Evidence That a Bioactive Lipid, Ceramide-1 Phosphate (C1P), Is Upregulated In Bone Marrow Microenvironment After Myeloablative Therapy and Is a Potential Novel Homing Factor for Hematopoietic Stem Cells

Abstract 401

Stromal derived factor-1 (SDF-1), when employed at supra-physiological concentrations, is a potent in vitro chemoattractant for hematopoietic stem progenitor cells (HSPCs). However, because this chemokine is extremely sensitive to degradation by proteolytic enzymes (e.g., MMP-2) and as we have observed myeloablative conditioning for hematopoietic transplantation induces a highly proteolytic microenvironment in bone marrow (BM), SDF-1 secreted by stromal cells and osteoblasts must be rapidly degraded under these conditions. While a role for the SDF-1–CXCR4 axis in retention of HSPCs in BM is undisputed, the role of SDF-1 in the homing of HSPCs in a highly proteolytic microenvironment is somewhat less certain and some redundant homing mechanisms may exist. This latter notion is supported by several observations, such as that i) CXCR4^-/- fetal liver HSPCs may home to BM in an SDF-1- independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced, and iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy are able to engraft even in lethally irradiated recipients. To reappraise the role of SDF-1 and other new potential factors in homing of HSPCs, we employed several complementary strategies. First we measured expression of SDF-1 mRNA in BM at 24 and 48 hours after lethal irradiation and observed a ∼3-fold increase. By contrast, the SDF-1 protein level in BM, evaluated by ELISA, surprisingly decreased as compared to non-irradiated mice. Next, we found that after blocking SDF-1 with AMD3100 treatment, conditioned media (CM) from irradiated BM cells still chemoattracted HSPCs. This SDF-1- independent chemotactic activity was resistant to heat inactivation, but was eliminated after stripping by activated charcoal, suggesting the possible involvement of bioactive lipids. Therefore, we began a search for unknown chemoattractants that could direct trafficking of HSPCs, with bioactive lipids as strong candidates, because, as small molecules, they are resistant to proteases. We focused especially on ceramide-1 phosphate (C1P) and sphingosine-1 phosphate (S1P), which are products of membrane-lipids metabolism. It is known that C1P, in contrast to S1P, is retained intracellularly and can be released mostly from damaged cells. Mass spectrometry (MS) analysis revealed that the major isoforms of C1P were detected at higher concentration in supernatant from irradiated BM when compared to supernatant from non-irradiated BM, which suggests that this bioactive lipid and chemoattractant is released from “leaky” BM cells damaged by myeloablative irradiation. We report here for the first time that C1P i) is a strong chemoatttractant for murine and human HSPCs, ii) activates phosphorylation of MAPKp42/44 and AKT in these cells, iii) induces expression of matrix metallopeptidases (MMPs), and iv) modulates adhesion of HSPCs to stroma and endothelium. Furthermore, in direct clonogenic studies, we did not observe any toxic effect of C1P on proliferation of murine and human clonogenic progenitors. We therefore propose a novel paradigm in which C1P is a chemoattractant for HSPCs that, in contrast to SDF-1, is highly resistant to proteolysis. In the proteolytic microenvironment induced in BM after myeloablative radio/chemotherapy, it could play along with SDF-1 an important and, until now, unrecognized role in the homing of HSPCs after transplantation. Furthermore, C1P secreted by damaged cells in other organs (e.g., infarcted myocardium) may in these highly proteolytic or necrotic microenvironments play a similar role in the homing of circulating stem cells involved in regeneration.