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 6

Extracellular DEK does not require its nuclear function to regulate hematopoiesis.

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Extracellular DEK does not require its nuclear function to regulate hema...
(A) Analysis of H3K9me3 levels in the nucleus of Dek–/– LSK cells incubated with vehicle, WT rmDEK, GST-tagged WT rhDEK (GST WT rhDEK), GST-tagged mutant DNA-binding motif rhDEK (GST rhDBM-DEK), or GST-tagged mutant NLS rhDEK (GST rhNLS-DEK). Data are mean ± SD of triplicate tubes. *P < 0.05 compared with percent H3K9me3 levels in the nucleus of the vehicle group. (B) HPC colony assay examining the effect of different doses of WT or mutant DEK. Data are mean ± SD of triplicate plates. *P < 0.05, **P < 0.01 compared with vehicle control. (C) Number of LT-HSCs in day 0 input of C57BL/6 Lin– BM cells and number of LT-HSCs 4 days after culture in expansion media with vehicle or WT or mutant DEK was determined. Data are ± SEM of fold change from input LT-HSC numbers of 3 pools of 2 mice. *P < 0.05, **P < 0.01 compared with vehicle control; ‡P < 0.05 compared with WT rmDEK or GST WT rhDEK. (D–F) C57BL/6 mice were injected s.c. with vehicle or 10 μg dialyzed WT or mutant DEK once a day for 2 days. BM was harvested 48 hours after final injection, and immunophenotyping of LT-HSCs (D), ST-HSCs (E), and MPPs (F) was performed. Data are mean ± SEM of 6 mice per group. *P < 0.05 compared with vehicle-treated mice. (G–I) HPC number from the mice used in D–F was determined by CFU assay. The percentage of HPCs at the time of isolation in cycle was determined by 3HTdr assays (J–L). Data are mean ± SEM of 6 mice per group plated in triplicate. ***P < 0.001 compared with vehicle-treated WT mice. For A–L, 1-way ANOVA with post hoc Tukey’s multiple-comparisons test was used.