The Chemotactic Lipid S1P Regulates Hematopoietic Progenitor Cell Egress and Mobilization Via Its Major Receptor S1P₁ and by SDF-1 Inhibition In a p38/Akt/mTOR Dependent Manner

Abstract 553

Egress of hematopoietic stem and progenitor cells (HSPC) from the bone marrow (BM) reservoir to the circulation in steady state conditions is a key requirement for normal hematopoiesis. It is dramatically enhanced by G-CSF-induced mobilization, which is widely used for clinical HSPC transplantation. An interplay between cytokines, chemokines (mainly SDF-1 (CXCL12) and its major receptor CXCR4), adhesion molecules, matrix metalloproteinases and neurotransmitters, tightly regulate HSPC egress and mobilization. Recent observations indicate an essential role for sphingolipids, and particularly sphingosine-1-phosphate (S1P) and its major receptor S1P₁ in leukocyte trafficking in vivo. Furthermore, several pharmacological agents that target S1P and S1P₁ attenuate development of autoimmune and cardiovascular diseases as well as cancer. Based on these findings, we hypothesized that HSPC motility, both in steady state and in stress-induced conditions, is regulated by S1P/S1P₁ signaling. We found that cells expressing S1P₁ receptor are mainly located near sinusoids in the murine BM, suggesting involvement of S1P/S1P₁ axis in HSPC steady state egress. To identify the role of S1P₁ in HSPC homeostatic release, we injected mice with the inhibitor FTY720 and discovered a significant decrease in primitive Sca-1+/c-Kit+/Lineage- (SKL) cell numbers in the peripheral blood along with their accumulation in the BM, 24 hr post a single i.p injection. To examine the S1P/S1P₁ axis involvement in stress induced mobilization, we tested S1P levels following G-CSF administration. S1P concentrations were decreased in BM supernatants and increased in the peripheral blood, suggesting the formation of a gradient towards the blood, with a potential HSPC mobilization capacity. Accordingly, a 5-fold decreased transcription level of sphingosine kinase 1 (Sphk1, S1P producing enzyme) and a milder increased transcription level of sphingosine phosphatase 1 (SPP1, S1P degrading enzyme) were observed in the BM of G-CSF treated mice. These changes in both S1P modulating enzymes expression levels were mediated by mTOR signaling, independent of the PI3K pathway. Another effect of G-CSF mobilization was enhancing the percentage of BM HSPC expressing surface S1P₁ receptor, which was abolished upon inhibition of mTOR by Rapamycin. These findings imply that the reduction in S1P BM levels enabled increased S1P₁ receptor expression and HSPC recruitment to the blood. Co-injections of FTY720 with G-CSF revealed decreased numbers of primitive SKL and immature colony-forming cells in the blood, indicating reduced HSPC mobilization. Accordingly, administration of G-CSF to Sphk1 KO mice, which have low S1P plasma concentrations, led to decreased mobilization of primitive SKL cells and progenitors to the blood. We also investigated the cross talk between S1P/S1P₁ and SDF-1/CXCR4 axes. Disruption of the S1P/S1P₁ axis during G-CSF administration (by co-injections of FTY720 or by using Sphk1 KO mice) reduced HSPC mobilization however, BM mononuclear cells obtained from these mice exhibited enhanced migration to a gradient of SDF-1 in vitro. These results imply that SDF-1/CXCR4 activation is not sufficient for HSPC mobilization. Previously, we have shown that CXCR4 neutralizing antibodies co-administrated on days 4 and 5 of G-CSF treatment, significantly but not completely inhibited HSPC mobilization (Petit et al., Nat Immunol, 2002). Interestingly, such treatment in Sphk1 KO mice completely inhibited mobilization and increased primitive SKL cells in the BM. These results suggest that S1P/S1P₁ axis has an important role in parallel to SDF-1/CXCR4 axis during stress-induced mobilization since inactivation of both pathways resulted in total abrogation of HSPC recruitment to the blood. Finally, we show that S1P can inhibit SDF-1 transcription in murine BM stromal cells via activation of the p38/Akt/mTOR signaling pathway. Since SDF-1 reduction in the BM is essential for HSPC mobilization, S1P-induced inhibition of its transcription allows the progenitor cells to detach and migrate. Taken together, our findings reveal involvement of S1P and its major receptor S1P₁ in HSPC egress and stress-induced mobilization. These findings may help broaden our understanding regarding the mechanisms behind HSPC motility and thus improve clinical mobilization protocols and drug development based on targeting the S1P/S1P₁ axis.