Abstract
Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB(1) and CB(2). The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB(1) receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10(7) cells), and 2-AG (75.2 ng/10(7) cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10(7) cells) and 2-AG (98.8 ng/10(7) cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB(1) agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1(-/-) showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)(-/-) mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1(-/-), as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.
Highlights
Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB1 and CB2
There was a synergistic effect of LPS, when co-administered with the fatty acid amide hydrolase (FAAH) inhibitor URB597, in mobilization of hematopoietic stem and progenitor cells (HSPCs) (Fig. 1B)
These results suggest that endocannabinoids induce migration and trafficking of HSPCs, and that this migration is enhanced under stress conditions
Summary
Antibodies and Chemical and Biological Compounds—Rabbit polyclonal anti-CB1 antibodies (ABR-Affinity BioReagents, Golden, CO) were used for immunofluorescent staining. Human CD34-positive cells were isolated from mononuclear cells derived from human bone marrow by using the CD34 immunomagnetic bead separation method of the mini-MACS system following the manufacturer’s guidelines (Miltenyi Biotec). Bone marrow cells were stained with Hoechst 33342 (Sigma), as described previously [29]. RT-PCR Analysis of CB1 and CB2 Expression—RNA from total bone marrow mononuclear cells or as indicated was extracted using the RNeasy Mini Kit along with the DNA Shredder Kit (both from Qiagen, Valencia, CA) following the manufacturer’s protocol. Western Blotting—For the Western blotting procedure, isolated HSPCs (1 ϫ 104) derived from bone marrow of WT, Cnr1Ϫ/Ϫ, or Cnr2Ϫ/Ϫ mice were used to prepare protein lysates for Western blot analysis, using specific antibodies for CB1 and CB2. Statistical Analysis—The statistical significance of the results reported here was determined by a two-tailed t test. p Ͻ 0.01 and p Ͻ 0.05 were considered significant, as indicated
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