Neuropeptides Orexin A and B Are Funktionally Aktive in CD34+ Hematopoietic Stem and Progenitor Cells.

Abstract 4593

Orexin receptors are involved in the regulation of sleep-wake-rhythm, food intake and energy homeostasis and it was still recently believed that their expression is restricted to the nervous system. But, during the last years orexin receptors have been detected in an increasing number of peripheral tissues. We have earlier found orexin receptor 1 and 2 expression on human CD34⁺ hematopoietic stem and progenitor cells. Still, the sources of their physiological ligands, the peptides orexin A and B, seemed so far to be restricted to the central nerve system. Ca²⁺-dependent signaling and activation of mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (ERK1/2) pathways are considered as main downstream signaling pathways of the orexin receptors.

In this study, we investigated the signaling and functional role of orexin receptors in CD34⁺ hematopoietic stem and progenitor cells.

Using confocal fluorescence microscopy and flow cytometry we found that stimulation of purified CD34⁺ cells with orexin A and B led to an increase of the intracellular calcium concentration due to both calcium influx and calcium release from intracellular stores. Of interest, incubation with orexin reduces the SDF-1β-induced calcium influx. Furthermore orexin receptor stimulation led to a decrease of the intracellular cAMP concentration. Following orexin receptor stimulation with orexin A and B, we observed an initial increase of ERK1/2 phosphorylation up to 30 minutes upon incubation with orexin followed by a decrease at several time points up to 8 hours in comparison to the unstimulated control.

To investigate a potential impact on the functional properties of human CD34⁺ cells we performed proliferation and apoptosis assays, migration and adhesion assays as well as colony forming and long-term culture assays. Remarkably, stimulation with orexin A and B led to a significant higher proportion of early pluripotent hematopoietic progenitor (CFU-GEMM) colonies and a significant reduction of erythroid precursors. A more immature phenotype of orexin-stimulated CD34⁺ cells is also reflected by array-based gene expression profiling. Long-term culture assays revealed a significant higher frequency of LTC-IC indicating also a more immature phenotype of orexin-stimulated cells. In line, orexin receptor stimulation led to a significant increase of the proportion of Lin^-, CD34⁺, CD38^- HSC in the G0-phase of the cell cycle. Furthermore, stimulation with orexin A and B increased the number of apoptotic cells in the Lin^-, CD34⁺, CD38^- HSC fraction and the total hematopoietic stem and progenitor population determined by flowcytometric analysis of intracellular cleaved caspase 3 content. The adhesive capacity of CD34⁺ cells to fibronectin and collagen coated dishes and the migratory capacity was significantly decreased upon orexin receptor stimulation. Concurrent incubation with the selective G_i-protein inhibitor pertussis toxin abrogated these effects. Given the functional impact of the orexin system on CD34⁺ cells, we asked if orexins are secreted locally in the bone marrow or autocrine by CD34⁺ cells or if they are humorally transported to the bone marrow cavity. Using FACS analysis, immunfluorescent staining and western blotting we could detect prepro-Orexin in CD34⁺ cells and using ELISA orexin was found in the serum obtained by bone marrow biopsies and peripheral blood. Taken together, the phenotype of orexin-stimulated hematopoietic stem and progenitor cells suggest a mobilizing effect of the orexin receptor stimulation as well as an increased repopulation capacity which might be of relevance in clinical stem cell mobilization and transplantation and is currently verified in murine models.