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

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5-Mediated ERK Activation and Adhesion-Independent Growth of Human Pre-B Cell Lines Leukemia (2009) 23, 1807–1817 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE 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 prolifera- tion 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).
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