Secreted nuclear protein DEK regulates hematopoiesis through CXCR2 signaling
Maegan L. Capitano, … , David M. Markovitz, Hal E. Broxmeyer
Maegan L. Capitano, … , David M. Markovitz, Hal E. Broxmeyer
Published June 3, 2019; First published May 20, 2019
Citation Information: J Clin Invest. 2019;129(6):2555-2570. https://doi.org/10.1172/JCI127460.
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Research Article Hematology Stem cells

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 5

Extracellular DEK’s ability to regulate hematopoiesis requires HSPGs.

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Extracellular DEK’s ability to regulate hematopoiesis requires HSPGs. (A...
(A) C57BL/6 BM cells were pretreated with Xyl-PheNO2 or heparinase III to neutralize HSPGs and then utilized in a CFU assay. Anti-CXCR2 antibody served as a positive control, while vehicle or isotype control served as a negative control. Data are mean ± SD of triplicate plates. **P < 0.01 compared with vehicle control. (B) HSC expansion assays using C57BL/6 Lin– BM cells pretreated with anti-CXCR2 blocking antibody or heparin (blocks HSPGs). Isotype antibody or vehicle control served as negative control. Number of LT-HSCs in day 0 input and number of LT-HSCs 4 days after culture in expansion media with either vehicle control or rmDEK were determined. Three pools of 2 mice were utilized. Data are mean ± SEM. *P < 0.05 compared with vehicle control. (C) Representative images from Amnis ImageStream analysis of H3K9me3 in the nucleus of WT and vehicle- or rmDEK-treated Dek–/– LSK cells. Images were taken at ×40. Numbers in the upper left are object image numbers. Scale bars: 10 μm. (D) ImageStream analysis of H3K9me3 in the nucleus of Dek–/– LSK cells pretreated with HSPG inhibitor (heparin), endocytosis inhibitor (Pitstop 2), the Gαi protein signaling inhibitor PT, or an agent that kills cycling cells (hydroxyurea [HU]), then incubated for 16 hours with either vehicle or rmDEK. (E) H3K9me3 expression in the nucleus of Dek–/– LSK cells pretreated with anti-CXCR2 blocking antibody, anti-CXCR4 antibody, isotype antibody, or vehicle, then incubated for 16 hours with either vehicle or rmDEK pretreated with vehicle, aptamer library, or DEK-targeted aptamers DTA-64 and DTA-85+ends. For D and E, IDEAs software was used for analysis. Data are mean ± SD of triplicate tubes. *P < 0.05, **P < 0.01 compared with percent H3K9me3 levels in the nucleus of the vehicle group. For A–E, 1-way ANOVA with post hoc Tukey’s multiple-comparisons test was used.