MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Abstract 3536

Poster Board III-473

Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34⁺ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34⁺ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34⁺ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34⁺ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34⁺ cells is necessary for proMMP-2 activation as media conditioned by CD34⁺ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation.