The mechanism(s) underlying the release of stem/progenitor cells from bone marrow into the circulation is poorly understood. We hypothesized that matrix metalloproteinases (MMPs), especially gelatinases, which are believed to participate in the proteolysis of basement membranes and in the migration of leukocytes, may facilitate this process. First, we investigated whether CD34+stem/progenitor cells express gelatinases A (MMP-2) and/or B (MMP-9) and whether growth factors and cytokines (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], stem cell factor [SCF], macrophage colony-stimulating factor [M-CSF], interleukin-3 [IL-3], IL-6, IL-8, and tumor necrosis factor- [TNF-]) are able to modulate their expression. Next, we examined the transmigration of these stem/progenitor cells through reconstituted basement membrane (Matrigel) and its modulation by growth factors and cytokines. CD34+ cells were obtained from steady-state bone marrow and peripheral blood (from leukapheresis products collected either in steady-state hematopoiesis or after mobilization with G-CSF plus chemotherapy or G-CSF alone). We found that peripheral blood CD34+ cells, regardless of whether they were mobilized or not, strongly expressed both gelatinases (MMP-2 and MMP-9) in contrast to steady-state bone marrow CD34+ cells, which did not. However, all the growth factors and cytokines tested could induce MMP-2 and MMP-9 secretion by the latter cells. Moreover, the stimulatory effects of G-CSF and SCF on both MMP-2 and MMP-9 secretion were found to be significantly higher in CD34+ cells isolated from bone marrow than in those from peripheral blood. In addition TNF-, GM-CSF, and IL-6 increased the secretion of a partially active form of MMP-2. Basal transmigration of bone marrow CD34+ cells through Matrigel was lower than that of peripheral blood CD34+ cells (P < .0001), but growth factors and cytokines increased it by 50% to 150%. Positive correlations were established between expression of gelatinases and CD34+cell migration (r > .9). The stimulatory effect of G-CSF was significantly greater on the migration of CD34+ cells from bone marrow than on those from peripheral blood (P = .004). Moreover, CD34+ cell migration was reduced to approximately 50% by antibodies to MMP-2 and MMP-9, tissue inhibitors of metalloproteinases (rhTIMP-1 and -2), ando-phenanthroline. TNF-–induced gelatinase secretion and migration of CD34+ cells and of clonogenic progenitors (colony-forming unit–granulocyte-macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM], and colony-forming unit-megakaryocyte [CFU-MK]) were dose-dependent. Therefore, this study demonstrated that CD34+ cells that are circulating in peripheral blood express both MMP-2 and MMP-9 and transmigrate through Matrigel. In contrast, CD34+ cells from steady-state bone marrow acquire similar properties after exposure to growth factors and cytokines, which upregulate expression of gelatinases and transmigration of these cells when they enter the bloodstream. Hence, we suggest that growth factors and cytokines induce release of stem/progenitor cells from bone marrow into peripheral blood during mobilization, as well as during steady-state hematopoiesis, by signaling through gelatinase pathways.
Summary. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate the turnover of the extracellular matrix and may modulate the biology of haematopoietic cells. We investigated whether MMPs and TIMPs are produced in long-term marrow cultures (LTMCs) established from normal donors and acute myelogenous leukaemia (AML) patients, and by fibroblast-(F), granulocyte macrophage-(GM) and megakaryocyte-(Meg) colony-forming unit (CFU) and erythroid burst-forming unit (BFU-E)-derived precursor cells. ProMMP-9 levels were highest (. 400 ng/ml) at week 1 of normal LTMC, whereas proMMP-2, TIMP-1, TIMP-2 and TIMP-3 levels peaked (up to 1000 ng/ml) after the establishment of the adherent layer. In LTMC from AML patients, these patterns of secretion were reversed. Moreover, we found that after a 24 h incubation in serum-free media, normal CFU-GM-, BFU-E-and CFU-Meg-derived cells secreted proMMP-9 and CFU-F-derived cells proMMP-2, in contrast to cells from LTMC adherent layer which secreted both active and latent forms of MMP-2 and MMP-9 under serum-free conditions. However, when these adherent cells were incubated in 12´5% fetal calf or horse serum or complete LTMC growth media, active forms of MMP-2 and MMP-9 were no longer detectable, and TIMP levels increased. Hence, we concluded that (i) MMPs/TIMPs are secreted by normal human bone marrow haematopoietic and stromal cells and may play an important role in intercellular cross-talk in haematopoiesis; and (ii) only latent forms of MMPs are present under LTMC conditions, indicating that the specific media used for weekly re-feeding of LTMC can block activation of MMP-2 and MMP-9, maintaining the integrity of the stromal layer and supporting haematopoiesis in vitro.Keywords: matrix metalloproteinases, tissue inhibitors of metalloproteinases, long-term marrow cultures, haematopoietic progenitor cells, acute myelogenous leukaemia.Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) not only regulate the turnover of the extracellular matrix (ECM) but also cell survival, proliferation, differentiation, adhesion and migration (Werb, 1997). On the basis of their domain structure and substrate specificity, MMPs are classified into four major subgroups: collagenases, gelatinases, stromelysins and membrane-type MMPs (MT-MMPs) (Forget et al, 1999;Nagase & Woessner, 1999;Janowska-Wieczorek et al, 2000a;Lenz et al, 2000).The gelatinases (MMP-2/72-kDa type IV collagenase and MMP-9/92-kDa type IV collagenase) degrade denatured collagens (gelatins), native collagen types II, IV, V, VII, X and XI, fibronectin, vitronectin, elastin and aggrecan (Forget et al, 1999). The primary mechanism for MMP regulation occurs at the transcriptional level and involves a variety of extracellular factors including cytokines, growth factors and cell contact with the ECM (Nagase & Woessner, 1999;Westermarck & Kahari, 1999). Another level of regulation of MMP activity is through proenzyme activation, as most MMPs are secreted as latent enzymes. MT...
Hyaluronic acid (HA), the bone marrow (BM) extracellular matrix microenvironment (ECM) component, not only supports cell adhesion but also promotes migration and homing of hematopoietic stem/progenitor cells (HSPC) by interacting with its cell surface receptor CD44. CD44 has been shown to co-localize with matrix metalloproteinases (MMPs), particularly membrane-type (MT)-1 MMP and MMP-9, at the leading edge of migrating tumor cells, and the cleavage of CD44 by MT1-MMP is critical for tumor cell migration and invasion. MT1-MMP has strong pericellular proteolytic activity and also activates latent forms of MMP-2 and MMP-9. In this study we examined the effect of HA on MT1-MMP expression and migration of BM, peripheral blood and cord blood CD34+ cells. We found that HA upregulates mRNA for MT1-MMP and MMP-9, increases MT1-MMP protein (as evaluated by Western blotting) and stimulates MMP-9 and MMP-2 activity (as determined by zymography) in CD34+ cells. In chemotaxis assays HA alone did not show any chemotactic activity but primed the chemotaxis of CD34+ cells to a low SDF-1 gradient (10 ng/mL) and their trans-Matrigel chemoinvasion to a low SDF-1 gradient. Similarly, SDF-1 besides stimulating MMP-2 and MMP-9 (as we previously described in Exp Hematol2000; 28:1274), also upregulated MT1-MMP in CD34+ cells. In addition, we found that the HA enhanced activation of latent MMP-2 in co-cultures of CD34+ cells with HUVEC. In conclusion, we demonstrate for the first time that (i) HA primes CD34+ cell chemotaxis and chemoinvasion to a low SDF-1 gradient; (ii) that both HA and SDF-1 stimulate MT1-MMP, MMP-2 and MMP-9 in CD34+ cells, and (iii) HA enhances activation of proMMP-2 in the ECM. Hence the interaction of HA and SDF-1 with MT1-MMP could play an important role in HSPC migration and homing and we postulate that pretreatment of HSPC with HA before transplantation could enhance their homing by inducing expression of MMPs that is optimal for stem cell engraftment.
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