DEK Regulates Hematopoietic Stem and Progenitor Cell Fate By Activating AKT and ERK Mediated Signaling through CXCR2/Heparan Sulfate Proteoglycans

DEK is a nuclear DNA-binding protein that has been implicated in the regulation of transcription, DNA repair, mRNA processing, and chromatin remodeling. Endogenous DEK regulates hematopoiesis, as bone marrow (BM) from DEK^-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term (LT) and secondary hematopoietic stem cell (HSC) engrafting capability (Broxmeyer et al., 2012 & 2013). DEK is also secreted and found in extracellular spaces. We recently demonstrated that extracellular DEK decreases the number and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo and in vitro. Of importance, in vivo administration of recombinant mouse (rm)DEK significantly increased the number of phenotypic LT-HSC.Moreover, DEK significantly enhanced the ex vivo expansion of rigorously-defined and functional HSC of human CD34⁺ cord blood (CB) and mouse lineage negative BM cells. This suggests that DEK may enhance HSC numbers by blocking the production of HPC and thus acting as a fate determinant.

Upon finding that DEK has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we explored whether exogenous DEK functioned by signaling through CXCR2, the receptor that binds and mediates ELR+ CXC chemokine function. By blocking CXCR2 and utilizing CXCR2^-/- BM, we demonstrated that DEK regulation of HPC and HSC numbers is CXCR2-dependent. New studies now show that DEK signals through Gai-protein coupled receptor signaling; pretreating BM cells with pertussis toxin inhibited DEK's regulatory function in HPCs, a process unique to DEK as other ELR+ CXC chemokines (i.e. IL-8) were insensitive to the inhibitory effects of pertussis toxin. To determine which CXCR2-mediated signaling pathway DEK utilizes, we performed intracellular staining of mouse BM cells for phosphorylated AKT, ERK1/2, p38MAPK, and STAT3 following 15, 30, and 60 minute stimulation with rmDEK; rmSCF, rhSDF1a and rhIL-8 were used as positive controls. DEK stimulated phosphorylation of AKT and ERK1/2, but not p38MAPK or STAT3 in phenotypically-defined HSCs, a process blocked when BM cells were pretreated with CXCR2 neutralizing antibody, suggesting that DEK induces signaling through CXCR2. Activation of AKT can lead to NF-kB p65 translocation from the cytoplasm to the nucleus and intracellular DEK has been implicated in regulating this process in HeLa cells (Sammons et al., 2006). However, using ImageStream we saw no NF-kB p65 translocation in DEK-treated BM HSCs as compared to a TNFa positive control.

We previously showed that DEK binds heparan sulfate proteoglycans (HSPGs; Saha et al., 2014). DEK also functions in an HSPG-dependent manner to regulate HPC and HSC numbers. HSPGs are important in chemokine signaling as they help in chemokine presentation. Blocking DEK's ability to bind to HSPGs by pretreating BM cells with heparin blocked DEK-mediated phosphorylation of ERK1/2 and AKT in HSCs, suggesting that HSPGs are also important in DEK-mediated CXCR2 signaling. DEK^-/- BM LSK cells can internalize extracellular rmDEK, resulting in an increase in the heterochromatin marker H3K9Me3 in the nucleus. We now know that this process is dependent on the ability of DEK to bind to HSPGs and be internalized since the trimethylation of H3K9 is inhibited by pretreating cells with heparin or with Pitstop2, an inhibitor of HSPG-mediated endocytosis. This process is independent of CXCR2, as blocking CXCR2 does not inhibit the increases in H3K9Me3 seen in the nucleus of DEK^-/- LSK cells following DEK treatment. These data suggest that DEK utilizes HSPGs in two separate ways: 1) as a co-receptor to mediate CXCR2 signaling and 2) as a means to endocytose DEK, leading to heterochromatin stabilization.