Secreted nuclear protein DEK regulates hematopoiesis through CXCR2 signaling
Maegan L. Capitano, Nirit Mor-Vaknin, Anjan K. Saha, Scott Cooper, Maureen Legendre, Haihong Guo, Rafael Contreras-Galindo, Ferdinand Kappes, Maureen A. Sartor, Christopher T. Lee, Xinxin Huang, David M. Markovitz, Hal E. Broxmeyer
Maegan L. Capitano, Nirit Mor-Vaknin, Anjan K. Saha, Scott Cooper, Maureen Legendre, Haihong Guo, Rafael Contreras-Galindo, Ferdinand Kappes, Maureen A. Sartor, Christopher T. Lee, Xinxin Huang, David M. Markovitz, Hal E. Broxmeyer
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Research Article Hematology

Secreted nuclear protein DEK regulates hematopoiesis through CXCR2 signaling

Abstract

The nuclear protein DEK is an endogenous DNA-binding chromatin factor regulating hematopoiesis. DEK is one of only 2 known secreted nuclear chromatin factors, but whether and how extracellular DEK regulates hematopoiesis is not known. We demonstrated that extracellular DEK greatly enhanced ex vivo expansion of cytokine-stimulated human and mouse hematopoietic stem cells (HSCs) and regulated HSC and hematopoietic progenitor cell (HPC) numbers in vivo and in vitro as determined both phenotypically (by flow cytometry) and functionally (through transplantation and colony formation assays). Recombinant DEK increased long-term HSC numbers and decreased HPC numbers through a mechanism mediated by the CXC chemokine receptor CXCR2 and heparan sulfate proteoglycans (HSPGs) (as determined utilizing Cxcr2–/– mice, blocking CXCR2 antibodies, and 3 different HSPG inhibitors) that was associated with enhanced phosphorylation of ERK1/2, AKT, and p38 MAPK. To determine whether extracellular DEK required nuclear function to regulate hematopoiesis, we utilized 2 mutant forms of DEK: one that lacked its nuclear translocation signal and one that lacked DNA-binding ability. Both altered HSC and HPC numbers in vivo or in vitro, suggesting the nuclear function of DEK is not required. Thus, DEK acts as a hematopoietic cytokine, with the potential for clinical applicability.

Authors

Maegan L. Capitano, Nirit Mor-Vaknin, Anjan K. Saha, Scott Cooper, Maureen Legendre, Haihong Guo, Rafael Contreras-Galindo, Ferdinand Kappes, Maureen A. Sartor, Christopher T. Lee, Xinxin Huang, David M. Markovitz, Hal E. Broxmeyer

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Figure 4

Inhibition of HPC numbers by DEK is CXCR2 and Gαi dependent.

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Inhibition of HPC numbers by DEK is CXCR2 and Gαi dependent. (A–C) HPC C...
(A–C) HPC CFU assays with rmDEK (A), rhIL-8 (B), or rmMIP2 (C) using either Dek–/– or WT BM cells. **P < 0.01, ***P < 0.001 comparing doses between WT and Dek–/– BM cells. (D) HPC CFU assay examining the effect of pretreating rmDEK, rhIL-8, rmMIP2, or rmMIP1α with DEK-targeting aptamers (DTA-64 and DTA-85+ends) or control vehicle and DTA library. (E) HPC CFU assay examining whether pretreatment of WT BM cells with anti-CXCR2 blocking antibody inhibits rmDEK, rhIL-8, rmMIP2, or rmMIP1α effects. Vehicle, isotype antibody, and anti-CXCR4 blocking antibody were used as controls. **P < 0.01 compared with control. In A–E, data are mean ± SD of triplicate plates. (F–H) Cxcr2–/– or WT mice were injected s.c. with 10 μg dialyzed rmDEK or vehicle once a day for 2 days. BM was harvested 48 hours after final injection. HPC number was determined by CFU assay. Data are mean ± SEM of 4 mice per group plated in triplicate. *P < 0.05, **P < 0.01 compared with vehicle-treated WT mice. (I) HPC CFU assay examining whether full-length (FL) or DPP4-truncated (T) rmDEK or IL-8 alone or mixed at a 1:1 ratio affects C57BL/6 BM colony formation when pretreated with anti-CXCR2 blocking antibody or isotype control. Percent inhibition was calculated based on counts from plates with control media added. (J) HPC CFU colony assay using C57BL/6 BM cells that were pretreated with PT (Gai inhibitor) or vehicle control, then cultured with rmDEK, rhIL-8, rmMIP2, rmMIP1α, or rm6Ckine. In I and J, data are mean ± SD of triplicate plates. **P < 0.01, ***P < 0.001 compared with control. For A–J, 1-way ANOVA with post hoc Tukey’s multiple-comparisons test was used.