Recombinant DEK Regulates Hematopoietic Stem and Progenitor Cells in a CXCR2- GαI Protein-Dependent Manner

DEK, a non-histone nuclear phosphoprotein involved in heterochromatin remodeling, is released from cells and can regulate hematopoiesis (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Marrow from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cells (HSC) engrafting capability. Moreover, recombinant mouse (rmu) DEK inhibited HPC colony formation in vitro. We now show that rmuDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being much more sensitive than control mice to this suppression. In addition, rmuDEK treatment of WT mice in vivo reversibly reduces the phenotypic number of ST-HSC, MPP, CMP, GMP, and MEP in the marrow. In vivo administration of rmuDEK to DEK-/- mice greatly enhanced the number of phenotypic LT-HSC and functional HSC in these mice (competitive HSC repopulation in lethally irradiated mice). DEK-/- mice are also more sensitive to 5-FU than control mice resulting in slower hematopoietic cell recovery, possibly due to the enhanced cycling status of HPC in DEK-/- mice. To determine the biological role of extracellular DEK, we utilized a reagent that could inactivate its function. This inactivating agent, but not its control, neutralized the inhibitory effect of rmuDEK. In addition, treating marrow cells in vitro with truncated rmuDEK by pretreating the DEK with the enzyme DPP4 (which DEK has targeted truncation sites for) also blocked the inhibitory effects of DEK suggesting that DEK must be in its full length form in order to perform its function

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. To determine whether rmuDEK’s inhibitory function is mediated through the CXCR2 receptor, a neutralizing monoclonal antibody against CXCR2 was utilized. Marrow treated in vitro with rmuDEK, rhIL-8, or rmuMIP-2 inhibited colony formation; however pretreating marrow cells with the neutralizing CXCR2 antibody blocked the inhibitory effect of these proteins. Marrow treated with rmuMIP-1α (a chemokine that does not bind to CXCR2) also inhibited colony formation; however neutralizing CXCR2 antibody had no effect on the ability of MIP-1α to inhibit colony formation. Neutralizing CXCR4, a chemokine receptor that binds SDF-1 but not IL-8, MIP-2 or MIP-1α, had no effect on the inhibition of colony formation. DEK inhibition of CFU-GM colony formation is dependent on Gαi-protein-coupled receptor signaling as determined through the use of pertussis toxin. This is a unique mechanism since IL-8 and MIP-1α had been previously reported by us to be insensitive to the inhibitory effects of pertussis toxin.

As extracellular DEK can remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 25: 673; Saha et al., 2013, PNAS, 110: 6847), we next assessed the effects of DEK on the heterchromatin marker H3K9Me3 in the nucleus of purified mouse Lineage negative, Sca-1 positive, c-kit positive (LSK) marrow cells by imaging flow cytometry. RmuDEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK marrow cells indicating that rmuDEK can be internalized by LSK cells and mediate heterchromatin formation. These results add to emerging evidence that DEK, a unique nuclear protein, plays a role in regulating hematopoiesis through a CXCR2/Gαi protein signaling pathway. Its modulation may be of practical use.