Mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4

Adhesion molecules and stromal cell-derived factor-1 (SDF-1)/CXCR4 signaling play key roles in homing and mobilization of hematopoietic stem cells (HSC). Active signaling through SDF-1/CXCR4 and upregulation of adhesion molecules are required for homing, whereas downregulation of adhesion molecules and disruption of SDF-1/CXCR4 signaling are required for mobilization of HSC. We studied the surface expression of CXCR4 very late activation antigen (VLA)-4 and VLA-5 on myeloma cells mobilized with cyclophosphamide and GM-CSF in 12 multiple myeloma patients undergoing HSC mobilization for autologous transplantation. We also studied the plasma levels of SDF-1 in apheresis collection of these patients. We observed a statistically significant decrease in the levels of SDF-1 and surface expression of CXCR4 on myeloma cells in four consecutive apheresis collections compared with premobilization bone marrow specimens. We also observed a statistically significant decrease in surface expression of VLA-4 in myeloma cells in the apheresis collections compared with premobilization bone marrow samples. Furthermore, myeloma cells derived from apheresis collections had decreased adhesion and trans-stromal migration in response to SDF-1, which could be reversed by short incubation with interleukin-6. Hence, mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4.     

The Involvment of Hematopoietic-Specific PLC -β2 in Homing and Engraftment of Hematopoietic Stem/Progenitor Cells

Migration and bone marrow (BM) homing of hematopoietic stem progenitor cells (HSPCs) is regulated by several signaling pathways, and here we provide evidence for the involvement in this process of hematopoietic-specific phospholipase C-β2 (PLC-β2). This enzyme is involved in release of intracellular calcium and activation of protein kinase C (PKC). Recently we reported that PLC-β2 promotes mobilization of HSPCs from BM into peripheral blood (PB), and this effect is mediated by the involvement of PLC-β2 in the release of proteolytic enzymes from granulocytes and its role in disintegration of membrane lipid rafts. Here we report that, besides the role of PLC-β2 in the release of HSPCs from BM niches, PLC-β2 regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including SDF-1, S1P, C1P, and ATP. Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release in PLC-β2-KO mice and a high baseline level of heme oxygenase 1 (HO-1), an enzyme that negatively regulates cell migration.     

Chemokine receptor CXCR4-dependent internalization and resecretion of functional chemokine SDF-1 by bone marrow endothelial and stromal cells

Regulation of the availability of chemokine SDF-1 (CXCL12) in bone marrow is still not fully understood. Here we describe a unique function for the chemokine receptor CXCR4 expressed on bone marrow endothelial cells, which efficiently internalize circulating SDF-1, resulting in its translocation into the bone marrow. Translocated SDF-1 increased the homing of transplanted human CD34(+) hematopoietic progenitors to the bone marrow. The chemokine transporter function of CXCR4 was a characteristic of endothelial and stromal cells but not of hematopoietic cells. Thus, chemokine translocation across the blood-bone marrow barrier allows effective transfer of functional SDF-1 from the periphery to the stem cell niche in the bone marrow during both homeostasis and 'alarm' situations.     

Cationic liposome-mediated CXCR4 gene delivery into hematopoietic stem/progenitor cells: implications for clinical transplantation and gene therapy

The chemokine stromal cell-derived factor (SDF)-1α/CXCL12 and its receptor CXC chemokine receptor 4 (CXCR4) play a crucial role in the homing/engraftment and retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. It has been shown using the viral gene transfer technique that CXCR4 overexpression on human CD34(+) HSPC significantly improves their engraftment in murine models. However, clinical trials with gene therapy have revealed safety concerns related to the immunogenicity of the viral carriers, due to the random integration of viral genes into the host genome. Therefore, a method for CXCR4 gene delivery into HSPC that is safe, nonviral, and highly efficient is needed to improve clinical transplantation and gene therapies. In this work, we investigated the nonviral CXCR4 gene delivery into HSPC using the cationic liposome agent IBAfect. We used CD34(+) cells from cord blood and the models of immature hematopoietic cells expressing CD34 antigen, namely, leukemic cell lines KG-1a and KG-1. Transfection efficiency was determined by flow cytometric analysis 12, 24, 48, and 72 h after transfection, and the viability of cells analyzed by trypan blue exclusion and MTS assays. The functional response of CXCR4-transfected HSPC toward an SDF-1α gradient was determined by chemotaxis assay. We found that ~25% transfection is achieved for KG-1a and KG-1 cells and 20% for HSPC, and that the viability of CXCR4-transfected HSPC is not significantly altered. More importantly, overexpression of CXCR4 using IBAfect significantly increased the chemotaxis of KG-1 cells and HSPC toward SDF-1α. However, we tested 2 other commercially available cationic liposomes (Lipofectamine 2000 and 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]) in parallel, and we found that they failed to deliver the CXCR4 gene into cells under the same conditions. These results suggest that IBAfect-mediated in vitro gene delivery to overexpress CXCR4 on HSPC is a safe and efficient technique with great potential for improving the efficacy of HSPC transplantation and gene therapy protocols.     

G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4

Granulocyte colony-stimulating factor (G-CSF) induced hematopoietic stem cell mobilization is widely used for clinical transplantation; however, the mechanism is poorly understood. We report here that G-CSF induced a reduction of the chemokine stromal cell derived factor 1 (SDF-1) and an increase in its receptor CXCR4 in the bone marrow (BM), whereas their protein expression in the blood was less affected. The gradual decrease of BM SDF-1, due mostly to its degradation by neutrophil elastase, correlated with stem cell mobilization. Elastase inhibition reduced both activities. Human and murine stem cell mobilization was inhibited by neutralizing CXCR4 or SDF-1 antibodies, demonstrating SDF-1 CXCR4 signaling in cell egress. We suggest that manipulation of SDF-1 CXCR4 interactions may be a means with which to control the navigation of progenitors between the BM and blood to improve the outcome of clinical stem cell transplantation.     

归巢因子基质细胞衍生因子1/CXCR4在角膜上的分布

背景：基质细胞衍生因子1/CXCR4在介导骨髓移植等组织器官的干细胞归巢中起着重要的作用。对于角膜干细胞移植的归巢和目前角膜上基质细胞衍生因子1/CXCR4的具体分布及含量较为少见。 目的：荧光定量PCR检测大鼠角膜组织中基质细胞衍生因子1/CXCR4基因mRNA的表达水平。 方法：取完整正常大鼠角膜组织,细分为角膜中央区,角膜旁中央及角膜缘区,用荧光定量PCR法(染料法)检测相应部位基质细胞衍生因子1/CXCR4基因mRNA的表达水平。 结果与结论：角膜中央区基质细胞衍生因子1及CXCR4基因mRNA的表达水平分别为0.213±0.015,0.189± 0.02;角膜旁中央区基质细胞衍生因子1及CXCR4基因mRNA的表达水平分别为0.529±0.077,0.285± 0.015;角膜缘区基质细胞衍生因子1及CXCR4基因mRNA的表达水平分别为0.666±0.069,0.258±0.067。结果表明,在大鼠角膜组织中,角膜中央区,周边区及边缘区相应基质细胞衍生因子1及CXCR4的基因mRNA的表达水平均存在着显著性的差异,这种差异性对于角膜缘干细胞缺乏症干细胞移植治疗中细胞的归巢及移行起着重要的作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

S1P(1) overexpression stimulates S1P-dependent chemotaxis of human CD34+ hematopoietic progenitor cells but strongly inhibits SDF-1/CXCR4-dependent migration and in vivo homing

The CXC chemokine receptor 4 (CXCR4) and its ligand stromal derived factor 1 (SDF-1) regulate egress and homing of hematopoietic stem cells. Activation of sphingosine-1-phosphate (S1P) receptors (S1P(1-5)) modulates chemokine-induced migration of lymphocytes and hematopoietic stem cells. To analyze the influence of S1P(1) on SDF-1-dependent chemotaxis and trafficking, we overexpressed S1P(1) in CD34+ mobilized peripheral blood progenitor cells (PBPCs). Using a gamma-retroviral vector, transgene overexpression was achieved in more than 90% of target cells. S1P(1) transgene positive PBPCs showed enhanced chemotaxis towards S1P. S1P(1) overexpression resulted in reduced CXCR4 surface expression levels and strong inhibition of SDF-1-dependent ERK1/2 phosphorylation and Ca(2+) flux. Furthermore, SDF-1-dependent migration of S1P(1) overexpressing PBPCs or Jurkat cells was reduced up to 10-fold. Sublethally irradiated NOD/SCID mice were transplanted with 6-day cultured PBPCs overexpressing either S1P(1)-IRES-GFP or GFP alone. Screening for GFP positive human cells in the mouse bone marrow 20h after transplantation revealed an eightfold reduction in bone marrow homing of S1P(1) transgene expressing cells. Our data suggest that S1P(1) acts as an inhibitor of CXCR4-dependent migration of hematopoietic cells to sites of SDF-1 production.     

Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis

The alpha-chemokine stromal-derived factor (SDF)-1 and the G-protein-coupled seven-span transmembrane receptor CXCR4 axis regulates the trafficking of various cell types. In this review, we present the concept that the SDF-1-CXCR4 axis is a master regulator of trafficking of both normal and cancer stem cells. Supporting this is growing evidence that SDF-1 plays a pivotal role in the regulation of trafficking of normal hematopoietic stem cells (HSCs) and their homing/retention in bone marrow. Moreover, functional CXCR4 is also expressed on nonhematopoietic tissue-committed stem/progenitor cells (TCSCs); hence, the SDF-1-CXCR4 axis emerges as a pivotal regulator of trafficking of various types of stem cells in the body. Furthermore, because most if not all malignancies originate in the stem/progenitor cell compartment, cancer stem cells also express CXCR4 on their surface and, as a result, the SDF-1-CXCR4 axis is also involved in directing their trafficking/metastasis to organs that highly express SDF-1 (e.g., lymph nodes, lungs, liver, and bones). Hence, we postulate that the metastasis of cancer stem cells and trafficking of normal stem cells involve similar mechanisms, and we discuss here the common molecular mechanisms involved in these processes. Finally, the responsiveness of CXCR4+ normal and malignant stem cells to an SDF-1 gradient may be regulated positively/primed by several small molecules related to inflammation which enhance incorporation of CXCR4 into membrane lipid rafts, or may be inhibited/blocked by small CXCR4 antagonist peptides. Consequently, strategies aimed at modulating the SDF-1-CXCR4 axis could have important clinical applications both in regenerative medicine to deliver normal stem cells to the tissues/organs and in clinical hematology/oncology to inhibit metastasis of cancer stem cells.     

Expression of functional CXCR4 by muscle satellite cells and secretion of SDF-1 by muscle-derived fibroblasts is associated with the presence of both muscle progenitors in bone marrow and hematopoietic stem/progenitor cells in muscles

We found that the murine cell lines C2C12 and G7 derived from muscle satellite cells, which are essential for muscle regeneration, express the functional CXCR4 receptor on their surface and that the specific ligand for this receptor, alpha-chemokine stromal-derived factor 1 (SDF-1), is secreted in muscle tissue. These cell lines responded to SDF-1 stimulation by chemotaxis, phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT serine-threonine kinase, and calcium flux, confirming the functionality of the CXCR4 receptor. Moreover, supernatants derived from muscle fibroblasts chemoattracted both satellite cells and human CD34(+) hematopoietic stem/progenitor cells. In a similar set of experiments, supernatants from bone marrow fibroblasts were found to chemoattract CXCR4(+) satellite cells just as they chemoattract CD34(+) cells. Moreover, preincubation of both muscle satellite cells and hematopoietic stem/progenitor CD34(+) cells before chemotaxis with T140, a specific CXCR4 inhibitor, resulted in a significantly lower chemotaxis to media conditioned by either muscle- or bone marrow-derived fibroblasts. Based on these observations, we postulate that the SDF-1-CXCR4 axis is involved in chemoattracting circulating CXCR4(+) muscle stem/progenitor and circulating CXCR4(+) hematopoietic CD34(+) cells to both muscle and bone marrow tissues. Thus, it appears that tissue-specific stem cells circulating in peripheral blood could compete for SDF-1(+) niches, and this would explain, without invoking the concept of stem cell plasticity, why hematopoietic colonies can be cultured from muscles and early muscle progenitors can be cultured from bone marrow.     

Blockade of alpha6-integrin reveals diversity in homing patterns among human, baboon, and murine cells

Our understanding of the mechanisms by which intravenously transplanted hematopoietic stem/progenitor cells (HSPCs) home to and engraft the bone marrow (BM) remains incomplete, but participation of adhesion molecules has been documented. We here demonstrate that blockade of the alpha6-integrin enhanced BM homing of human and nonhuman primate BM-derived HSPCs by >60% in the xenogeneic transplant model and led to significantly improved engraftment. The effect was limited to BM-derived HSPCs, as granulocyte-colony-stimulating factor mobilized peripheral blood or cord blood HSPCs express little or no alpha6 integrin. By contrast, despite high alpha6 integrin expression, no effect of alpha6 blockade on murine BM-HSPCs homing/engraftment was observed.     

Syngenic bone marrow cells restore hepatic function in carbon tetrachloride-induced mouse liver injury

Progenitor cells in bone marrow have been explored for the treatment of liver injury. Stem cell homing to the injured tissue is regulated through stromal cell derived factor-1 (SDF-1) and its receptor CXCR4. We hypothesized that syngenic bone marrow cells (BMCs) would restore hepatic function in the injured liver through the regulation by SDF-1/CXCR4 system. After injecting carbon tetrachloride (CCl(4)), the mice were injected with syngenic BMCs or normal saline. Morphological and functional analysis of the liver was performed. Flow cytometry for the stem cell markers and CXCR4 was done with the liver, BM, and spleen cells from each group. Carboxyfluorescein diacetate succinimidyl ester was used to trace the homing of transplanted BMCs. The SDF-1 expression of the liver was assessed by immunohistochemistry. Hepatosplenomegaly and necrosis of the CCl(4)-injected mouse liver were improved after BMCs transplantation The hepatic enzymes were increased after injury and then decreased after BMCs transplantation. The expression of stem cell markers and CXCR4 was exclusively increased in the damaged liver compared to the BM and spleen, and even more elevated after BMCs transplantation. SDF-1 expression in the liver was observed after CCl(4) injection and it was elevated after BMCs transplantation. The intrinsic and extrinsic BMCs migrate specifically to the injured liver rather than BM or spleen, and the transplanted BMCs contribute to the repair of the damaged liver. SDF-1/CXCR-4 interaction plays a role in stem cell homing toward the damaged organ, and transplanted BMCs are involved in the up-regulated SDF-1 expression seen in the injured liver.     

SDF-1/CXCR4增强骨髓间质干细胞的造血支持作用

目的探讨基质细胞衍生因子-1(SDF-1)/CXCR4在骨髓间质干细胞(MSCs)支持CD34 造血干/祖细胞(HSPCs)扩增中的作用。方法在长期培养基(LTC)中,以大鼠骨髓MSCs作为饲养层体外扩增骨髓CD34 细胞,每周分别加入SDF-1、SDF-1抗体或CXCR4抗体至5周。计算CD34 细胞数和集落形成细胞(CFC)数,以评价造血支持功能。为评估SDF-1/CXCR4对CD34 细胞增殖周期的影响,进行了杀伤试验以计算增殖指数。流式细胞术检测MSCs和CD34 细胞中SDF-1与CXCR4的表达;ELISA检测MSCs和CD34 细胞培养基中SDF-1的含量。结果CD34 细胞数、CFC数和增殖指数在加入SDF-1后明显增加(P<0.01),加入SDF-1抗体或CXCR4抗体后明显减少(分别为P<0.05,P<0.01)。CD34 细胞表面表达CXCR4,MSCs则不表达;MSCs细胞内表达SDF-1,而CD34 细胞不表达。在MSCs培养基中检测到SDF-1,在CD34 细胞培养基中未发现。结论SDF-1/CXCR4在骨髓MSCs支持HSPCs扩增中起重要作用。     

Small peptide analogue of SDF-1alpha supports survival of cord blood CD34+ cells in synergy with other cytokines and enhances their ex vivo expansion and engraftment into nonobese diabetic/severe combined immunodeficient mice

The SDF-1/CXCR4 axis has been implicated in the chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. We studied the effects of a SDF-1 peptide analogue CTCE-0214 on the survival of cord blood CD34+ cells in culture, expansion, and engraftment of expanded cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Our results demonstrated that CTCE-0214 synergized with thrombopoietin (TPO), stem cell factor (SCF), or flt-3 ligand (FL) on the survival of stem and progenitor cells in culture. Adding CTCE-0214 at a low concentration (0.01 ng/ml) for 4 days together with TPO, SCF, and FL significantly enhanced ex vivo expansion of CD34+ cells to subsets of primitive (CD34+CD38- cells, colony-forming unit-mixed [CFU-GEMMs]), erythroid (CFU-Es), myeloid (CFU-GMs), and megakaryocytic (CD61+CD41+ cells, CFU-MKs) progenitors, as well as their multilineage engraftment in NOD/SCID mice. Interestingly, the short exposure of expanded cells to CTCE-0214 (100 and 500 ng/ml) for 4 hours did not increase the quantity of progenitor cells but enhanced their engraftment capacity. The proportion of CD34+ cells expressing surface CXCR4 was decreased, but the overall number of this population increased upon expansion. The small peptide analogue of SDF-1 could be developed for ex vivo expansion and improving engraftment of cord blood transplantation.     

Novel Strategy for Successful Long-Term Hematopoietic Recovery after Transplanting a Limited Number of Hematopoietic Stem/Progenitor Cells

Various investigators have attempted to overcome the shortage of available hematopoietic stem/progenitor cells (HSPCs) by facilitating their engraftment after transplantation. Preconditioning of HSPCs with the granulocyte-derived cationic peptide LL-37 has been suggested as a useful strategy to facilitate engraftment of transplanted cells by enhancing their responsiveness to CXCL12. In this study, we evaluated whether LL-37 preconditioning is acceptable for clinical application. We found that the effect of LL-37 preconditioning was specific to clonogenic cells and was mediated specifically by increased calcium influx with the activation of downstream signaling through mammalian target of rapamycin complex 1 (mTORC1). Because hyperactivation of mTORC1 and the disruption of 5′ adenosine monophosphate-activated protein kinase (AMPK) are known to deplete HSPC pools, we compared the repopulation capacity of HSPCs preconditioned with LL-37 and those preconditioned with AMPK activator (AICAR). In vivo competitive repopulation experiments revealed that LL-37 preconditioning impairs long-term repopulation of transplanted HSPCs, suggesting that this strategy might not acceptable for clinical applications in which long-term repopulation capacity is a prerequisite. AICAR preconditioning dramatically enhanced the long-term repopulation of transplanted HSPCs, however. Taken together, these results suggest that future strategies to ensure successful transplantation outcomes should focus on protecting HSPCs from various stimuli during their homing to the bone marrow niches rather than activating them before transplantation.     

Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence

In this study we provide evidence that the SDF-1alpha/CXCR4 chemokine axis is involved in both the retention of neutrophils within the bone marrow and the homing of senescent neutrophils back to the bone marrow. We show that the functional responses of freshly isolated human and murine neutrophils to CXCR2 chemokines are significantly attenuated by SDF-1alpha, acting via CXCR4. As a consequence, the mobilization of neutrophils from the bone marrow in vivo by the CXCR2-chemokine, KC, was dramatically enhanced by blocking the effects of endogenous SDF-1alpha using a specific CXCR4 antagonist. As neutrophils age, they upregulate expression of CXCR4 and acquire the ability to migrate toward SDF-1alpha. We show here that these senescent CXCR4(high) neutrophils preferentially home to the bone marrow in vivo in a CXCR4-dependent manner, suggesting a previously undefined mechanism for the clearance of senescent neutrophils from the circulation.     

Cell surface engineering to enhance mesenchymal stem cell migration toward an SDF-1 gradient

Mesenchymal stem cell (MSC) therapy for the treatment of myocardial infarction (MI) has shown considerable promise in clinical trials. A billion MSCs need to be administered for therapeutic efficacy, however, because only ∼1% of the cells reach the ischemic myocardium after systemic infusion. This is due to the loss of the homing signal on the surface of the MSCs during their expansion in culture. Stromal-derived factor-1 (SDF-1) is up-regulated immediately after infarction and is released into the peripheral blood. This SDF-1 reaches the bone marrow and recruits CXC chemokine receptor 4 (CXCR4)-positive stem cells. The CXCR4/SDF-1 axis plays an important role in MSC homing to the ischemic myocardium. Since SDF-1 is highly expressed for only 48 h after infarction, the current approaches requiring long-term culture of MSCs to induce CXCR4 expression are not clinically useful. To provide a clinically viable means to improve the homing of MSCs, we have developed a surface modification method to incorporate recombinant CXCR4 protein on the membrane of MSCs within 10 min. Using this method, we have confirmed the improved migration of MSCs toward an SDF-1 gradient.     

Stromal-derived factor-1 and its receptor, CXCR4, are constitutively expressed by mouse liver sinusoidal endothelial cells: implications for the regulation of hematopoietic cell migration to the liver during extramedullary hematopoiesis

Stromal-derived factor (SDF)-1 is the main regulating factor for trafficking/homing of hematopoietic stem cells (HSC) to the bone marrow (BM). It is possible that this chemokine may also play a fundamental role in regulating the migration of HSC to several organs during extramedullary hematopoiesis. Because liver sinusoidal endothelial cells (LSEC) constitute an extramedullary niche for HSC, it is possible that these cells represent one of the main cellular sources of SDF-1 at the liver. Here, we show that LSEC express SDF-1 at the mRNA and protein level. Biological assays showed that conditioned medium from LSEC (LSEC-CM) stimulated the migration of BM progenitor lineage-negative (BM/Lin?) cells. This effect was significantly reduced by AMD3100, indicating that the SDF-1/CXCR4 axis is involved in the stimulatory migrating effect induced by LSEC-CM. Early localization of HSC in SDF-1-expressing LSEC microenvironment together with increased levels of this chemokine in hepatic homogenates was found in an experimental model of liver extramedullary hematopoiesis. Flow cytometry studies showed that LSEC express the CXCR4 receptor. Functional assays showed that activation of this receptor by SDF-1 stimulated the migration of LSEC and increased the expression of PECAM-1. Our findings suggest that LSEC through the production of SDF-1 may constitute a fundamental niche for regulation of HSC migration to the liver. To our knowledge, this is the first report showing that LSEC not only express and secrete SDF-1, but also its receptor CXCR4.     

Control of human hematopoietic stem/progenitor cell migration by the extracellular matrix protein Slit3

Patients whose hematopoietic system is compromised by chemo- and/or radiotherapy require transplantation of hematopoietic stem and progenitor cells (HSPCs) to restore hematopoiesis. Successful homing of transplanted HSPCs to the bone marrow (BM) largely depends on their migratory potential, which is critically regulated by the chemokine CXCL12. In this study, we have investigated the expression and function of Slit proteins and their corresponding Roundabout (Robo) receptors in human HSPC migration. Slit proteins are extracellular matrix proteins that can modulate the (chemoattractant-induced) migration of mature leukocytes. We show that mRNAs for all Slits (Slit1-3) are expressed in primary BM stroma and BM-derived endothelial and stromal cell lines, but not in CD34? HSPCs. Human CD34? HSPCs expressed mRNAs for all Robos (Robo1-4), but only the Robo1 protein was detected on their cell surface. Functionally, Slit3 treatment increased the in vivo homing efficiency of CD34? HSPCs to the BM in NOD/SCID mice, whereas Slit3-exposed HSPC migration in vitro was inhibited. These effects do not appear to result from modulated CXCL12 responsiveness as CXCR4 expression, CXCL12-induced actin polymerization or the basal and CXCL12-induced adhesion to fibronectin or BM-derived endothelial cells of CD34? HSPC were not altered by Slit3 exposure. However, we show that Slit3 rapidly reduced the levels of active RhoA in HL60 cells and primary CD34? HSPC, directly affecting a pathway involved in actin cytoskeleton remodeling and HSPC migration. Together, our results support a role for Slit3 in human HSPC migration in vitro and homing in vivo and might contribute to the design of future approaches aimed at improving transplantation efficiency of human CD34? HSPCs.     

SDF-1 increases recruitment of osteoclast precursors by upregulation of matrix metalloproteinase-9 activity

Although chemokines play essential roles in the trafficking and homing of many circulating hematopoietic cell types, their potential influences on osteoclast (OC) recruitment or bone remodeling are not well known. Therefore, chemokine receptor expression was analyzed by RNase protection assay during OC formation induced by RANKL in a murine mononuclear cell line (RAW 264.7). Relatively high CXCR4 expression was detected in RAW cells (pre-OCs), whereas CXCR4 levels were downregulated during RAW-OC development. SDF-1, the unique ligand for CXCR4, stimulated RAW cell production of matrix metalloproteinase (MMP)-9 activity, a matrix-degrading enzyme essential for pre-OC migration into the developing bone marrow cavity. Induced MMP-9 activity in RAW cells was associated with their increased MMP-dependent transmigration through a collagen gel in response to SDF-1. We conclude that SDF-1 stimulation of MMP-9 activity in pre-OCs may be a key aspect of their recruitment to bone and migration within the marrow to sites for OC differentiation and bone resorption.     

SDF-1 Increases Recruitment of Osteoclast Precursors by Upregulation of Matrix Metalloproteinase-9 Activity

Although chemokines play essential roles in the trafficking and homing of many circulating hematopoietic cell types, their potential influences on osteoclast (OC) recruitment or bone remodeling are not well known. Therefore, chemokine receptor expression was analyzed by RNase protection assay during OC formation induced by RANKL in a murine mononuclear cell line (RAW 264.7). Relatively high CXCR4 expression was detected in RAW cells (pre-OCs), whereas CXCR4 levels were downregulated during RAW-OC development. SDF-1, the unique ligand for CXCR4, stimulated RAW cell production of matrix metalloproteinase (MMP)-9 activity, a matrix-degrading enzyme essential for pre-OC migration into the developing bone marrow cavity. Induced MMP-9 activity in RAW cells was associated with their increased MMP-dependent transmigration through a collagen gel in response to SDF-1. We conclude that SDF-1 stimulation of MMP-9 activity in pre-OCs may be a key aspect of their recruitment to bone and migration within the marrow to sites for OC differentiation and bone resorption.