localization of GSK3 (66). Phosphorylation of GSK3 leads to its inactivation (49). We found that following SDF-1 stimulation, GSK3 coimmunoprecipitated with Akt in CD34+ cells, and inhibition of GSK3 impaired chemotaxis. Additionally, expression of CAAkt impaired chemotaxis, and CAAkt-expressing CD34+ cells had higher levels of pGSK3. This suggests that PP2A activity is primarily required for optimal level and duration of activation of Akt, which in turn regulates activation of GSK3 by regulating its phosphorylation status. SDF-1 stimulation of OA-pretreated cells leads to robust and prolonged Akt activation, which in turn leads to prolonged phosphorylation and inactivation of GSK3, thereby impairing chemotaxis. In addition to its association with Akt following SDF-1 stimulation, GSK3 was also found to be constitutively associated with PP2A-C in CD34+ cells and phosphorylated in CD34+ cells in the absence of SDF-1 stimulation when the cells were pretreated with 1 µM OA. Therefore, the profound decrease in chemotaxis when cells are pretreated with higher concentration of OA (e.g., 1 µM) is most likely due to strong inhibition of GSK3 that is mediated by two pathways. One pathway may be due to the direct effect of PP2A inhibition on GSK3 phosphorylation, and the other due to phosphorylation of GSK by activated Akt in response to SDF-1 stimulation. GSK3 inhibition impaired the chemotaxis response, but not as profoundly as 1 µM OA. Effects of inhibitors in whole cells are limited by various factors, including permeability, and it is possible that inhibition of GSK3 activity by GSK3 inhibitor IX in CD34+ cells at the dose used is not as strong as when cells are pretreated with 1 µM OA. Based on our current findings, we propose the following model to describe the intracellular role of PP2A for effective chemotaxis of CD34+ cells toward SDF-1 (Fig. 9).


Figure 9
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  		FIGURE 9. Proposed model for the role of PP2A in SDF-1-induced chemotaxis of CD34+ cells. Upon binding of SDF-1 to CXCR4, G-coupled proteins activate both phosphatidylinositol phospholipase C and PI3K, which activate Akt. pAkt in turn phosphorylates GSK-3 and inactivates GSK3. Chemotaxis is achieved by orchestrating different signals that alternate between switch-on and switch-off mode. During SDF-1-directed CD34+ cell chemotaxis, Akt phosphorylation status, which in turn regulates GSK-3 phosphorylation, is regulated by balance of PI3K and PP2A activity. The right side of the diagram is based on new information based on our study. The broken arrow indicates pathways that allow the proteins to oscillate between switch-on and switch-off mode during chemotaxis.

 
Homing to bone marrow and engraftment in a proper microenvironment is required for a transplanted HSPC to be able to eventually repopulate and give rise to different lineages of blood cells. SDF-1 influences both homing and retention of transplanted HSPC to marrow probably by being a chemoattractant for HSPC and also by its ability to activate various adhesion molecules that are required for the interaction and retention of HSPC in the niche (67). We have found that repopulation of NOD-SCID mice transplanted with CD34+ cells pretreated with OA was significantly reduced on average greater than 60% less than mice transplanted with control CD34+ cells. The reduced SDF-1-directed chemotaxis and adhesion of HSPC to fibronectin in response to OA pretreatment are likely reasons for this phenomenon. Of possible relevance, it has been reported that chronic myeloid leukemia blast cells have reduced chemotactic activity and adhesion to fibronectin in response to SDF-1 (68). A more recent study showed that PP2A activity in chronic myeloid leukemia blast progenitors is significantly reduced (69). Taking these studies together with our findings suggests that PP2A may play an important role in SDF-1-mediated responses of both normal and malignant hemopoietic cells. Thus, further studies to understand the role of PP2A in normal and diseased hemopoiesis are warranted.

Our data, which demonstrate a role for PP2A in efficient chemotaxis of CD34+ CB cells primarily via modulation of Akt activation, may have potential therapeutic implications in disease states and for use in enhancing the homing and engraftment of HSPC.


   Acknowledgments
 
We thank Dr. Exing Wang for his help in acquiring confocal microscope images and Tapash Paul for his help in analysis of polarization data. We also thank Dr. Barbara Graham-Evans for her help in conducting transplantation experiments and Dr. Janardhan Sampath for critically reading the manuscript and for his valuable suggestions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 These studies were supported by U.S. Public Health Service Grants RO1 HL 67384 and RO1 HL 56416 and a project in PO1 HL053586 to H.E.B. Back

2 Address correspondence and reprint requests to Dr. Sunanda Basu, Walther Oncology Center, Indiana University School of Medicine, Research Institute No. 2 Building, Room 302, 950 West Walnut Street, Indianapolis, IN 46202-5181. E-mail address: sunbasu{at}iupui.edu Back

3 Abbreviations used in this paper: CB, cord blood; CAAkt, constitutively active Akt; DNPP2A-C, dominant-negative mutant of protein phosphatase 2A-catalytic subunit; GPCR, G protein-coupled receptor; GSK, glycogen synthase kinase; HSPC, hemopoietic stem and progenitor cell; OA, okadaic acid; PH, pleckstrin homology; PKB, protein kinase B; PP, protein phosphatase; PP2A-C, PP2A-catalytic subunit; PTEN, phosphatase and tensin homologue; SDF-1, stromal cell-derived factor-1; siRNA, small interfering RNA; WT, wild type. Back

Received for publication June 1, 2007. Accepted for publication June 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Broxmeyer, H. E., F. O. Smith. 2004. Cord blood hematopoietic cell transplantation. K. G. Blume, and S. J. Forman, and F. R. Applebaum, eds. Thomas’ Hematopoietic Cell Transplantation 550 Blackwell Scientific Publications, Cambridge.
  2. Christopherson, K. W., G. Hangoc, C. R. Mantel, H. E. Broxmeyer. 2004. Modulation of hematopoietic stem cell homing and engraftment by CD26. Science 305: 1000-1003. [Abstract/Free Full Text]
  3. Lapidot, T., O. Kollet. 2002. The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2mnull mice. Leukemia 16: 1992-2003. [Medline]
  4. Ma, Q., D. Jones, T. A. Springer. 1999. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 10: 463-471. [Medline]
  5. Peled, A., I. Petit, O. Kollet, M. Magid, T. Ponomaryov, T. Byk, A. Nagler, H. Ben-Hur, A. Many, L. Shultz, et al 1999. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 283: 845-848. [Abstract/Free Full Text]
  6. Dutt, P., J. F. Wang, J. E. Groopman. 1998. Stromal cell-derived factor-1{alpha} and stem cell factor/kit ligand share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis. J. Immunol. 161: 3652-3658. [Abstract/Free Full Text]
  7. Lee, Y., A. Gotoh, H. J. Kwon, M. You, L. Kohli, C. Mantel, S. Cooper, G. Hangoc, K. Miyazawa, K. Ohyashiki, H. E. Broxmeyer. 2002. Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines. Blood 99: 4307-4317. [Abstract/Free Full Text]
  8. Ganju, R. K., S. A. Brubaker, J. Meyer, P. Dutt, Y. Yang, S. Qin, W. Newman, J. E. Groopman. 1998. The {alpha}-chemokine, stromal cell-derived factor-1{alpha}, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J. Biol. Chem. 273: 23169-23175. [Abstract/Free Full Text]
  9. Nagasawa, T., S. Hirota, K. Tachibana, N. Takakura, S. Nishikawa, Y. Kitamura, N. Yoshida, H. Kikutani, T. Kishimoto. 1996. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382: 635-638. [Medline]
  10. Zou, Y. R., A. H. Kottmann, M. Kuroda, I. Taniuchi, D. R. Littman. 1998. Function of the chemokine receptor CXCR4 in hematopoiesis and in cerebellar development. Nature 393: 595-599. [Medline]
  11. Ma, Q., D. Jones, P. R. Borghesani, R. A. Segal, T. Nagasawa, T. Kishimoto, R. T. Bronson, T. A. Springer. 1998. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in C. Proc. Natl. Acad. Sci. USA 95: 9448-9453. [Abstract/Free Full Text]
  12. Sipkins, D. A., X. Wei, J. W. Wu, J. M. Runnels, D. Cote, T. K. Means, A. D. Luster, D. T. Scadden, C. P. Lin. 2005. In vivo imaging of specialized bone marrow endothelial microdomains for tumor engraftment. Nature 435: 969-973. [Medline]
  13. Funamoto, S., K. Milan, R. Meili, R. A. Firtel. 2001. Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium. J. Cell Biol. 153: 795-810. [Abstract/Free Full Text]
  14. Meili, R., C. Ellsworth, S. Lee, T. B. Reddy, H. Ma, R. A. Firtel. 1999. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in dictyostelium. EMBO J. 18: 2092-2105. [Medline]
  15. Wang, F., P. Herzmark, O. D. Weiner, S. Srinivasan, G. Servant, H. R. Bourne. 2002. Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils. Nat. Cell Biol. 4: 513-518. [Medline]
  16. Huang, Y. E., M. Iijima, C. A. Parent, S. Funamoto, R. A. Firtel, P. Devreotes. 2003. Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells. Mol. Biol. Cell 14: 1913-1922. [Abstract/Free Full Text]
  17. Hunter, T.. 1995. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80: 225-236. [Medline]
  18. Funamoto, S., R. Meili, S. Lee, L. Parry, R. A. Firtel. 2002. Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 109: 611-623. [Medline]
  19. Sotsios, Y., G. C. Whittaker, J. Westwick, S. G. Ward. 1999. The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes. J. Immunol. 163: 5954-5963. [Abstract/Free Full Text]
  20. Sotsios, Y., S. G. Ward. 2000. Phosphoinositide 3-kinase: a key biochemical signal for cell migration in response to chemokines. Immunol. Rev. 177: 217-235. [Medline]
  21. Berthebaud, M., C. Riviere, P. Jarrier, A. Foudi, Y. Zhang, D. Compagno, A. Galy, W. Vainchenker, F. Louache. 2005. RGS16 is a negative regulator of SDF-1-CXCR4 signaling in megakaryocytes. Blood 106: 2962-2968. [Abstract/Free Full Text]
  22. Xu, J., F. Wang, K. A. Van, P. Herzmark, A. Straight, K. Kelly, Y. Takuwa, N. Sugimoto, T. Mitchison, H. R. Bourne. 2003. Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114: 201-214. [Medline]
  23. Gao, P., R. L. Wange, N. Zhang, J. J. Oppenheim, O. M. Howard. 2005. Negative regulation of CXCR4-mediated chemotaxis by the lipid phosphatase activity of tumor suppressor PTEN. Blood 106: 2619-2626. [Abstract/Free Full Text]
  24. Resjo, S., O. Goransson, L. Harndahl, S. Zolnierowicz, V. Manganiello, E. Degerman. 2002. Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes. Cell. Signal. 14: 231-238. [Medline]
  25. Lee, J., Y. Chen, T. Tolstykh, J. Stock. 1996. A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc. Natl. Acad. Sci. USA 93: 6043-6047. [Abstract/Free Full Text]
  26. Millward, T. A., S. Zolnierowicz, B. A. Hemmings. 1999. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. 24: 186-191. [Medline]
  27. Andjelkovic, M., T. Jakubowicz, P. Cron, X. F. Ming, J. W. Han, B. A. Hemmings. 1996. Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. Proc. Natl. Acad. Sci. USA 93: 5699-5704. [Abstract/Free Full Text]
  28. Basu, S., H. E. Broxmeyer. 2005. Transforming growth factor-β1 modulates responses of CD34+ cord blood cells to stromal cell-derived factor-1/CXCL12. Blood 106: 485-493. [Abstract/Free Full Text]
  29. Campbell, R. A., P. Bhat-Nakshatri, N. M. Patel, D. Constantinidou, S. Ali, H. Nakshatri. 2001. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor {alpha}: a new model for anti-estrogen resistance. J. Biol. Chem. 276: 9817-9824. [Abstract/Free Full Text]
  30. Zicha, D., G. Dunn, G. Jones. 1997. Analyzing chemotaxis using the Dunn direct-viewing chamber. Methods Mol. Biol. 75: 449-457. [Medline]
  31. Broxmeyer, H. E., G. Hangoc, S. Cooper, R. C. Ribeiro, V. Graves, M. Yoder, J. Wagner, S. Vadhan-Raj, L. Benninger, P. Rubinstein. 1992. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc. Natl. Acad. Sci. USA 89: 4109-4113. [Abstract/Free Full Text]
  32. Petit, I., P. Goichberg, A. Spiegel, A. Peled, C. Brodie, R. Seger, A. Nagler, R. Alon, T. Lapidot. 2005. Atypical PKC-{zeta} regulates SDF-1-mediated migration and development of human CD34+ progenitor cells. J. Clin. Invest. 115: 168-176. [Medline]
  33. Bialojan, C., A. Takai. 1988. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases: specificity and kinetics. Biochem. J. 256: 283-290. [Medline]
  34. Favre, B., P. Turowski, B. A. Hemmings. 1997. Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin. J. Biol. Chem. 272: 13856-13863. [Abstract/Free Full Text]
  35. Namboodiripad, A. N., M. L. Jennings. 1996. Permeability characteristics of erythrocyte membrane to okadaic acid and calyculin A. Am. J. Physiol. 270: C449-C456. [Medline]
  36. Stampwala, S. S., R. H. Bunge, T. R. Hurley, N. E. Willmer, A. J. Brankiewicz, C. E. Steinman, T. A. Smitka, J. C. French. 1983. Novel antitumor agents CI-920, PD 113,270 and PD 113,271. II. Isolation and characterization. J. Antibiot. 36: 1601-1605. [Medline]
  37. Scheithauer, W., D. D. Von Hoff, G. M. Clark, J. L. Shillis, E. F. Elslager. 1986. In vitro activity of the novel antitumor antibiotic fostriecin (CI-920) in a human tumor cloning assay. Eur. J. Cancer Clin. Oncol. 22: 921-926. [Medline]
  38. Ingebritsen, T. S., A. A. Stewart, P. Cohen. 1983. The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur. J. Biochem. 132: 297-307. [Medline]
  39. Yan, Y., M. C. Mumby. 1999. Distinct roles for PP1 and PP2A in phosphorylation of the retinoblastoma protein: PP2a regulates the activities of G1 cyclin-dependent kinases. J. Biol. Chem. 274: 31917-31924. [Abstract/Free Full Text]
  40. Myles, T., K. Schmidt, D. R. Evans, P. Cron, B. A. Hemmings. 2001. Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells. Biochem. J. 357: 225-232. [Medline]
  41. Peled, A., O. Kollet, T. Ponomaryov, I. Petit, S. Franitza, V. Grabovsky, M. M. Slav, A. Nagler, O. Lider, R. Alon, et al 2000. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 95: 3289-3296. [Abstract/Free Full Text]
  42. Borisy, G. G., T. M. Svitkina. 2000. Actin machinery: pushing the envelope. Curr. Opin. Cell Biol. 12: 104-112. [Medline]
  43. Li, L., C. H. Ren, S. A. Tahir, C. Ren, T. C. Thompson. 2003. Caveolin-1 maintains activated Akt in prostate cancer cells through scaffolding domain binding site interactions with and inhibition of serine/threonine protein phosphatases PP1 and PP2A. Mol. Cell. Biol. 23: 9389-9404. [Abstract/Free Full Text]
  44. Miller, W. E., R. J. Lefkowitz. 2001. Expanding roles for β-arrestins as scaffolds and adapters in GPCR signaling and trafficking. Curr. Opin. Cell Biol. 13: 139-145. [Medline]
  45. Pierce, K. L., R. J. Lefkowitz. 2001. Classical and new roles of β-arrestins in the regulation of G-protein-coupled receptors. Nat. Rev. Neurosci. 2: 727-733. [Medline]
  46. Vlahos, C. J., W. F. Matter, K. Y. Hui, R. F. Brown. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269: 5241-5248. [Abstract/Free Full Text]
  47. Manoukian, A. S., J. R. Woodgett. 2002. Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways. Adv. Cancer Res. 84: 203-229. [Medline]
  48. Farooqui, R., S. Zhu, G. Fenteany. 2006. Glycogen synthase kinase-3 acts upstream of ADP-ribosylation factor 6 and Rac1 to regulate epithelial cell migration. Exp. Cell Res. 312: 1514-1525. [Medline]
  49. Plyte, S. E., K. Hughes, E. Nikolakaki, B. J. Pulverer, J. R. Woodgett. 1992. Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim. Biophys. Acta 1114: 147-162. [Medline]
  50. Sutherland, C., I. A. Leighton, P. Cohen. 1993. Inactivation of glycogen synthase kinase-3β by phosphorylation: new kinase connections in insulin and growth-factor signalling. Biochem. J. 296: 15-19. [Medline]
  51. Ivaska, J., L. Nissinen, N. Immonen, J. E. Eriksson, V. M. Kahari, J. Heino. 2002. Integrin {alpha}2β1 promotes activation of protein phosphatase 2A and dephosphorylation of Akt and glycogen synthase kinase 3β. Mol. Cell. Biol. 22: 1352-1359. [Abstract/Free Full Text]
  52. Bodine, D. M.. 2004. Animal models for the engraftment and differentiation of human hematopoietic stem and progenitor cells. H. E. Broxmeyer, ed. Cord Blood Biology, Immunology, Banking and Clinical Transplantation 47 AABB Press, Bethesda.
  53. Kohn, A. D., F. Takeuchi, R. A. Roth. 1996. Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J. Biol. Chem. 271: 21920-21926. [Abstract/Free Full Text]
  54. Welch, H., A. Eguinoa, L. R. Stephens, P. T. Hawkins. 1998. Protein kinase B and rac are activated in parallel within a phosphatidylinositide 3OH-kinase-controlled signaling pathway. J. Biol. Chem. 273: 11248-11256. [Abstract/Free Full Text]
  55. Van Weering, D. H., J. de Rooij, B. Marte, J. Downward, J. L. Bos, B. M. Burgering. 1998. Protein kinase B activation and lamellipodium formation are independent phosphoinositide 3-kinase-mediated events differentially regulated by endogenous Ras. Mol. Cell. Biol. 18: 1802-1811. [Abstract/Free Full Text]
  56. Gu, Y., M. D. Filippi, J. A. Cancelas, J. E. Siefring, E. P. Williams, A. C. Jasti, C. E. Harris, A. W. Lee, R. Prabhakar, S. J. Atkinson, et al 2003. Hematopoietic cell regulation by Rac1 and Rac2 guanosine triphosphatases. Science 302: 445-449. [Abstract/Free Full Text]
  57. Parent, C. A., B. J. Blacklock, W. M. Froehlich, D. B. Murphy, P. N. Devreotes. 1998. G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95: 81-91. [Medline]
  58. Anzelon, A. N., H. Wu, R. C. Rickert. 2003. Pten inactivation alters peripheral B lymphocyte fate and reconstitutes CD19 function. Nat. Immunol. 4: 287-294. [Medline]
  59. Fox, J. A., K. Ung, S. G. Tanlimco, F. R. Jirik. 2002. Disruption of a single Pten allele augments the chemotactic response of B lymphocytes to stromal cell-derived factor-1. J. Immunol. 169: 49-54. [Abstract/Free Full Text]
  60. Beaulieu, J. M., T. D. Sotnikova, S. Marion, R. J. Lefkowitz, R. R. Gainetdinov, M. G. Caron. 2005. An Akt/β-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 122: 261-273. [Medline]
  61. Fong, A. M., R. T. Premont, R. M. Richardson, Y. R. Yu, R. J. Lefkowitz, D. D. Patel. 2002. Defective lymphocyte chemotaxis in β-arrestin2- and GRK6-deficient mice. Proc. Natl. Acad. Sci. USA 99: 7478-7483. [Abstract/Free Full Text]
  62. Etienne-Manneville, S., A. Hall. 2003. Cdc42 regulates GSK-3β and adenomatous polyposis coli to control cell polarity. Nature 421: 753-756. [Medline]
  63. Li, R.. 2005. Neuronal polarity: until GSK-3 do us part. Curr. Biol. 15: R198-R200. [Medline]
  64. Kim, D., S. Kim, H. Koh, S. O. Yoon, A. S. Chung, K. S. Cho, J. Chung. 2001. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production. FASEB J. 15: 1953-1962. [Abstract/Free Full Text]
  65. Koivisto, L., K. Alavian, L. Hakkinen, S. Pelech, C. A. McCulloch, H. Larjava. 2003. Glycogen synthase kinase-3 regulates formation of long lamellipodia in human keratinocytes. J. Cell Sci. 116: 3749-3760. [Abstract/Free Full Text]
  66. Kobayashi, T., S. Hino, N. Oue, T. Asahara, M. Zollo, W. Yasui, A. Kikuchi. 2006. Glycogen synthase kinase 3 and h-prune regulate cell migration by modulating focal adhesions. Mol. Cell. Biol. 26: 898-911. [Abstract/Free Full Text]
  67. Simmons, P. J., A. Zannettino, S. Gronthos, D. Leavesley. 1994. Potential adhesion mechanisms for localization of hemopoietic progenitors to bone marrow stroma. Leuk. Lymphoma 12: 353-363. [Medline]
  68. Peled, A., I. Hardan, L. Trakhtenbrot, E. Gur, M. Magid, M. rash-Yahana, N. Cohen, V. Grabovsky, S. Franitza, O. Kollet, et al 2002. Immature leukemic CD34+CXCR4+ cells from CML patients have lower integrin-dependent migration and adhesion in response to the chemokine SDF-1. Stem Cells 20: 259-266. [Medline]
  69. Neviani, P., R. Santhanam, R. Trotta, M. Notari, B. W. Blaser, S. Liu, H. Mao, J. S. Chang, A. Galietta, A. Uttam, et al 2005. The tumor suppressor PP2A is functionally inactivated in blast crisis CML through the inhibitory activity of the BCR/ABL-regulated SET protein. Cancer Cells 8: 355-368.



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