Novel Evidence That a Lipolytic Enzyme - Hematopoietic-Specific Phospholipase C Beta 2 - Promotes Mobilization of Hematopoietic Stem Cells By Decreasing Their Lipid Raft-Mediated Bone Marrow Retention and Increasing the Pro-Mobilizing Effects of Granulocytes

Background. Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α₄β₁ integrin VLA-4 receptor, whose specific ligands, stromal-derived factor 1 (SDF-1) and vascular cell adhesion molecule 1 (VCAM-1), respectively, are expressed in BM stem cell niches. The integrity of the membrane lipid rafts containing these BM-retention receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, plays an important role in mobilization of HSPCs into peripheral blood (PB) by i) inducing degranulation of BM-residing granulocytes and ii) promoting their egress from BM into blood so that they permeabilize the endothelial barrier for egress of HSPCs (Leukemia 2010, 24, 976-985). It is known that a hematopoietic-specific lipolytic enzyme, phospholipase C-β2 (PLC-β2), is an intracellular enzyme involved in signaling through the C5a receptor and the chemotactic response of leucocytes to a C5a gradient. On the other hand, if released from the cells, PLC-β2 affects GPI-A by enzymatic digestion and disrupts lipid raft integrity and may affect cell-surface expression of proteins that contain GPI-A, including a truncated form of VCAM-1, the complement cascade inhibitors CD55 and CD59, and uPAR.

Hypothesis.Based on the foregoing, we asked whether, in addition to proteolytic enzymes, the lipolytic enzyme PLC-β2 is released from granulocytes during mobilization andplays a role in pharmacological mobilization by disintegrating lipid raft-mediated CXCR4 and VLA-4-dependent BM retention mechanisms and by enhancing the C5a-mediated pro-mobilizing effects of granulocytes.

Materials and Methods. To address this novel concept, we evaluated the level of PLC-β2 in BM during mobilization, the effect of this enzyme on adhesion and migration of HSPCs, as well as the status of lipid raft integrity on HSPCs and performed G-CSF- and AMD 3100-induced mobilization studies in PLC-β2-KO and WT animals.

Results. We found that PLC-β2, as an intracellular enzyme in granulocytes, mediates the pro-mobilizing responses of these cells in a C5a receptor-dependent manner, specifically, by increasing degranulation of granulocytes and enhancing their C5a-dependent egress from BM into PB. PLC-β2 is also released from granulocytes during their degranulation, and thus the level of this enzyme increases in the BM microenvironment. After its release from granulocytes, PLC-β2 digests GPI-A and thus disintegrates membrane lipid rafts and impairs CXCR4-SDF-1- and VLA-VCAM-1-mediated retention of HSPCs in BM niches. It is also responsible for digestion of a shorter truncated isoform of VCAM-1 in stem cell niches and generation of soluble uPAR. In support of this dual intracellular and extracellular role of PLC-β2, we found that PLC-β2-KO mice are poor mobilizers. These novel and pleotropic effects of this lipolytic enzyme are shown in Figure 1.

Conclusions. PLC-β2 is the first lipolytic enzyme identified so far that plays a crucial role in pharmacological mobilization of HSPCs, and modification of its activity may lead to better mobilization strategies, which is currently being tested in our laboratories. Moreover, we propose that, in addition to PLC-β2, other lipolytic enzymes are involved in the mobilization process, and this is also currently being investigated in our laboratories.