Extracellular DEK Is a Novel Chemotactic Agent of Lineage Negative, Sca-1 Positive, c-Kit Positive Bone Marrow Cells

DEK, a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is known to be secreted by macrophages and act as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK is known to function as a chemotactic factor that attracts neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells, SDF-1 being the most potent of proteins with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative, sca-1 positive, c-kit positive (LSK) mouse bone marrow and neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced migration of LSK cells at a dose of 100ng/mL, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ cells. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with the maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration was observed when IL-8 or MIP-2 was utilized. When Ly6G+ cells were used CXCR2 neutralizing antibodies blocked the migration of Ly6G+ cells towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ migration towards SDF-1. CXCR2 is known to be a G protein-coupled receptor and this interaction can be blocked through the use of pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro. Thus, DEK may be involved in migration and homing of HSCs and HPCs.