Novel Evidence That, in Addition to Proteolytic Enzymes, Lipolytic Enzymes Are Involved in Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs) - an Important Pro-Mobilizing Role Identified for Hematopoietic-Specific Phospholipase C (PLCβ2)

Background. Hematopoietic stem/progenitor cells (HSPCs) express the chemokine receptor CXCR4 and the very late antigen 4 receptor (VLA-4, also known as alpha₄beta₁ integrin) on their cell surface and are retained in bone marrow (BM) niches by interaction of these receptors with their respective ligands, α-chemokine stromal-derived growth factor 1 (SDF-1) and vascular adhesion molecule 1 (VCAM-1, also known as CD106), which are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). Mobilization studies employing small molecule antagonists of CXCR4 or VLA-4 indicate the importance of both axes in retention of HSPCs in the BM microenvironment. Furthermore, it has been postulated that a crucial role in the mobilization process plays activation of a proteolytic microenvironment (J Clin Invest. 2003;111:187-96) and complement cascade (ComC) (Blood 2004;103:2071-8) in the BM microenvironment. However, to our surprise no attention has been paid so far to the role of lipolytic enzymes. Phospholipase C (PLC) is an enzyme released by neutrophils that cleaves the phosphoglycerol bond in glycosylphospahtidylinositol (GPI anchor), a glycolipid that is attached to the C-termini of several important proteins during posttranslational modification. GPI-linked proteins are thought to be preferentially located in lipid rafts. The most important GPI-anchored proteins include VCAM-1, complement inhibitors CD55 and CD59, and uPAR. We have reported that for retention of HSPCs in BM, both CXCR4 and VLA-4 have to be incorporated into membrane lipid rafts (Blood 2005;105:40-48).

Hypothesis. Based on our previous observation that the HSPCs of paroxysmal nocturnal hemoglobinuria patients show defective retention in BM due to lack of functional GPI anchor (Leukemia 2012; 26:1722—5) we have hypothesized that PLC in normal BM may facilitate mobilization of HSPCs by perturbing expression of GPI anchor, which is so crucial for proper retention of HSPCs in BM.

Materials and Methods. PLC activity has been measured by ELISA in the BM of mobilized mice as well in conditioned media from neutrophils exposed to several pro-mobilizing factors (G-CSF, AMD3100, C3a, C5a, _desArgC5a, and S1P). We also tested the effect of PLC on the expression of VCAM-1 on BM-derived stroma and CD55 and CD59 antigens on BM mononuclear cells. The effect of PLC on incorporation of CXCR4 and VLA-4 into membrane lipid rafts has been studied by confocal microscopy employing murine Sca-1⁺ and human CD34⁺ cells. Here, to test our hypothesis, mobilization studies using AMD3100 and G-CSF have been performed in PLCβ2-KO (PLCβ2^–/–) mice and their wild type (WT) littermates.

Results. Our data indicate that the PLC level increases in BM during mobilization and is released from neutrophils in response to several pro-mobilization factors (G-CSF, AMD3100, C3a, C5a, _desArgC5a, and S1P). PLC efficiently cleaves VCAM-1 expressed on BM stromal cells and thus perturbs the VCAM-1–VLA4 interaction as well as removes CD55 and CD59 from BM mononuclear cells, which enhances the pro-mobilizing effects of the ComC. PLC also inhibits lipid raft formation on HSPCs and by this means impairs the normal BM-retention function of CXCR4 and VLA-4. Finally, what is most important, we observed a mobilization defect in PLCβ2^–/– mice, as evaluated by the number of mobilized leucocytes, SKL cells, and CFU-GM.

Conclusions. We have established for the first time that, in addition to proteolytic enzymes, lipolytic enzymes, including LPC, are upregulated in the BM microenvironment, and that PLC promotes mobilization of HSPCs by perturbing the BM-retention function of GPI-anchored proteins. These data support an important role for GPI anchor-dependent proteins in the retention of HSPCs in BM niches.