5-Mediated ERK Activation and Adhesion-Independent Growth of Human Pre-B Cell Lines

SDF-1 and PDGF enhance avb5-mediated ERK activation and adhesion-independent growth of human pre-B cell lines

M Acharya1,3, AL Edkins1,4, BW Ozanne2 and W Cushley1

1Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, UK and 2Cancer Research-UK Beatson Laboratories, Garscube Estate, Glasgow, Scotland, UK

CD23 acts through the avb5 integrin to promote growth of and differentiation of centrocytes by binding to CD21.7–9 human pre-B cell lines in an adhesion-independent manner. Plasma sCD23 levels are low in normal subjects, but high avb5 is expressed on normal B-cell precursors in the bone levels are reported in inflammatory disorders such as rheuma- marrow. Soluble CD23 (sCD23), short CD23-derived peptides 10 containing the arg-lys-cys (RKC) motif recognized by avb5 and toid arthritis and systemic lupus erythmatosus. Particularly anti-avb5 monoclonal antibodies (MAbs) all sustain growth of high levels of sCD23 with prognostic value are a hallmark of pre-B cell lines. The chemokine stromal cell-derived factor-1 B-cell chronic lymphoblastic leukaemia.11 The avb5 integrin, (SDF-1) regulates key processes during B-cell development. a member of the vitronectin receptor family, is expressed by SDF-1 enhanced the growth-sustaining effect driven by ligation bone marrow B-cell precursors.2 The vitronectin receptor family of avb5 with anti-avb5 MAb 15F-11, sCD23 or CD23-derived a RKC-containing peptides. This effect was restricted to B-cell members comprise the v chain in non-covalent association precursors and was specific to SDF-1. The enhancement in with one of five b-subunits (b1, b3, b5, b6 and b8) and bind growth was associated with the activation of extracellular a broad spectrum of ligands, including fibrinogen, fibronectin signal-regulated kinase (ERK) and both these responses were and vitronectin, typically by the recognition of arg-gly-asp attenuated by the MEK inhibitor U0126. Finally, platelet-derived motifs.12–14 Vitronectin receptors have diverse roles in cell growth factor also enhanced both avb5-mediated cell growth adhesion, migration and survival and are implicated in the and ERK activation. The data suggest that adhesion-indepen- growth of a number of cancer cells. The vitronectin receptors dent growth-promoting signals delivered to B-cell precursors 2,6 through the avb5 integrin can be modulated by cross-talk with avb5 and avb3 are recognized CD23 receptors and avb5 receptors linked to both G-protein and tyrosine kinase-coupled recognizes an arg-lys-cys (RKC) motif on sCD23 in an adhesion- signalling pathways. independent interaction.2 Leukemia (2009) 23, 1807–1817; doi:10.1038/leu.2009.126; The growth and survival of normal and leukaemic B-cell published online 16 July 2009 progenitors is dependent on intimate contact with bone marrow Keywords: B lymphocyte; development; SDF-1; av integrin; PDGF stromal layers15–17 and the factors responsible for the proliferation of progenitor cells include both cellular adhesion molecules and soluble proteins.15 The avb5–CD23 interaction could represent an important mechanism regulating the growth of Introduction B-cell precursors, in combination with other factors present in the bone marrow microenvironment. B lymphopoiesis is a tightly regulated multi-stage process. The Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a member bone marrow microenvironment provides a variety of cytokines, of the CXC chemokine family18 that binds to CXCR4 and CXCR7 chemokines, growth factors and adhesion molecules that receptors.19,20 Gene inactivation of SDF-1 or CXCR4 in vivo co-ordinately regulate B-cell development. Perturbation of causes embryonic lethality due to abnormal cerebral and gastro- B-cell differentiation in the bone marrow leads to malignancies intestinal vasculatures and haematopoietic development.21–24 including acute lymphoblastic leukaemia (ALL).1 SDF-1 is the most powerful chemoattractant for undifferentiated The interaction between soluble CD23 (sCD23) and human CD34 þ haematopoietic progenitors.25,26 Several studies the integrin avb5 sustains growth of human pre-B cell lines have shown that SDF-1 activates the integrins LFA-1, VLA-4 and derived from ALL patients.2,3 Human CD23, a 45-kDa type II VLA-5,27–31 and facilitates transendothelial migration, homing transmembrane glycoprotein, can be cleaved from the cell and engraftment of precursor B cells in the bone marrow.31,32 In surface to release a range of sCD23 proteins with cytokine- addition to its established role in regulating cell motility, SDF-1 like activities. The cytokine-like activities include promoting also plays a role in cell survival33,34 and proliferation,35 often the release of cytokines from monocytic cells, mediated by the in synergy with other cytokines.36 CXCR4 ligation by SDF-1 aMb2, aXb24,5 and avb3 integrins,6 and influencing the survival induces receptor internalization, elevation of cytoplasmic Ca2 þ levels, activation of phosphatidylinositol 3-kinase (PI3K) and Correspondence: Professor W Cushley, Division of Molecular and phosphorylation of MEK, extracellular signal-regulated kinase Cellular Biology, Faculty of Biomedical and Life Sciences, University (ERK) and components of focal adhesion complexes in many of Glasgow, Glasgow G12 8QQ, Scotland, UK. 37–43 E-mail: [email protected] cell types including B-cell precursors. 3Current Address: Department of Pediatrics, Developmental Immuno- The well-established role of SDF-1 in B lymphopoiesis and in logy Laboratory, Massachusetts General Hospital, Boston MA 02114, influencing integrin function makes it an attractive candidate for USA. 4 modulating avb5 function. The data in this report indicate that Current address: Chaperone Research Group, Department of SDF-1 modulates the growth-sustaining function of avb5, by Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown 6139, South Africa. influencing ERK signalling downstream of both CXCR4 and Received 4 April 2008; revised 5 May 2009; accepted 6 May 2009; avb5; platelet-derived growth factor (PDGF) also enhances published online 16 July 2009 avb5-mediated signalling. PDGF has been extensively studied in SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1808 the context of integrin-growth factor receptor cross-talk44 and Cell growth assays we therefore assessed the role of PDGF together with other SMS-SB cells were washed and then plated at a density of cytokines, in modulating avb5 function. The data show that 2500 cells/100 ml culture (low cell density), a seeding density at biological responses activated through the avb5–CD23 inter- which the cells are prone to apoptosis,3 in 96-well microtitre actions in early B-cell precursors are modulated by inputs from assay plates in PFHM. All other cell lines were washed both G-protein-coupled and tyrosine kinase-linked receptors. extensively in PFHM before culture at 5000 cells/100 ml cultures. Cultures were propagated in the presence or absence of cytokines, MAbs or peptides, at 37 1C for 72 h followed by the Materials and methods addition of 0.3 mCi/well [3H]-TdR for 18 h before harvest; incorporation was determined by liquid scintillation spectro- Antibodies and reagents metry. For assays with bone marrow B cells or ALL cells, Recombinant cytokines (interleukin (IL)-7, IL-3, IL-11 and IL-4), g-irradiated (30 Gy (3000 rad)) hMSC-tert stromal cells were anti-CXCR7 (clone 358426, IgG2a), biotinylated anti-CXCR4 plated at a density of 2 Â 105 cells/well at least 7 days before (12G5, IgG2a) antibody and recombinant human 25-kDa the addition of mononuclear cells. Fluorescence-activated sCD23, encompassing residues Met151-Ser231 with an N-terminal cell-sorted human bone marrow B cells were plated at a His6 tag, were purchased from R&D Systems (Abingdon, UK). density of 3000 cells/well on the stromal cells in RPMI-1640 Anti-avb5 (P1F6, IgG1; 15F11, IgG2a), biotinyl-murine IgG1 and medium supplemented with 2% (v/v) heat-inactivated foetal recombinant SDF-1b were from Chemicon (Hampshire, UK), calf serum, 2 mM fresh glutamine, and penicillin and strepto- and anti-phospho-p44/42 MAP kinase, anti-p44/42 MAP kinase, mycin. ALL cells were plated at the same cell density in AIMV PI3K inhibitor LY294002 and PDGF-B were supplied by Cell serum-free medium. All cultures were established in triplicate. Signalling Technologies (Beverly, MA, USA). Streptavidin-phy- Cytokines were used at 0.5–32 nM and sCD23 was employed at coerythrin, annexinV-fluorescein isothiocyanate, anti-CD19-PE, 0.4–6 nM. In experiments using inhibitors, cells were incubated anti-k-fluorescein isothiocyanate and anti-CXCR4-PE were from with the inhibitors (5–10 mM U0126, 50 mM LY294002 and BD Bioscience (Oxford, UK). Isotype control IgG2a antibody, 50 mM AMD3100) for 30 min at 37 1C before the introduction anti-actin antibody, horseradish peroxidase-conjugated anti- of stimuli. rabbit immunoglobulins, AMD3100, propidium iodide and Dulbecco’s modified Eagle’s medium were from the Sigma Chemicals Co. (Poole, UK). Biotinylated isotype control IgG2a Peptide biochemistry antibody was from Serotec (Oxford, UK). The MEK inhibitor CD23-derived peptides containing the RKC motif recognized by U0126 was purchased from Promega (Southampton, UK). the avb5 integrin are biologically active. The peptides were used Tritiated thymidine ([3H]-TdR) was obtained from Amersham at 0.5–3.33 mM and are numbered according to their position International plc (Amersham, Buckinghamshire, England). RPMI- in the original screening library.2 The sequences of the 1640 medium, AIMV serum-free medium and protein-free peptides used in this study are no. 9, KWINFQRKC; no. 11, hybridoma medium-II (PFHM) were from GIBCO-BRL (Paisley, FQRKCYYFG; LP long peptide, KWINFQRKCYYFGKG. Peptide Scotland). Anti-ERK/2 antibody was purchased from Santa Cruz no. 8, PEKWINFQR; no. 41, SGSGGEFIWVDGS; and no. 34, Biotechnology Inc. (Santa Cruz, CA, USA). SGSGHTGSWIGLR that lack the RKC motif and do not bind to avb5 were used as negative control peptides.

Cell lines and cell culture The SMS-SB cell line was derived from a female patient Flow cytometry presenting with ALL;45 SMS-SB cells were cultured in PFHM Cells were harvested and washed twice with ice-cold phos- 6 supplemented with 2 mM fresh glutamine and 1% (w/v) phate-buffered saline and resuspended at 5 Â 10 cells/ml. One penicillin and streptomycin at 37 1Cin5%CO2 in air in a hundred microlitres of cell suspension was stained with either humidified atmosphere, at 2.5–5 Â 105 cells/ml. RS4;11,46 biotinylated, unlabelled primary MAb or biotinylated CD23- Blin-1, RAJI, Daudi and IB4 B cell lines were from laboratory derived peptides (10 mg/ml) for 30–60 min; unlabelled primary stocks. Cell lines were maintained in RPMI-1640 medium antibody was visualized using a secondary phycoerythrin- supplemented with 10% (v/v) heat-inactivated foetal calf serum, conjugated anti-mouse IgG or, in the case of biotinylated 2mM fresh glutamine, and penicillin and streptomycin, at 37 1C antibody or peptides, streptavidin-phycoerythrin. For assay of 6 in a 5% CO2 in air in a humidified atmosphere. hMSC-tert- apoptosis, approximately 1 Â 10 cells were washed twice with telomerized human stromal cell line was a gift from Dr M ice-cold phosphate-buffered saline and stained with 100 ml 47 Kassem (Denmark) and was maintained in Dulbecco’s labelling buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethane- modified Eagle’s medium supplemented with 10% (v/v) heat- sulphonic acid-KOH, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.5 mg inactivated foetal calf serum, 2 mM fresh glutamine, and propidium iodide and 5 ml annexinV-fluorescein isothiocyanate) penicillin and streptomycin. Normal human bone marrow was and incubated at room temperature for 20 min. The labelling obtained from Lonza Biologics (Slough, UK). Aliquots were buffer was aspirated and the cells were resuspended in 400 ml subjected to sterile sorting on a FACSAria instrument (BD wash buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesul- Bioscience) using fluorescein isothiocyanate anti-k chain and phonic acid-KOH, pH 7.4, 150 mM NaCl, 5 mM CaCl2). Cells PE-anti-CD19 monoclonal antibodies (MAbs) to obtain a were analysed on an FACScan flow cytometer, using CellQuest fraction of cells enriched for B-cell precursors. For cultures software (Becton Dickinson, San Jose, CA, USA). with sCD23, peptides and anti-avb5 MAbs, the cells were propagated in medium supplemented with 2% (v/v) foetal calf serum and hMSC-tert stromal cells. Mononuclear cells were Western blot analysis isolated from samples from ALL patients and were cultured Approximately 3 Â 107 cells in PFHM were stimulated with in AIMV serum-free medium, hMSC-tert stromal cells and either SDF-1 (250 ng/ml), sCD23 (160 ng/ml), LP (1 mg/ml), stimulants. peptide no. 8 or 9 (5 mg/ml) or 15F-11 MAb (5 mg/ml) alone

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1809 or in combination for specified time periods at 37 1C. Stimula- Results tion was stopped by the addition of RIPA buffer without detergents (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM SDF-1 enhances growth of pre-B cell lines induced by ethylene glycol tetraacetic acid and 1 mM Na3VO4). Cell the ligation of avb5 integrin pellets were lysed in RIPA buffer containing detergents The avb5–sCD23 interaction sustains the growth of human and protease inhibitors (RIPA buffer as above plus 1% (w/v) precursor B cell lines.2 CD23-derived peptides containing the NP40, 1 mM Na deoxycholate, 1 mM PMSF and 2 mg/ml RKC motif recognized by the avb5 integrin and the 15F-11 MAb, leupeptin) and clarified by centrifugation at 13 000 g for which binds the assembled avb5 heterodimer, all sustain growth 15 min. Protein concentrations were determined using the in a manner similar to sCD23.2 SMS-SB cells treated with sCD23 Bradford reagent (BioRad Laboratories Ltd, Hampstead, UK) show a dose-dependent increase in [3H]-TdR incorporation with bovine serum albumin as a standard. Equal amounts (Figure 1a). Other pre-B cell lines such as RS4;11 and 697 show of protein (30–40 mg) were loaded per lane on 4–12% a similar response to sCD23 stimulation (data not shown). SMS- NuPAGE Novex Bis-Tris polyacrylamide gradient gels SB cell growth induced by a suboptimal concentration of sCD23 (Invitrogen, Paisely, UK) and transferred onto nitrocellulose. is enhanced by SDF-1 treatment (Figure 1b) in a dose-dependent Phosphorylated and total protein profiles were detected manner; SDF-1 alone has a minimal effect on the growth of sequentially on the same membrane using specific primary SMS-SB cells at any concentration used. Growth of SMS-SB cells antibodies, secondary antibodies conjugated to horse- stimulated by the 15F-11 MAb is also enhanced by SDF-1 radish peroxidase and enhanced chemiluminescence (Pierce, (Figure 1c), confirming that the growth-promoting effect is Rockford, USA). Bands were quantified by densitometry using specific to avb5 ligation. SDF-1 enhances the growth of SMS-SB ImageJ software (HIN, Bethesda, MD, USA). cells stimulated with CD23-derived peptide no. 9, a nonameric

Figure 1 SDF-1 enhances avb5-mediated growth of SMS-SB cells. (a) Low cell density (LCD) cultures of SMS-SB cells were propagated with the indicated concentrations of recombinant sCD23 and [3H]-TdR incorporation assessed after 72 h. (b) Growth of LCD cultures of SMS-SB cells in response to a suboptimal concentration of sCD23 (158 ng/ml), together with indicated concentrations of SDF-1 (black bars) and SDF-1 alone (hatched bars), is shown. (c–e) The growth response of SMS-SB cells to stimulation with 5 mg/ml 15F-11 MAb (c), 5 mg/ml peptide no. 9 (d) and 1 mg/ml long peptide (LP) (e), respectively, in combination with SDF-1. In each panel, the treatment with SDF-1 alone is shown as a hatched bar, the combination of SDF-1 and stimulus as a black bar and the combination of SDF-1 with either isotype control IgG2a (c) or irrelevant peptide no. 34 (d) as a white bar. (f) Growth with a suboptimal concentration of LP (1 mg/ml) and SDF-1 (250 ng/ml) in untreated cells and cells treated with 50 mM AMD3100 for 30 min before SDF-1 addition. In both cases, no stimulus is represented as grey bars, LP treatment is represented as black bars, the LP and SDF-1 combination as white bars and SDF-1 alone as hatched bars. Bars represent the s.d. of triplicate determinations. In the figures, *Po0.05 or **Po0.01 indicate the statistical significance of the increase in growth of cells treated with different concentrations of SDF-1 and avb5 stimulants (sCD23, 15F-11MAb, peptide no. 9 or LP) in comparison with SDF-1 treatment alone, and #Po0.05 and ##Po0.01 indicate the significance in enhancement of proliferation with 250 ng/ml SDF-1 and avb5 stimulants when compared with avb5 stimulants alone. Data are representative of three independent experiments. (g) SMS-SB cells cultured in the presence or absence of 250 ng/ml SDF-1 (24 h) were stained for avb5 expression using the anti-avb5 MAbs P1F-6 and 15F-11. Fluorescence intensity histograms for the relevant avb5 MAb are represented by the grey shaded area and isotype control antibody staining is represented by the black line. LCD, low cell density; SDF-1, stromal cell-derived factor-1; [3H]-TdR, tritiated thymidine; sCD23, soluble CD23.

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1810 peptide containing the RKC motif, but has no effect on the avb5 expression (Figure 2b) illustrates that expression of the ability of peptide no. 34, which lacks the RKC tripeptide, to integrin is highest in cell lines representative of B-cell precursors promote cell growth (Figure 1d). SDF-1 also enhances cell and decreases in mature B cell lines (Figure 2b). Cell lines growth stimulated by the LP, a particularly potent CD23-derived representing earlier stages of B-cell development (RS4;11 and peptide containing the RKC motif (Figure 1e). The CXCR4- SMS-SB) have low levels of CXCR4 expression and show the specific antagonist AMD3100 was used to inhibit binding most robust growth response following avb5 ligation; SDF-1 of SDF-1 to CXCR4. Figure 1f shows that after AMD3100 enhances avb5-mediated cell growth induced by LP in these treatment, there is no difference in cell growth induced by LP in cells (Figure 2c). Despite higher CXCR4 expression, SDF-1 does the presence or absence of SDF-1 in SMS-SB cells, indicating not affect the growth of either Blin-1 cells, which show a modest that the enhancement in LP-induced cell growth is through increase in cell growth after avb5 ligation (by sCD23), or Daudi SDF-1 and CXCR4. There was no change in avb5 expression cells, which show little or no growth response through avb5 in cells treated with SDF-1 compared with untreated cells (as shown by LP treatment). Finally, the cell line IB4, which is (Figure 1g), nor was there any increase in peptide binding to low/negative for both CXCR4 and avb5 expression, shows no treated cells (Supplementary Figure 1A). Furthermore, the increase in cell growth following avb5 ligation with LP in the stimulation of avb5 with either LP or sCD23 did not influence presence or absence of SDF-1 (Figure 2c). CXCR4 expression (Supplementary Figure 1B). Although the The cytokines IL-7, IL-11, IL-3 and IL-4 were also assessed to data show that SDF-1 can cooperate with soluble avb5-directed determine if any of them could enhance avb5-induced cell ligands to enhance growth of SMS-SB cells, it remains to be growth; none of these cytokines mimicked the ability of SDF-1 determined whether this reflects either inhibition of apoptosis, to enhance avb5-mediated cell growth (Figure 3). IL-7 has no stimulation of cell division or a combination of both processes. effect on LP-induced SMS-SB cell growth (Figure 3a), and whereas IL-11 alone induces an increase in [3H]-TdR incorporation (Figure 3b), it has no effect on avb5-regulated cell growth; Enhancement of avb5-driven cell growth by SDF-1 is neither IL-3 nor IL-4 enhances avb5-induced growth (Figure 3c specific to B-cell precursors and d). Growth responses to the different cytokines were tested CXCR4 expression varies in cell lines representing distinct stages using all of the different avb5-directed stimuli and none of the of B-cell development (Figure 2a), and a similar assessment of cytokines tested modulated avb5 expression (data not shown).

Figure 2 Levels of CXCR4 expression do not influence cell growth. (a and b) Staining of CXCR4 (12G5) and avb5 (P1F6), respectively, in five cell lines. Antibody staining is shown in the grey shaded area and the isotype control staining by the black line. (c) In the different cell lines shown, [3H]-TdR incorporation driven by avb5 ligation (either with 2 ng/ml sCD23 or with 1 mg/ml LP) and SDF-1 is shown by black bars, and the growth response induced with SDF-1 alone is shown as hatched bars. Bars represent the s.d. of triplicate determinations; *Po0.05 or **Po0.01 indicates a statistically significant different increase in the growth of cells treated with different concentrations of SDF-1 and avb5 stimulants (sCD23 or LP) in comparison with SDF-1 treatment alone, and #Po0.05 and ##Po0.01 indicate a significant enhancement of cell growth with 250 ng/ml SDF-1 and avb5 stimulants when compared with avb5 stimulants alone. Data are representative of three independent experiments. LP, long peptide; sCD23, soluble CD23; SDF-1, stromal cell-derived factor-1.

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1811

Figure 3 Regulation of avb5-mediated cell growth by other cytokines. (a–d) Growth of LCD cultures of SMS-SB cells in response to 1 mg/ml LP or 5 mg/ml peptide no. 9, together with the indicated concentrations of IL-7, IL-11, IL-3 and IL-4, respectively. In each case, growth induced by the peptides and the cytokine is shown in black bars, by cytokine alone is shown as hatched bars and by cytokine with negative control peptide no. 41 or 8 is shown as white bars. The concentrations of each cytokine are equivalent molar concentrations. Data are representative of three independent experiments. LCD, low cell density; LP, long peptide.

SDF-1 enhances ERK phosphorylation induced by in ERK phosphorylation when added together with a soluble sCD23 and other avb5 ligands avb5 ligand. The signalling pathways that might regulate synergistic activa- We used the MEK inhibitor U0126 and the PI3K inhibitor tion of cell growth by avb5 ligation and SDF-1 treatment were LY294002 to further investigate the specificity of the ERK evaluated, with a focus on ERK phosphorylation as SDF-1 is signalling pathway in avb5-mediated functions. Pre-treatment of known to target this pathway.41 Treatment of SMS-SB cells with SMS-SB cells with U0126 completely abolishes the effect of sCD23 promotes robust phosphorylation of ERK at 30–60 min sCD23- and CD23-derived peptides on cell growth (Figure 5a). (Figure 4a). SDF-1 alone provokes an early, modest increase in Moreover, U0126 inhibits basal survival of SMS-SB cells in low- phospho-ERK levels (Figure 4a). By contrast, co-stimulation of density cultures within a few hours of initiation of the culture. SMS-SB cells with SDF-1 and sCD23 stimulates a very robust U0126 treatment also abrogates the ERK phosphorylation driven ERK phosphorylation that is sustained until 10 min post- by SDF-1 or sCD23 individually and also completely prevents the stimulation, yielding phospho-ERK levels that are some 15-fold enhancement of ERK phosphorylation observed with SDF-1 and greater than either stimulus alone (Figure 4a and b). The 15F11 sCD23 co-stimulation (Figure 5c). The PI3K inhibitor LY294002 MAb induces ERK phosphorylation at 10–30 min post-stimula- also attenuates growth of SMS-SB cells in response to avb5 tion, whereas co-stimulation with SDF-1 and 15F11 promotes ligation, but this inhibition is less complete than that observed an earlier onset of ERK phosphorylation that is sustained to with U0126 (Figure 5b). Thus, after 48 h of LP stimulation, a small 30 min (Figure 4c). The RKC-containing CD23-derived peptide increase in [3H]-TdR incorporation remains evident in cells pre- no. 9 behaves similarly, inducing a modest response by itself treated with LY294002, whereas no growth is detectable after 12 h and a more rapid and sustained response when added together of U0126 treatment (Figure 5a and b). Analysis of SMS-SB cell with SDF-1; peptide no. 8, which lacks the RKC motif, fails to survival using annexinV/propidium iodide staining showed that induce significant phosphorylation either alone or in association U0126 treatment results in a higher proportion of annexinV/ with SDF-1 (Figure 4d). Finally, the RKC-containing pentadeca- propidium iodide double-positive cells than LY294002 treatment meric LP induces a sustained and robust ERK phosphorylation after a 12 h period (Figure 5d), illustrating that U0126 leads to that is enhanced by SDF-1 co-stimulation (Figure 4e). LP has an increased apoptosis in SMS-SB cells relative to LY294002 affinity for avb5 that is an order of magnitude greater than that of treatment. This underscores the importance of the ERK signalling CD23 itself (B10À7 vs 6 Â 10À6 M),2 and this greater affinity pathway in the survival of SMS-SB cells. may explain the higher and more sustained levels of ERK phosphorylation noted with LP. The data are consistent with the interpretation that ligation of avb5 by a soluble ligand alone PDGF enhances avb5-driven growth of pre-B cell lines promotes a modest level of ERK phosphorylation that is PDGF stimulates SMS-SB cell growth and the cells express enhanced by SDF-1; SDF-1 also drives a more rapid elevation PDGF receptor-b.48 Figure 6a and b, respectively, show that

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1812 Untreated sCD23 15F-11 25 pERK sCD23+SDF-1 pERK 20 sCD23 only SDF-1 only ERK 15 Actin

10 pERK +SDF-1 +SDF-1 pERK 5 Fold increase pERK Actin ERK 0 025103060 0 2 5 10 30 60 0 2 5 10 30 60 Time (min) 0 2 5 10 30 60

Time (min) Time (min)

Peptide 9 Peptide 8 Long Peptide

pERK pERK

ERK ERK

pERK +SDF-1 pERK + SDF-1

ERK Actin

0 2 5 10 30 60 0 2 5 10 30 60 0 2 5 10 30 60 Time (min) Time (min)

Figure 4 SDF-1 enhances ERK phosphorylation in cells treated with avb5 ligands. Lysates from treated SMS-SB cells were separated by SDS- polyacrylamide gel electrophoresis and transferred to nitrocellulose. Membranes were probed with antibodies to phosphorylated ERK and subsequently stripped and reprobed with antibodies to total protein (either ERK or actin). (a) Western blots of phosphorylated and total ERK in untreated SMS-SB cells and cells treated with sCD23 (160 ng/ml), SDF-1 (250 ng/ml) or a combination of sCD23 and SDF-1, at the time points shown. (b) The increase in phosphorylation was quantitated by densitometric analysis of the bands. (c) Western blots for phosphorylation of ERK and for total actin in SMS-SB cells treated with 15F-11 MAb alone or in combination with SDF-1. (d) Western blots for phosphorylated and total ERK in SMS-SB cells treated with peptide no. 9 (5 mg/ml), negative control peptide no. 8 (5 mg/ml). (e) Western blots for phosphorylation of ERK and for total actin in SMS-SB cells treated with LP (1 mg/ml) alone or in combination with SDF-1. LP, long peptide; sCD23, soluble CD23; SDF-1, stromal cell-derived factor-1.

PDGF-b significantly enhances both LP- and sCD23-induced cells (avb5 þ ) above background levels (Figure 7b), and growth of SMS-SB cells. As observed with SDF-1, PDGF co-stimulation with SDF-1 (Figure 7c) or PDGF (Figure 7d) with treatment does not alter surface expression of avb5 (Figure 6c) lower concentrations of sCD23 resulted in significant enhance- and, furthermore, the increase in [3H]-TdR incorporation ment in cell growth. The data show that normal bone marrow- promoted by sCD23 and PDGF treatment correlates well with derived B-cell precursors and patient-derived ALL cells can enhancement in ERK phosphorylation. Stimulation of SMS-SB respond to sCD23 and related ligands. The growth responses cells with PDGF alone results in significant ERK phosphory- were not as robust as those noted in the cell line models and lation, and co-stimulation with PDGF and sCD23 strikingly although we observed enhancement in cell growth with the enhances this ERK phosphorylation (Figure 6d). addition of SDF-1 and PDGF, the [3H]-TdR incorporation induced by suboptimal concentration of sCD23 in these cases was minimal. Response of normal bone marrow B cells and ALL cells to soluble avb5-specific stimulation B-cell precursors from the bone marrow that are positive for Discussion avb5 integrin also express CXCR4, and cells from patients with ALL also express the avb5 integrin and CXCR4. Growth of It is well established that chemokines dynamically regulate the BM-derived B-cell precursors and ALL cells was investigated positioning of cells of the immune system by influencing the to relate the data from pre-B cell lines with their normal and structure of integrin molecules to ensure that cells attracted to a malignant counterparts in the bone marrow. The ability of specific niche are retained there through adhesion reactions CD19 þ /kÀ fluorescence-activated cell-sorted B-cell precursors with tissue matrix proteins. Chemokines can also positively isolated from bone marrow aspirates to respond to stimulation influence signalling through integrins in such adhesion-depen- with soluble avb5-directed ligands was assessed. A robust dent interactions, as exemplified by the action of SDF-1 on incorporation of thymidine was stimulated by both LP (5 mg/ml) haematopoietic precursors at the level of the VLA-4–ICAM1 and by sCD23 (250 ng/ml) (Figure 7a), whereas a control peptide interaction.31,32 This report shows that SDF-1 positively devoid of the RKC motif, peptide no. 8, was without effect; influences signalling events delivered to integrins in a soluble, the cells also responded to the 15F11 MAb, indicating the adhesion-independent manner. Thus, ligation of avb5 using specificity of avb5 integrin in mediating cell growth (data not specific MAbs, sCD23 or peptides derived from sCD23 provokes shown). Second, both sCD23 and LP promote growth of ALL ERK phosphorylation and growth responses in B-cell precursors

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1813

Figure 5 Effect of inhibition of ERK and PI3K signalling on avb5-mediated growth of SMS-SB cells. SMS-SB cells were treated with 10 mM U0126 3 or 50 mM LY294002 or left untreated for 30 min before plating at LCD; cell growth was assessed by [ H]-TdR incorporation after 72 h of culture. (a) Cell proliferation with LP and/or SDF-1 in untreated cells and cells treated with U0126. (b) Cell growth stimulated by LP and/or SDF-1 in untreated cells or cells treated with LY294002. Unstimulated cells are represented as grey bars, LP treated as black bars, the LP and SDF-1 combination as white bars and SDF-1 treatment alone as hatched bars. Bars represent the s.d. of triplicate determinations, with *Po0.05 or **Po0.01 indicating a statistically signiﬁcant increase in proliferation of cells treated with different concentrations of SDF-1 and avb5 stimulants (LP) in comparison with SDF-1 treatment alone; #Po0.05 and ##Po0.01 denote the signiﬁcance of enhancement in cell growth with 250 ng/ml SDF-1 and avb5 stimulants when compared with avb5 stimulants alone. Data are representative of three independent experiments. (c) Western blot analysis of phosphorylated ERK and total actin in SMS-SB cells pre-treated with 10 mM U0126, 30 min before stimulation with SDF-1 (250 ng/ ml) or sCD23 (160 ng/ml) or a combination of SDF-1 and sCD23. (d) Contour plots for PI/annexinV staining of cells exposed to 10 mM U0126, 50 mM LY294002, dimethyl sulphoxide (vehicle) or no treatment for 12 h. Quadrant gates in the insets are based on unstained, annexinV or PI stains alone. LCD, low cell density; LP, long peptide; PI3K, phosphatidylinositol 3-kinase; sCD23, soluble CD23; SDF-1, stromal cell-derived factor-1.

that are enhanced by SDF-1. PDGF also enhances signalling antagonist; however, it has also been suggested to be a delivered by soluble ligation of avb5, suggesting that both weak partial agonist,51 which might partly explain the small tyrosine kinase- and G-protein-coupled receptor-linked path- apparent growth effect with AMD3100 alone. However, in the ways modulate adhesion-independent signalling by avb5. presence of SDF-1, AMD3100 clearly acts as a CXCR4 Growth of SMS-SB cells stimulated with sCD23 was enhanced antagonist. Importantly, AMD3100 has no effect on LP-induced by the addition of SDF-1 and similar responses were noted with cell growth but blocks the enhancement in growth with LP and sCD23-derived peptides (containing the RKC motif recognized SDF-1. Finally, the assessment of CXCR7 expression illustrated by avb5) and the anti-avb5 MAb 15F-11, all of which promote that SMS-SB cells have a modest level of CXCR7, Blin-1 cells growth of these cells. SDF-1 acts through CXCR7 as well as have no CXCR7 but express CXCR4, whereas IB4 cells lack through CXCR4, and to address the speciﬁcity of CXCR4 in CXCR4 but express CXCR7; IB4 cells do not respond to SDF-1 enhancing avb5-mediated signals, the binding of SDF-1 to stimulation (data not shown). Taken together, the data are CXCR4 was blocked using the CXCR4 antagonist consistent with the interpretation that SDF-1 mediates its effect AMD3100.49,50 AMD3100 completely abolished the ability of on avb5-stimulated cell growth by CXCR4. SDF-1 to enhance avb5-mediated SMS-SB cell growth Higher levels of CXCR4 expression do not inﬂuence the lack (Figure 1f); pre-treatment of SMS-SB cells with the anti-CXCR4 of response to avb5 ligation noted in the more mature cell lines, MAb (12G5) also inhibited the action of SDF-1 (data not shown). and this is consistent with data showing that although B-cell There is growth of SMS-SB cells with AMD3100 treatment alone precursors express higher levels of CXCR4 with increasing (Figure 1f). AMD3100 has been extensively used as CXCR4 maturation, the cell’s chemotactic response to SDF-1 itself is

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1814

Figure 6 PDGF enhances avb5-driven growth and ERK phosphorylation in SMS-SB cells. LCD SMS-SB cultures were stimulated with the indicated concentrations of PDGFb and 1 mg/ml LP (a) or 160 ng/ml sCD23 (b) for 72 h before determination of [3H]-TdR incorporation. Growth induced by PDGF and LP or sCD23 in combination with PDGF is shown as black bars and that driven by PDGF alone is shown as hatched bars. Bars represent the s.d. of triplicate determinations, with *Po0.05 or **Po0.01 indicating the statistical signiﬁcance of the increase in growth of cells treated with different concentrations of SDF-1 and avb5 stimulants (sCD23 or LP) in comparison with SDF-1 treatment alone; #Po0.05 and ##Po0.01 indicate the signiﬁcance of the enhancement of cell growth with 250 ng/ml SDF-1 and avb5 stimulants when compared with avb5 stimulants alone. (c) SMS-SB cells untreated or treated with 250 ng/ml PDGF (24 h) were stained for avb5 expression using the anti-avb5 MAbs P1F-6 and 15F-11. Staining with the relevant avb5 MAbs is represented by the grey shaded area and isotype control antibody staining is represented by the black line. (d) Western blots for phosphorylated ERK and ERK2 in SMS-SB cells treated with PDGF (250 ng/ml) alone or PDGF (250 ng/ml) with sCD23 (160 ng/ml). LCD, low cell density; LP, long peptide; PDGF, platelet-derived growth factor; [3H]-TdR, tritiated thymidine; sCD23, soluble CD23; SDF-1, stromal cell-derived factor-1.

apparently blunted. Thus, CXCR4 is continuously expressed by IL-4 and IL-11 at the concentrations used. It has been shown B cells and, as the cells mature and begin to express surface IgM, that SDF-1, IL-3 and IL-7 induce proliferation of ALL cells the chemotactic response to SDF-1 is lost despite the sustained co-cultured with stromal cells; IL-7 alone did not induce ERK expression of CXCR4.52–54 In agreement with these results, our activation but there was a synergistic ERK activation following data support the notion that avb5-mediated cell growth is IL-7 and SDF-1 co-stimulation.57 However, in the co-culture important at particular stages of B-cell development and is studies, the ALL cells were propagated on stromal cells and modulated by SDF-1 in a stage-specific manner. effects on ERK and other signalling pathways would have been SMS-SB cells undergo apoptosis in low-density culture affected by multiple adhesion contacts between the ALL cells conditions.3 The inhibition of cell growth by U0126 shows that and stromal cells. Therefore, the data from the ALL-stromal cell basal ERK phosphorylation is necessary for the survival of SMS- co-culture model illustrate convincingly that ERK phosphory- SB cells; in the presence of U0126, the cells rapidly undergo lation can occur in an adhesion-dependent manner.57 apoptosis. ERK phosphorylation observed following sCD23, In our own studies of normal bone marrow B cells and ALL- RKC-containing peptide or 15F-11 treatment shows that growth derived cells, cultures that lacked serum or stromal cell support induced by the ligation of avb5 with sCD23 is regulated through failed to support any growth of the cells. Using bone marrow the ERK signalling pathway. The enhancement in cell growth B-cell precursors propagated on irradiated stromal cells, we noted with SDF-1 and sCD23 correlated well with a sustained were able to show that avb5 ligation by sCD23 induces growth increase in ERK phosphorylation. This was verified by similar response in these precursors, similar to that seen in cell lines. enhancements observed when CD23-derived peptides and The importance of this was further assessed in ALL cells to 15F-11 antibody were used as stimuli. However, it cannot be investigate if SDF-1 could enhance this avb5-mediated growth excluded that other signalling pathways might be involved in response. Cultures with stromal cells supported modest growth regulating SMS-SB cell growth, and the observation that of ALL cells exposed to avb5 stimulants alone and some degree LY294002 inhibited the enhancement of avb5-mediated cell of enhancement of sCD23-driven growth of ALL cells by SDF-1 growth by SDF-1 indicates that AKT has the potential to regulate was achieved. The data indicate that normal marrowcytes adhesion-independent signalling by avb5. This would be respond to sCD23 and that in ALL cells, some additive effect of consistent with studies that show that SDF-1 activates both avb5-directed ligands and other receptors can be elicited. The ERK and AKT38,55,56 and merits further investigation. finding that sCD23 can induce ERK phosphorylation in B-cell Many cytokines are known to modulate integrin function and precursor cell lines is consistent with data from studies of B-cell precursor survival, but no similar enhancement of avb5- monocytes that showed strong ERK responses to integrin ligation mediated cell growth was promoted by the cytokines IL-7, IL-3, and sCD23 stimulation,58,59 and it remains to be determined

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1815

Figure 7 Response of normal B-cell precursors and ALL cells to avb5 ligands. Aliquots of kÀ ﬂuorescence-activated cell-sorted human bone marrow B cells (a) or cells derived from samples from ALL patients (b) were stimulated with 5 mg/ml peptide no. 8, LP or with 250 ng/ml sCD23 for 48 h before pulse with tritiated thymidine. Aliquots of ALL cells were also stimulated with 150 ng/ml sCD23 in the presence or absence of 250 ng/ml SDF-1 (c) or PDGF (d), before assessment of cell growth. Bars represent the s.d. of triplicate determinations, with *Po0.05. LP, long peptide; PDGF, platelet-derived growth factor; sCD23, soluble CD23; SDF-1, stromal cell-derived factor-1

whether sCD23 and other avb5 ligands can induce ERK phos- A range of adhesion and signalling molecules controlling each phorylation in normal B-cell precursors and ALL cells. stage are not only key to understanding the progression into the Finally, PDGF enhanced the cell growth induced by avb5ina developmental pathway but also in developing therapies for manner similar to that observed with SDF-1. Interestingly, a neoplasias that arise in different stages of B-cell development. previous study shows that early pre-B cells, but not mature B cells, express the PDGF receptor-b, and PDGF was shown to promote proliferation of these cells but not immature B cells.60 Acknowledgements A number of studies, particularly in adherent cell systems, have shown a cross-talk between growth factors and integrin MA and ALE were postgraduate scholars supported by the signalling, and synergy between growth factors and cell Wellcome Trust four-year PhD programme, Molecular Functions adhesion has been observed in the activation of the MAPK61,62 in Disease. BWO is supported by Cancer Research UK. and PI3K/AKT pathways.63 Moreover, PDGF has previously been shown to interact physically with the avb3 integrin to References stimulate proliferation of oligodendrocytes by the activation of the integrin in an adhesion-dependent manner.64 Therefore, it is 1 LeBien TW. Fates of human B-cell precursors. Blood 2000; 96: plausible that PDGF and avb5 also interact to promote 9–23. adhesion-independent proliferation in B-cell precursors. Our 2 Borland G, Edkins AL, Acharya M, Matheson J, White LJ, Allen JM et al. alphavbeta5 integrin sustains growth of human pre-B cells data indicate that avb5 synergizes with two distinct signalling through an RGD-independent interaction with a basic domain of pathways, through a tyrosine kinase receptor (PDGF receptor-b) the CD23 protein. J Biol Chem 2007; 282: 27315–27326. and a G-protein-coupled receptor (CXCR4), to promote ERK 3 White LJ, Ozanne BW, Graber P, Aubry JP, Bonnefoy JY, Cushley phosphorylation and pre-B-cell growth. It will be important to W. Inhibition of apoptosis in a human pre-B-cell line by CD23 is determine the common molecules downstream of both these mediated via a novel receptor. Blood 1997; 90: 234–243. pathways leading to ERK phosphorylation to understand further 4 Lecoanet-Henchoz S, Gauchat JF, Aubry JP, Graber P, Life P, Paul- Eugene N et al. CD23 regulates monocyte activation through a the mechanisms involved in avb5-mediated cell growth. novel interaction with the adhesion molecules CD11b-CD18 and The model emerging from these data suggests a role for avb5 CD11c-CD18. Immunity 1995; 3: 119–125. integrin in B-cell precursor survival and growth that can be 5 Lecoanet-Henchoz S, Plater-Zyberk C, Graber P, Gretener D, modulated by other biological effectors such as SDF-1 and Aubry JP, Conrad DH et al. Mouse CD23 regulates monocyte PDGF. This may have important implications for growth of activation through an interaction with the adhesion molecule normal and neoplastic B cells. B-cell development in different CD11b/CD18. Eur J Immunol 1997; 27: 2290–2294. 6 Hermann P, Armant M, Brown E, Rubio M, Ishihara H, Ulrich D compartments of the haematopoietic microenvironment is et al. The vitronectin receptor and its associated CD47 molecule accompanied by stage-speciﬁc changes that are critical for mediates proinﬂammatory cytokine synthesis in human monocytes proliferation and differentiation into later developmental stages. by interaction with soluble CD23. J Cell Biol 1999; 144: 767–775.

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1816 7 Liu YJ, Cairns JA, Holder MJ, Abbot SD, Jansen KU, Bonnefoy JY 28 Hartmann TN, Burger JA, Glodek A, Fujii N, Burger M. CXCR4 et al. Recombinant 25-kDa CD23 and interleukin 1 alpha promote chemokine receptor and integrin signaling co-operate in mediating the survival of germinal center B cells: evidence for bifurcation in adhesion and chemoresistance in small cell lung cancer (SCLC) the development of centrocytes rescued from apoptosis. Eur J cells. Oncogene 2005; 24: 4462–4471. Immunol 1991; 21: 1107–1114. 29 Hidalgo A, Sanz-Rodriguez F, Rodriguez-Fernandez JL, Albella B, 8 Bonnefoy JY, Lecoanet-Henchoz S, Aubry JP, Gauchat JF, Blaya C, Wright N et al. Chemokine stromal cell-derived factor- Graber P. CD23 and B-cell activation. Curr Opin Immunol 1995; 1alpha modulates VLA-4 integrin-dependent adhesion to fibro- 7: 355–359. nectin and VCAM-1 on bone marrow hematopoietic progenitor 9 Aubry JP, Pochon S, Graber P, Jansen KU, Bonnefoy JY. CD21 is a cells. Exp Hematol 2001; 29: 345–355. ligand for CD23 and regulates IgE production. Nature 1992; 358: 30 Liesveld JL, Dipersio JF, Abboud CN. Integrins and adhesive receptors 505–507. in normal and leukemic CD34+ progenitor cells: potential regulatory 10 Bansal A, Roberts T, Hay EM, Kay R, Pumphrey RS, Wilson PB. checkpoints for cellular traffic. Leuk Lymphoma 1994; 14: 19–28. Soluble CD23 levels are elevated in the serum of patients with 31 Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V primary Sjogren’s syndrome and systemic lupus erythematosus. et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, Clin Exp Immunol 1992; 89: 452–455. and VLA-5 on immature human CD34(+) cells: role in transen- 11 Sarfati M. CD23 and chronic lymphocytic leukemia. Blood Cells dothelial/stromal migration and engraftment of NOD/SCID mice. 1993; 19: 591–596; discussion 597–9. Blood 2000; 95: 3289–3296. 12 Hynes RO. Integrins: bidirectional, allosteric signaling machines. 32 Shen W, Bendall LJ, Gottlieb DJ, Bradstock KF. The chemokine Cell 2002; 110: 673–687. receptor CXCR4 enhances integrin-mediated in vitro adhesion and 13 Kumar CC. Signaling by integrin receptors. Oncogene 1998; 17 facilitates engraftment of leukemic precursor-B cells in the bone (11 Reviews): 1365–1373. marrow. Exp Hematol 2001; 29: 1439–1447. 14 Ruoslahti E. RGD and other recognition sequences for integrins. 33 Broxmeyer HE, Kim CH. Regulation of hematopoiesis in a sea of Annu Rev Cell Dev Biol 1996; 12: 697–715. chemokine family members with a plethora of redundant 15 Ashley DM, Bol SJ, Kannourakis G. Human bone marrow stromal activities. Exp Hematol 1999; 27: 1113–1123. cell contact and soluble factors have different effects on the 34 Nishii K, Katayama N, Miwa H, Shikami M, Masuya M, Shiku H survival and proliferation of paediatric B-lineage acute lympho- et al. Survival of human leukaemic B-cell precursors is supported blastic leukaemic blasts. Leuk Res 1994; 18: 337–346. by stromal cells and cytokines: association with the expression of 16 Bradstock K, Makrynikola V, Bianchi A, Byth K. Analysis of the bcl-2 protein. Br J Haematol 1999; 105: 701–710. mechanism of adhesion of precursor-B acute lymphoblastic 35 Juarez J, Bendall L. SDF-1 and CXCR4 in normal and malignant leukemia cells to bone marrow fibroblasts. Blood 1993; 82: hematopoiesis. Histol Histopathol 2004; 19: 299–309. 3437–3444. 36 Lataillade JJ, Clay D, Dupuy C, Rigal S, Jasmin C, Bourin P et al. 17 Manabe A, Coustan-Smith E, Behm FG, Raimondi SC, Campana D. Chemokine SDF-1 enhances circulating CD34(+) cell proliferation Bone marrow-derived stromal cells prevent apoptotic cell death in synergy with cytokines: possible role in progenitor survival. in B-lineage acute lymphoblastic leukemia. Blood 1992; 79: Blood 2000; 95: 756–768. 2370–2377. 37 Arai A, Jin A, Yan W, Mizuchi D, Yamamoto K, Nanki T et al. SDF- 18 Nagasawa T, Nakajima T, Tachibana K, Iizasa H, Bleul CC, 1 synergistically enhances IL-3-induced activation of the Raf-1/ Yoshie O et al. Molecular cloning and characterization of a murine MEK/Erk signaling pathway through activation of Rac and its pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 effector Pak kinases to promote hematopoiesis and chemotaxis. receptor, a murine homolog of the human immunodeficiency virus Cell Signal 2005; 17: 497–506. 1 entry coreceptor fusin. Proc Natl Acad Sci USA 1996; 93: 38 Bendall LJ, Baraz R, Juarez J, Shen W, Bradstock KF. Defective p38 14726–14729. mitogen-activated protein kinase signaling impairs chemotaxic but 19 Balabanian K, Lagane B, Infantino S, Chow KY, Harriague J, not proliferative responses to stromal-derived factor-1alpha in Moepps B et al. The chemokine SDF-1/CXCL12 binds to and acute lymphoblastic leukemia. Cancer Res 2005; 65: 3290–3298. signals through the orphan receptor RDC1 in T lymphocytes. J Biol 39 Cheng ZJ, Zhao J, Sun Y, Hu W, Wu YL, Cen B et al. Beta-arrestin Chem 2005; 280: 35760–35766. differentially regulates the chemokine receptor CXCR4-mediated 20 Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao signaling and receptor internalization, and this implicates multiple Z et al. A novel chemokine receptor for SDF-1 and I-TAC involved interaction sites between beta-arrestin and CXCR4. J Biol Chem in cell survival, cell adhesion, and tumor development. J Exp Med 2000; 275: 2479–2485. 2006; 203: 2201–2213. 40 Dutt P, Wang JF, Groopman JE. Stromal cell-derived factor-1 21 Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto alpha and stem cell factor/kit ligand share signaling pathways in T et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed hemopoietic progenitors: a potential mechanism for cooperative cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. induction of chemotaxis. J Immunol 1998; 161: 3652–3658. Proc Natl Acad Sci USA 1998; 95: 9448–9453. 41 Ganju RK, Brubaker SA, Meyer J, Dutt P, Yang Y, Qin S et al. The 22 Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, alpha-chemokine, stromal cell-derived factor-1alpha, binds to the Kitamura Y et al. Defects of B-cell lymphopoiesis and bone- transmembrane G-protein-coupled CXCR-4 receptor and activates marrow myelopoiesis in mice lacking the CXC chemokine PBSF/ multiple signal transduction pathways. J Biol Chem 1998; 273: SDF-1. Nature 1996; 382: 635–638. 23169–23175. 23 Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K, Kataoka 42 Lataillade JJ, Clay D, Bourin P, Herodin F, Dupuy C, Jasmin C et al. Y et al. The chemokine receptor CXCR4 is essential for vasculari- Stromal cell-derived factor 1 regulates primitive hematopoiesis by zation of the gastrointestinal tract. Nature 1998; 393: 591–594. suppressing apoptosis and by promoting G(0)/G(1) transition in 24 Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. CD34(+) cells: evidence for an autocrine/paracrine mechanism. Function of the chemokine receptor CXCR4 in haematopoiesis and Blood 2002; 99: 1117–1129. in cerebellar development. Nature 1998; 393: 595–599. 43 Wang JF, Park IW, Groopman JE. Stromal cell-derived factor- 25 Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC. The 1alpha stimulates tyrosine phosphorylation of multiple focal chemokine SDF-1 is a chemoattractant for human CD34+ adhesion proteins and induces migration of hematopoietic hematopoietic progenitor cells and provides a new mechanism progenitor cells: roles of phosphoinositide-3 kinase and protein to explain the mobilization of CD34+ progenitors to peripheral kinase C. Blood 2000; 95: 2505–2513. blood. J Exp Med 1997; 185: 111–120. 44 Eliceiri BP. Integrin and growth factor receptor crosstalk. Circ Res 26 Bleul CC, Fuhlbrigge RC, Casasnovas JM, Aiuti A, Springer TA. 2001; 89: 1104–1110. A highly efficacious lymphocyte chemoattractant, stromal cell- 45 Smith RG, Dev VG, Shannon Jr WA. Characterization of a novel derived factor 1 (SDF-1). J Exp Med 1996; 184: 1101–1109. human pre-B leukemia cell line. J Immunol 1981; 126: 596–602. 27 Bradstock KF, Gottlieb DJ. Interaction of acute leukemia cells 46 Stong RC, Korsmeyer SJ, Parkin JL, Arthur DC, Kersey JH. Human with the bone marrow microenvironment: implications for acute leukemia cell line with the t(4;11) chromosomal rearrange- control of minimal residual disease. Leuk Lymphoma 1995; 18: ment exhibits B lineage and monocytic characteristics. Blood 1–16. 1985; 65: 21–31.

Leukemia SDF-1 and PDGF enhance avb5 signalling in human pre-B cells M Acharya et al 1817 47 Simonsen JL, Rosada C, Serakinci N, Justesen J, Stenderup K, 56 Barbero S, Bonavia R, Bajetto A, Porcile C, Pirani P, Ravetti JL Rattan SI et al. Telomerase expression extends the proliferative life- et al. Stromal cell-derived factor 1alpha stimulates human span and maintains the osteogenic potential of human bone glioblastoma cell growth through the activation of both extra- marrow stromal cells. Nat Biotechnol 2002; 20: 592–596. cellular signal-regulated kinases 1/2 and Akt. Cancer Res 2003; 63: 48 Tsai LH, White L, Raines E, Ross R, Smith RG, Cushley W et al. 1969–1974. Expression of platelet-derived growth factor and its receptors by 57 Juarez J, Baraz R, Gaundar S, Bradstock K, Bendall L. Interaction of two pre-B acute lymphocytic leukemia cell lines. Blood 1994; 83: interleukin-7 and interleukin-3 with the CXCL12-induced proli- 51–55. feration of B-cell progenitor acute lymphoblastic leukemia. 49 Hendrix CW, Flexner C, MacFarland RT, Giandomenico C, Fuchs Haematologica 2007; 92: 450–459. EJ, Redpath E et al. Pharmacokinetics and safety of AMD-3100, a 58 Rezzonico R, Chicheportiche R, Imbert V, Dayer JM. Engagement novel antagonist of the CXCR-4 chemokine receptor, in human of CD11b and CD11c beta2 integrin by antibodies or soluble volunteers. Antimicrob Agents Chemother 2000; 44: 1667–1673. CD23 induces IL-1beta production on primary human monocytes 50 Schols D, Este JA, Henson G, De Clercq E. Bicyclams, a class of through mitogen-activated protein kinase-dependent pathways. potent anti-HIV agents, are targeted at the HIV coreceptor fusin/ Blood 2000; 95: 3868–3877. CXCR-4. Antiviral Res 1997; 35: 147–156. 59 Rezzonico R, Imbert V, Chicheportiche R, Dayer JM. Ligation of 51 Zhang WB, Navenot JM, Haribabu B, Tamamura H, Hiramatu K, CD11b and CD11c beta(2) integrins by antibodies or soluble Omagari A et al. A point mutation that confers constitutive activity CD23 induces macrophage inﬂammatory protein 1alpha (MIP- to CXCR4 reveals that T140 is an inverse agonist and that 1alpha) and MIP-1beta production in primary human monocytes AMD3100 and ALX40-4C are weak partial agonists. J Biol Chem through a pathway dependent on nuclear factor-kappaB. Blood 2002; 277: 24515–24521. 2001; 97: 2932–2940. 52 D’Apuzzo M, Rolink A, Loetscher M, Hoxie JA, Clark-Lewis I, 60 Trink B, Wang G, Shahar M, Meydan N, Roifman CM. Functional Melchers F et al. The chemokine SDF-1, stromal cell-derived factor platelet-derived growth factor-beta (PDGF-beta) receptor 1, attracts early stage B cell precursors via the chemokine receptor expressed on early B-lineage precursor cells. Clin Exp Immunol CXCR4. Eur J Immunol 1997; 27: 1788–1793. 1995; 102: 417–424. 53 Egawa T, Kawabata K, Kawamoto H, Amada K, Okamoto R, 61 Lin TH, Aplin AE, Shen Y, Chen Q, Schaller M, Romer L et al. Fujii N et al. The earliest stages of B cell development require a Integrin-mediated activation of MAP kinase is independent of FAK: chemokine stromal cell-derived factor/pre-B-cell growth-stimula- evidence for dual integrin signaling pathways in ﬁbroblasts. J Cell ting factor. Immunity 2001; 15: 323–334. Biol 1997; 136: 1385–1395. 54 Fedyk ER, Ryyan DH, Ritterman I, Springer TA. Maturation 62 Renshaw MW, Ren XD, Schwartz MA. Growth factor activation of decreases responsiveness of human bone marrow B lineage MAP kinase requires cell adhesion. EMBO J 1997; 16: 5592–5599. cells to stromal-derived factor 1 (SDF-1). J Leukoc Biol 1999; 66: 63 Green DR. Apoptotic pathways: the roads to ruin. Cell 1998; 94: 667–673. 695–698. 55 Bajetto A, Bonavia R, Barbero S, Florio T, Costa A, Schettini G. 64 Baron W, Shattil SJ, ffrench-Constant C. The oligodendrocyte Expression of chemokine receptors in the rat brain. Ann N Y Acad precursor mitogen PDGF stimulates proliferation by activation of Sci 1999; 876: 201–209. alpha(v)beta3 integrins. EMBO J 2002; 21: 1957–1966.

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