Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.
Bottom Line: A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis.We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants.Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.
Affiliation: Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Boston, MA 02115 Harvard Stem Cell Institute, Howard Hughes Medical Institute, and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138.Show MeSH
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Mentions: In many cell types, adenosine promotes the production of cytokines, chemokines, and growth factors and often exerts its cellular effects through or in association with these factors (Adair, 2005). In vascular endothelial cells, A2b activation modulates the production of several angiocrine factors (Feoktistov et al., 2002). One of the major factors is CXCL8 (also known as IL-8). CXCL8 is known as a potent stem cell–mobilizing agent (Laterveer et al., 1995), and it also increases the proliferation of hematopoietic progenitor cells in vitro (Hermouet et al., 2000). Human aortic endothelial cells (HAECs) express the A2b receptor (Iwamoto et al., 1994), and we confirmed A2b expression by RT-PCR (not depicted). We treated HAECs with BAY 60-6583, which increased CXCL8 protein production (Fig. 7 A). To demonstrate that A2b regulates CXCL8 in zebrafish embryos, we examined cxcl8 transcripts. Knockdown of A2b strongly decreased cxcl8 and runx1 expression in flk1:GFP+ endothelial cells, but did not affect the expression of endothelial marker fli1 (Fig. 7 B). We next examined the function of CXCL8 in HSC development. Inhibition of cxcl8 with MO (Stoll et al., 2011) in zebrafish embryos strongly reduced runx1+/cmyb+ HSPCs in the AGM (Fig. 7, C–F) and the CHT (Fig. 7, I and J). Loss of CXCL8 did not have an effect on the development of the dorsal aorta at the time when HSPCs start to emerge (Fig. 7, K and L) and did not interfere with primitive hematopoiesis (not depicted), which supports a specific role of cxcl8 in HSPC formation. In addition, we generated a CXCL8 mutation by a transcription activator–like effector nuclease (TALEN) approach. This mutation leads to the deletion of two amino acids in the conserved CXC domain (Fig. 7 M; Fernandez and Lolis, 2002), which has been shown to be important for its tertiary structure. We found that the HSPC staining is reduced in CXCL8 mutant embryos compared with those in control siblings (Fig. 7, N and O), in accordance with the defects we observed in CXCL8 MO knockdown embryos. Similar to loss of A2b, loss of CXCL8 also reduced scl+ hemogenic vascular endothelium (Fig. 7, P–R). More importantly, cxcl8 mRNA restored HSCs and progenitors in the embryos that lack the A2b (Fig. 7, G and H). These results suggest that adenosine signaling regulates CXCL8 production to mediate HSPC development.
Affiliation: Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Boston, MA 02115 Harvard Stem Cell Institute, Howard Hughes Medical Institute, and Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138.