Upregulation of MT1-MMP by Molecules Present in Leukaphereis Products Primes CD34+ Cell Homing.

Abstract

Hematopoietic stem/progenitor cells (HSPC) derived from mobilized peripheral blood engraft faster than those from bone marrow (BM) or cord blood (CB). We recently postulated that leukapheresis product supernatants (collected from G-CSF-mobilized patients) and their components (fibrinogen, fibronectin, thrombin, hyaluronic acid (HA) and C3 cleavage fragments) have a priming effect on HSPC homing by enhancing chemotactic responses to SDF-1 through increased incorporation of CXCR4 into membrane lipid rafts and stimulation of matrix metalloproteinase (proMMP-2 and proMMP-9) secretion in these cells (Blood 2005; 105:40). As MT1-MMP has been shown to activate latent forms of MMP-2 and (indirectly) MMP-9 and is known to localize proteolytic activity at the leading edge of migrating cells, we decided to investigate MT1-MMPs' role in the priming of HSPC homing. We observed that stimulation of BM and CB CD34+ cells with priming agents (HA, fibrinogen or thrombin) not only increased secretion of proMMP-2 and proMMP-9 (zymography) but also highly upregulated levels of MT1-MMP transcript (RT-PCR) and protein (Western blotting) in these cells. Moreover, trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), enhanced by priming agents, was inhibited by the potent MT1-MMP inhibitor epigallocatechin-3-gallate. Furthermore, priming agents (HA, fibrinogen and thrombin) increased levels of active MMP-2 in co-cultures of stromal cells (endothelial cells and BM fibroblasts) with CD34+ cells. To elucidate the mechanism of MT1-MMP upregulation by priming agents, we evaluated whether they affected MT1-MMP incorporation in ganglioside M1-enriched membrane lipid rafts in the same way as for CXCR4. We found that stimulation of hematopoietic and THP-1 cells with HA, fibrinogen and thrombin increased incorporation of MT1-MMP into membrane lipid rafts and these observations were confirmed using confocal microscopy. Hence it appears that MT1-MMP, like CXCR4, localizes in membrane lipid rafts, and this is enhanced by priming agents, leading to better homing of HSPC.