Hematopoietic stem and progenitor cell egress and mobilization by G-CSF

Hematopoietic stem and progenitor cells (HSPCs) as well as maturing leukocytes are typically distinguished by their motility capacity and ability to pave their way out from the bone marrow (BM) reservoir to the circulation, as part of host defense and repair mechanisms. HSPCs and maturing leukocytes are continuously released at low levels from the BM during steady-state homeostasis and at increased rates upon stress, such as bleeding or inflammation [1–4]. Current models of cell egress from the BM imply that HSPCs detach from their supporting stromal niches via adhesion interactions, translocate to the blood vessel, and extravasate through the endothelial barrier into the circulation. This multi-step process is orchestrated by a large number of cytokines, chemokines, proteolytic enzymes, as well as adhesion interactions [5, 6] that are all synchronized in parallel or in a reciprocal manner. These dynamic changes in HSPCs and their microenvironment during trafficking are achieved through a complex interplay between the immune and the nervous systems and bone remodeling (osteoblast and osteoclast activities) [1, 2, 7]. For instance, the sympathetic nervous system regulates the steady-state egress of HSPCs in murine, via circadian rhythms peaking 5 hours after the initiation of light and reaching a nadir 5 hours after darkness [8]. The sympathetic nervous system can directly stimulate human HSPC motility and proliferation [9] in addition to its indirect effect on the murine stroma microenvironment [10, 11]. Once HSPCs are found in the circulation, they may enter the spleen and non-lymphatic tissues or migrate back to the BM in a process called “homing”. As evident from the experiments with parabiotic mice, which share blood systems, donor peripheral blood HSPCs are cleared within minutes from the circulation of intravenously transplanted congeneic recipients [12, 13]. HSPC mobilization can be clinically or experimentally induced by a variety of cytokines and chemokines [5, 6]. At present, granulocyte colony-stimulating factor (G-CSF) is the most commonly used agent [14]. The mechanisms of G-CSF-induced mobilization consist of induction of quiescent HSPC proliferation thus increasing progenitor cell pool, accompanied by a decrease in retention of HSPCs in their BM microenvironment [15]. During the 5-day regimen of G-CSF administration, there is an increased release of proteolytic enzymes from BM neutrophils and other myeloid cells, correlating with the peak in HSPC mobilization [16]. Furthermore, following G-CSF administration, stromal cell-derived factor (SDF-1) (CXCL12, a chemokine able to strongly attract human and murine HSPCs [17–21]) levels in the BM are transiently increased followed by their downregulation at both protein [22, 23] and mRNA [24] levels. Thus, G-CSF administration results in proteolytic degradation of BM SDF-1 with subsequent mobilization of HSPCs. Most HSCs are found in contact with stromal cells that highly express SDF-1 within their specialized niches, which induce stem cell quiescence. Therefore, this chemokine is essential for maintaining a lifelong pool of HSCs by controlling the balance between HSC quiescence and self-renewal [25, 26]. SDF-1 downregulation allows quiescent HSCs to proliferate, differentiate, and to be recruited to the circulation.

MT1-MMP and RECK-mediated regulation of cell motility

Matrix metalloproteinases (MMPs) are a subfamily of zinc- and calcium-dependent enzymes, which all share a conserved methionine residue located C-terminal to the zinc ligands [27]. MMPs are implicated in a variety of physiological processes, including wound healing, uterine involution, and organogenesis, as well as in pathological conditions such as inflammatory, vascular and auto-immune disorders, and carcinogenesis [28–31]. While most MMPs are soluble proteins, six of them are membrane-bound MMPs (MT-MMPs). The proper functioning of membrane type 1-MMP (MT1-MMP) is essential for angiogenesis, wound healing, tissue remodeling [32], as well as tumor growth and metastasis [33, 34]. MT1-MMP-deficient mice develop multiple abnormalities due to defects in the remodeling of connective tissue [35, 36]. In accordance, osteogenic cells from MT1-MMP KO mice cannot degrade collagen and do not form bone when transplanted subcutaneously into host immunodeficient mice [35]. In terms of cell motility, MT1-MMP activates pro-MMP-2, which is involved in the invasion of cancer cells and spread of the metastasis [37]. Accordingly, MT1-MMP accumulates at invadopodia, which are specialized ECM-degrading membrane protrusions of invasive cells and thus facilitates tumor invasiveness [38]. Importantly, MT1-MMP is required during human monocyte migration and endothelial transmigration, thereby revealing a key role for MT1-MMP in monocyte recruitment during inflammation [39]. The induction of MT1-MMP activity in human mesenchymal stem cells by inflammatory cytokines promotes directed cell migration across reconstituted basement membranes [40, 41]. In addition to extracellular matrix degradation, MT1-MMP promotes cell invasion and motility by shedding of cell adhesion molecules, such as CD44 [32] and syndecan-1 [42]. Thus, MT1-MMP is considered a key player in cell trafficking by allowing proper migration of various cell types through mechanical barriers. The catalytic activity of MT1-MMP is regulated at the level of transcription as well as secretion by different endogenous activators and inhibitors such as furin, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 [27]. Another inhibitor is a membranal endogenous glycoprotein, named RECK (reversion-inducing cysteine-rich protein with Kazal motifs) [43]. Homozygous RECK-deficient murine embryos die at E10.5 and are characterized by small body size, reduced structural integrity, and frequent abdominal hemorrhage [44]. It has been previously demonstrated that RECK directly inhibits MT1-MMP via protein–protein interactions, subsequently interfering with the pro-MMP-2 activation cascade [45]. The mutual and reciprocal relationship between MT1-MMP and RECK seems to be necessary for regulation of cell motility.

Regulation of the HSPCs motility by MT1-MMP and RECK

Since MT1-MMP and RECK are essential for the motility and blood vessel intravasation of cancer and inflammatory cells, we and others studied their involvement in the trafficking of HSPCs between the BM and peripheral blood. We have found that both MT1-MMP and RECK are expressed on human and murine hematopoietic progenitor cells. Under steady-state conditions, human CD34+ progenitor cells from the peripheral blood express higher levels of MT1-MMP as compared to their BM counterparts, suggesting its involvement in the egress of these cells [46]. Neutralization of RECK activity in steady-state condition induces mobilization of human CD34+ progenitor cells in chimeric mice, emphasizing its role in retention of these cells in the BM [46]. Interestingly, MT1-MMP in mobilized peripheral blood cells is localized to lipid rafts [47], which are enriched in CXCR4, the major receptor for SDF-1 [48]. Since SDF-1 and lately also sphingosine 1-phosphate [49–51] were shown to be potent chemoattractants of HSPCs, MT1-MMP activity may affect the CXCR4/SDF-1-regulated trafficking of HSPCs. BM-and cord blood-derived mesenchymal stem cells, responsible for the development of stromal cells, also express MT1-MMP [41]. G-CSF-induced mobilization in clinical BM transplantation protocols is accompanied by an increase in MT1-MMP expression [46, 47] and a parallel decrease in RECK protein level on human and murine HSPCs [46]. The dynamic changes in MT1-MMP expression levels correlate with the numbers of mobilized CD34+ progenitor cells in healthy donors, suggesting a clinical relevant role for this molecule [46]. The reciprocal changes in the expression levels of MT1-MMP and RECK induced by G-CSF in human and murine progenitor cells are dependent on the PI3K/Akt pathway and antagonized by the treatment with rapamycin [an inhibitor of mammalian target of rapamycin (mTOR)] [46, 47]. PI3K/Akt pathway is essential for G-CSF-induced mobilization, since its inhibition by administration of rapamycin diminishes the level of HSPCs in the circulation [46], and abnormal PI3K activation interferes with HSPC retention in the BM [52]. In addition, PI3K/Akt signaling is essential for increased MT1-MMP incorporation into membranal lipid rafts and thus for its localization at the cellular migration front during G-CSF-induced mobilization [47]. Additional regulators implicated in G-CSF-induced mobilization are the cytokine hepatocyte growth factor (HGF) and its receptor c-Met, which were shown to play a crucial role in the motility of cancer cells [53]. HGF is increased in the plasma of mice upon treatment with G-CSF, while its receptor c-Met is upregulated on immature HSPCs [54]. The major regulator of c-Met transcription, HIF-1α was increased as well by G-CSF stimulations, correlating progenitor mobilization with an expansion of hypoxic murine BM areas [55]. HGF activates the mTOR pathway, thus inhibiting FOXO3a expression and leading to an increase in the reactive oxygen species (ROS) production during G-CSF-induced mobilization [54]. Accordingly, direct administration of HGF induces mobilization of HSPCs in humans as well as in mice, albeit at a lower level as compared to G-CSF [54, 56, 57]. Similarly to G-CSF, HGF treatment augments MT1-MMP expression on human CD34+ cells, revealing that activation of the HGF/c-Met pathway may contribute to the increase in MT1-MMP expression by G-CSF [56]. Additionally, HSPC mobilization is orchestrated by elements of the complement cleavage cascade, which is activated in the BM during G-CSF-induced mobilization [58]. As evident from the studies in C5-deficient mice, G-CSF-induced mobilization was significantly suppressed in the absence of functional complement system [58]. Activated C5a fragments (anaphylatoxin) decrease CXCR4 expression and chemotaxis toward an SDF-1 gradient of granulocytes and monocytes and promote proteolysis in the BM microenvironment through increased secretion of MMP-9 and expression of MT1-MMP and carboxypeptidase M in mononuclear and polymorphonuclear cells [58, 59]. Finally, HSPC egress and mobilization are affected by circadian oscillations due to the involvement of sympathetic nervous system in the regulation of hematopoietic cell trafficking [8]. G-CSF activation of peripheral noradrenergic neurons induces mobilization of HSPCs through suppression of endosteal bone-lining osteoblasts and reduction in BM SDF-1 levels [10]. However, our lab showed that this regulation is also through a direct effect on HSPCs since treatment with catecholamine increases the motility of normal and mobilized human progenitor cells, correlating with the increased expression of MT1-MMP and the activity of the metalloproteinase MMP-2 [9]. Interestingly, neurotransmitter stimulation activates the canonical Wnt signaling pathway, which mediates the increase in MT1-MMP activity on human CD34+ HSPCs [9].

How do MT1-MMP and RECK inversely regulate HSPC mobilization?

HSPCs are retained in the BM by adhesion interactions with different types of molecules such as: vascular cell adhesion molecule-1, intercellular adhesion molecule, β1 and β2 integrins, and the CD44 receptor [60]. Homing, as well as adhesion of immature human CD34+ cells to the BM microenvironment, depends on CD44 [61]. During G-CSF-induced mobilization, there is a reduction in CD44 membrane levels on BM HSPCs [61]. CD44-deficient mice display increased frequencies of circulating immature colony-forming cells, suggesting the importance of this adhesion molecule in HSPC retention. In accordance, increased activity of MT1-MMP and inhibition of RECK induces CD44 cleavage in the BM [46], implying that MT1-MMP facilitates progenitor cell release at least partly by antagonizing adhesion interactions, such as CD44-mediated retention. In addition, MT1-MMP affects the trafficking of HSPCs by modulating chemotaxis towards SDF-1 [17, 62, 63]. Blocking of MT1-MMP activity reduces SDF-1-induced chemotactic responses of human G-CSF-mobilized peripheral CD34+ cells [46, 47], whereas neutralization of RECK activity enhanced migration of steady-state human BM CD34+ cells via Matrigel. Accordingly, neutralization of MT1-MMP interferes with homing of human G-CSF-mobilized CD34+ cells to the BM of transplanted NOD/SCID mice, and the engraftment potential of BM progenitor cells from MT1-MMP deficient mice is significantly lower [46]. Interestingly, c-Met silencing on leukocytes obtained from the BM of G-CSF-treated mice impairs their ability of chemotaxis towards SDF-1 [54]. Thus, the emerging picture suggests a functional interaction between c-Met and SDF-1 pathways in BM leukocytes via MT1-MMP regulation of SDF-1 induced migration. In line with the aforementioned data, C5 and C5a complement cleavage fragments are released to the peripheral blood during mobilization and can directly chemoattract granulocytes and monocytes. These myeloid cells in contrast to stem cells are highly enriched in proteolytic enzymes and activated MT1-MMP and are the first cells that egress during mobilization from BM to pave a way for stem cells to follow [59, 64]. Of note, C3 and C3a complement cleavage fragments are also released in BM microenvironment and increase the responsiveness of HSPCs to SDF-1 [65], which is released from the BM to the blood, leading the way of HSPC [21, 23]. In addition, MT1-MMP can facilitate HSPC mobilization to the circulation by modulating MMP-2 and MMP-9 activity. During G-CSF-induced mobilization, there is an increase in the functional expression of MMP-2 and MMP-9 on circulating CD34+ cells comparing to their BM counterparts [66, 67]. RECK downregulates MMP-2 activation and MMP-9 expression in vivo [43, 44]. Accordingly, the inhibition of RECK activation in mice showed increased levels of secreted MMP-2 and MMP-9 [46]. This activation and secretion of MMP-2 and MMP-9 during G-CSF administration were found to be dependent on MT1-MMP activation [47].

Clinical aspects and future directions

The mobilization of HSPCs into the circulation constitutes the basis for modern BM transplantation procedures, which are routinely used to treat patients with hematological malignancies as well as inherited genetic disorders. Therefore, characterization of new regulators of HSPC development and mobilization in homeostasis and during stress became a major focus in the last decades. As MT1-MMP plays an essential role in motility while its inhibitor RECK plays an important role in retention, they both may become prime candidates for future clinical trials aimed at improving HSPC clinical mobilization in patients. Notwithstanding the foregoing, MMPs also play an important role in tumor invasion and metastasis [68, 69] and in particular MT1-MMP was found to have an essential role in the invasive capacity of acute myeloblastic leukemia (AML) cells [70]. The frequency of extramedullary infiltration (EMI) in AML is reported to be up to 40% and is most prevalent in the myelomonoblastic and monoblastic subtypes of AML. EMI patients have lower complete remission rates following induction chemotherapy and a shorter overall survival [71–74]. Therefore, clinical inhibition of MT1-MMP by drugs or neutralizing antibodies in addition to RECK activation in patients with AML or other types of cancer might be an important way to inhibit cancer cell invasiveness and subsequent metastasis formation [75, 76]. In summary, MT1-MMP and RECK are essential for the motility of HSPCs versus retention conditions respectively. During G-CSF-induced mobilization, there is a parallel increase in MT1-MMP and decrease in RECK levels on progenitor cells through the activation of PI3K/Akt pathway. The sympathetic nervous system as well as HGF and its receptor c-Met and also the complement system were all shown to have an important role in the regulation of MT1-MMP and RECK activity (Fig. 1, step 1). The HGF/c-Met axis activates PI3K/Akt pathway, leading to increased MT1-MMP activity and decreased RECK levels, both mediating the cleavage of the adhesion molecule CD44 to promote progenitor cell detachment from their BM niches (Fig. 1, step 2). Moreover, MT1-MMP and RECK promote the motility of HSPCs and chemotaxis towards SDF-1 thus increasing their migration to the blood (Fig. 1, step 3). Future directions in the study of MT1-MMP and RECK role in HSPC egress and mobilization must focus primarily on the molecular aspect of their activation. Though it was previously established by our group that the PI3K/Akt pathway plays an important role in this process, it is still unknown which downstream activators and transducers facilitate MT1-MMP and RECK upregulation both on the transcription and protein levels. In accordance, it is important to find whether MT1-MMP and RECK are both upregulated by the same molecular pathway or by different pathways and whether there is a mutual regulation between those two factors. Since the nervous system has an essential role in the trafficking of HSPCs, the cross talk and regulation of MT1-MMP and RECK by the sympathetic nervous system should be examined. In particular, it would be of great interest to identify a possible circadian oscillation rhythm in the expression of MT1-MMP and RECK on stem and progenitor cells which may correlate to the level of egress as previously described regarding SDF-1 levels in the BM [8]. Nevertheless, the molecular mechanism by which the sympathetic nervous system directly regulates the levels of MT1-MMP and RECK [9] during G-CSF-induced HSPC mobilization would be of great interest for future studies. We conclude by emphasizing that MT1-MMP and its inhibitor RECK play an essential role in HSPC retention versus mobilization from the BM to the peripheral blood.

Fig. 1
figure 1

MT1-MMP and its inhibitor RECK play opposite and essential roles in retention and mobilization of HSPCs. 1 G-CSF administration promote catecholamine secretion from the sympathetic nervous system as well as HGF secretion from PMN cells in the BM and activation of the complement cascade in the blood. 2 HGF binds to its receptor, c-Met, and activates the PI3K/Akt pathway, subsequently leading to ROS production. As a result of steps 1 and 2, MT1-MMP activation and RECK inhibition are induced in HSPCs, leading to cleavage of the adhesion molecule CD44 and detachment of HSPCs from stromal cells. 3 In parallel, MT1-MMP activity leads to increased motility of HSPCs towards SDF-1 which leads their way into the blood

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