G-CSF Induces Expression of Both Hepatocyte Growth Factor (HGF) and Its Receptor (c-Met) in Human Hematopoietic Stem/Progenitor Cells and Mature Myeloid Cells - Novel Evidence That during Mobilization the HGF-c-Met Axis Counterbalances G-CSF-Induced Attenuation of the SDF-1-CXCR4 Axis.

The stromal-derived factor-1 (SDF-1)-CXCR4 axis plays an important role in stem cell trafficking, and G-CSF- induced mobilization decreases SDF-1 expression in the bone marrow (BM) microenvironment and CXCR4 expression by CD34+ hematopoietic stem/progenitor cells (HSPC). Alternatively, the tyrosine kinase c-Met receptor-HGF axis was recently postulated to play an important role in the trafficking of non-hematopoietic cells; however, our previous research demonstrated that while HGF is an important constituent of the BM microenvironment, c-Met is not expressed by BM-derived steady state HSPC (

• expression of c-Met and HGF by hematopoietic and BM-derived stromal cells without and after exposure to G-CSF, and

• the chemotactic responses of steady state and mobilized hematopoietic cells to HGF.

We confirmed using RT-PCR and FACS analysis that the c-Met receptor is not expressed by steady-state BM CD34+ cells and mature mononuclear cells (MNC), but to our surprise we found that c-Met is expressed in G-CSF-mobilized CD34+ cells and MNC obtained from leukapheresis products. Supporting this was our finding that mPB but not steady state CD34+ cells responded to HGF stimulation by phosphorylation of MAPKp42/44 and AKT. HGF was found to highly expressed in mPB CD34+ cells and MNC but in steady state BM MNC. Moreover, G-CSF stimulation induced HGF expression in steady state BM MNC and BM-derived fibroblastic and mesenchymal stem cells. Importantly, when we compared c-Met expression on circulating PB leukocytes from patients during early and late stages of G-CSF mobilization we found that it increases in the later stages. When we compared the expression of CXCR4 and c-Met on leukocytes from the leukapheresis product vs circulating PB from the same patient we found that CXCR4 expression is similar but expression of c-Met is higher in the leukapheresis product. HGF was also found to be a strong chemoattractant for mPB leukocytes, but not for steady state leukocytes, and the chemotactic activity of HGF was totally inhibited by a c-Met antagonist. Additionally, we demonstrated that c-Met is incorporated into lipid rafts as shown by confocal microscopy and that G-CSF stimulation increases the secretion of matrix metalloproteinase (MMP)-9 from BM MNC, which was inhibited by the c-Met antagonist. A combination of G-CSF and HGF also upregulated membrane-type (MT)-MMPs such as MT1-MMP and MT6-MMP on leukocytes. Thus we demonstrate for the first time that G-CSF induces expression of functional c-Met and HGF in HSPC and leukocytes, and that the HGF-c-Met axis could play an important role in their mobilization and maintenance of high expression of matrix-degrading enzymes, allowing egress of HSPC from the BM.