ALBERT

Description: Current evidence suggests that hematopoietic stem/progenitor cell (HSPC) mobilization by granulocyte colony-stimulating factor (G-CSF) is mediated by induction of bone marrow proteases, attenuation of adhesion molecule function, and disruption of CXCL12/CXCR4 signaling in the bone marrow. The relative importance and extent to which these pathways overlap or function independently are uncertain. Despite evidence of protease activation in the bone marrow, HSPC mobilization by G-CSF or the chemokine Groβ was abrogated in CXCR4−/− bone marrow chimeras. In contrast, HSPC mobilization by a VLA-4 antagonist was intact. To determine whether other mobilizing cytokines disrupt CXCR4 signaling, we characterized CXCR4 and CXCL12 expression after HSPC mobilization with Flt3 ligand (Flt3L) and stem cell factor (SCF). Indeed, treatment with Flt3L or SCF resulted in a marked decrease in CXCL12 expression in the bone marrow and a loss of surface expression of CXCR4 on HSPCs. RNA in situ and sorting experiments suggested that the decreased CXCL12 expression is secondary to a loss of osteoblast lineage cells. Collectively, these data suggest that disruption of CXCR4 signaling and attenuation of VLA-4 function are independent mechanisms of mobilization by G-CSF. Loss of CXCL12 expression by osteoblast appears to be a common and key step in cytokine-induced mobilization.

Description: There is evidence that hematopoietic stem cells (HSC) are physically localized to specialized areas in the bone marrow termed the vascular and osteoblast niches. It is not clear if there are differences in the capacity of these niches to support HSC function. We and others previously showed that G-CSF treatment suppresses both osteoblast number and function, effectively eliminating the osteoblast niche. In contrast, G-CSF treatment has no apparent effect on the microvasculature in the bone marrow, suggesting that the vascular niche is intact. In this study, we utilized this system to assess the capacity of each niche to support HSC function. We previously reported that the competitive repopulation capacity of bone marrow isolated from G-CSF treated mice is markedly reduced. This is not due to a simple loss of HSC in the bone marrow, as the number of HSC, phenotypically defined as lineage-CD41-CD48-CD150+ (SLAM) or lineage-Kit+Sca+CD34- cells, was comparable to control mice. Moreover, the long-term repopulating activity of sorted SLAM cells from G-CSF treated mice was reduced. This repopulating defect is not secondary to impaired homing to the bone marrow, as direct intrafemoral injection of G-CSF treated bone marrow cells failed to rescue the engraftment defect. Since G-CSF is able to stimulate HSC proliferation, we predicted that the defect in repopulating activity might be secondary to loss of HSC quiescence. Contrary to our prediction, the percentage of quiescent HSC in the bone marrow was actually increased in G-CSF treated mice. Whereas 28.0 ± 3.4% of control SLAM cells were labeled after treatment with BrdU for 48 hours, only 7.5 ± 0.8% of SLAM cells isolated from G-CSF mice were labeled (p 〈 0.008). Moreover, the percentage of SLAM cells in G0, as determined by Hoechst and pyronin staining, was increased in G-CSF treated mice (80.3 ± 5.0% versus 65.5 ± 6.8% in untreated mice, p=0.104). To elucidate the molecular mechanisms by which disruption of the osteoblast niche leads to a loss of HSC activity, we performed RNA profiling experiments on SLAM cells sorted from G-CSF or saline-treated mice. Consistent with the quiescent phenotype, a significant increase in the expression of the cell cycle inhibitor, Cdkn1a (p21waf1), was observed in G-CSF treated SLAM cells. Collectively, these data show that the osteoblast and vascular niches are not functionally redundant and suggest that it is the osteoblast niche that is key to maintaining long-term repopulating activity of HSC.

Description: Accumulating evidence indicates that interaction of stromal cell-derived factor 1 (SDF-1/CXCL12 [CXC motif, ligand 12]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During granulocyte colony-stimulating factor (G-CSF)–induced HPC mobilization, CXCL12 protein expression in the bone marrow decreases. Herein, we show that in a series of transgenic mice carrying targeted mutations of their G-CSF receptor and displaying markedly different G-CSF–induced HPC mobilization responses, the decrease in bone marrow CXCL12 protein expression closely correlates with the degree of HPC mobilization. G-CSF treatment induced a decrease in bone marrow CXCL12 mRNA that closely mirrored the fall in CXCL12 protein. Cell sorting experiments showed that osteoblasts and to a lesser degree endothelial cells are the major sources of CXCL12 production in the bone marrow. Interestingly, osteoblast activity, as measured by histomorphometry and osteocalcin expression, is strongly down-regulated during G-CSF treatment. However, the G-CSF receptor is not expressed on osteoblasts; accordingly, G-CSF had no direct effect on osteoblast function. Collectively, these data suggest a model in which G-CSF, through an indirect mechanism, potently inhibits osteoblast activity resulting in decreased CXCL12 expression in the bone marrow. The consequent attenuation of CXCR4 signaling ultimately leads to HPC mobilization.

Description: The bone marrow microenvironment plays a key role in regulating hematopoietic stem cell (HSC) function. In particular, bone marrow stromal signals contribute to the maintenance of HSC quiescence, a property that is thought to be associated with long-term repopulating activity. We previously reported that G-CSF treatment disrupts the osteoblast niche by inducing osteoblast apoptosis and inhibiting osteoblast differentiation. In this altered bone marrow microenvironment, we also showed that the number of HSCs in the bone marrow after G-CSF treatment (as defined by CD34− Kit+ Sca+ lineage-cells or CD150+ CD48− CD41− lineage-[SLAM] cells) was unchanged and that the HSCs were more quiescent than HSCs from untreated mice. However, despite the quiescent phenotype, there was a marked loss of HSC long-term repopulating activity. To define mechanisms for this phenotype, we first asked whether G-CSF acts directly on HSCs to inhibit their long-term repopulating activity. Bone marrow chimeras containing wild type and G-CSFR−/− cells were established and treated with G-CSF. The contribution of G-CSFR−/− cells to hematopoiesis remained stable for at least 3 months after G-CSF treatment, demonstrating that the effects of G-CSF on HSC function are not direct. We next performed RNA expression profiling on sorted SLAM cells, a cell population highly enriched for HSCs. These data showed that expression of Cdkn1a (p21cip1/waf1) was increased in HSCs harvested from G-CSF treated mice. To define the contribution of Cdkn1a to HSC quiescence and loss of repopulating activity following treatment with G-CSF, Cdkn1a−/− mice (inbred on a C57BL/6 background) were studied. Wild-type or Cdkn1a−/− mice were treated with G-CSF for 7 days and pulse labeled with bromo-deoxyuridine (BrdU), and the percentage of SLAM cells that labeled with BrdU was determined. Consistent with our previous observations, treatment of wild-type mice with G-CSF resulted in a significant decrease in the percentage of BrdU+ SLAM cells in the bone marrow. In contrast, in Cdkn1a−/− mice, no change in the percentage of BrdU+ SLAM cells after G-CSF treatment was observed [10.08 ± 2.26% (untreated); 10.96 ± 2.80% (G-CSF treated); p = NS]. To assess HSC function, competitive repopulation assays were performed using untreated or G-CSF treated bone marrow from wild type or Cdkn1a−/− mice. Surprisingly, G-CSF had a similar deleterious effect on HSC repopulating activity in both wild type and Cdkn1a−/− mice. Collectively, these data show G-CSF treatment, possibly through disruption of the osteoblast niche, induces HSC quiescence and loss of long-term repopulating activity. HSC quiescence, but not loss of repopulating activity, is dependent upon Cdkn1a−/−. The mechanisms by which G-CSF treatment results in a loss of HSC function are under investigation.

Description: There is accumulating evidence that interaction of stromal cell derived factor-1 (SDF-1/CXCL12) with its cognate receptor, CXCR4, generates signals that regulate hematopoietic progenitor cell (HPC) trafficking in the bone marrow. During G-CSF induced HPC mobilization, SDF-1 protein expression in the bone marrow decreases, thereby attenuating CXCR4 signaling. We recently reported that G-CSF treatment induced a decrease in bone marrow SDF-1 mRNA that closely mirrored the fall in SDF-1 protein, suggesting that G-CSF targets one or more SDF-1 producing cell population in the bone marrow. However, the identity of cell populations in the bone marrow that express SDF-1 is controversial. In the present study, we address this issue by sorting cells into mature hematopoietic, hematopoietic progenitor, endothelial, and osteoblast cell populations. Real time RT-PCR analyses showed that osteoblasts and to a lesser degree endothelial cells are the major sources of SDF-1 production in the bone marrow. Surprisingly, on a per cell basis, SDF-1 expression per osteoblast was only modestly (less than two-fold) reduced in mice treated with G-CSF. These data raised the possibility that, rather than affecting SDF-1 expression per osteoblast, G-CSF regulated the number of osteoblasts in the bone marrow. To explore this possibility, osteoblast number in the bone marrow was measured by histomorphometry. Indeed, after 5 days of G-CSF treatment, a significant reduction in the number of endosteal osteoblasts was observed [number of osteoblasts per mm bone perimeter ± SEM: 74.8 ± 13.5 (untreated) versus 33.3 ± 3.8 (G-CSF)]. Moreover, expression of osteocalcin (a specific marker of mature osteoblasts) in the bone marrow was sharply reduced during G-CSF treatment: a 47 ± 12 fold reduction in osteocalcin mRNA (relative to b-actin mRNA) was observed in the bone marrow of G-CSF-treated mice compared with untreated mice. Finally, calcein double-labeling experiments showed that the mineral apposition rate was significantly reduced in G-CSF-treated mice. However, RT-PCR analyses showed that the G-CSF receptor is not expressed on osteoblasts. Accordingly, G-CSF had no direct effect on osteoblast activity in vitro. Collectively, these data show that G-CSF potently suppresses osteoblast number/activity in the bone marrow through an indirect mechanism. Since osteoblasts are thought to play a key role in establishing and maintaining the stem cell niche in the bone marrow, these data raise the possibility that G-CSF, by regulating osteoblast function (including SDF-1 expression), may have profound effects on the stem cell niche that ultimately contribute to HPC mobilization.

Description: G-CSF is the most widely used agent in the clinical setting to induce the mobilization of HPC. Several recent studies have shown that G-CSF treatment results in decreased SDF-1 protein expression in the bone marrow (BM). Moreover, functional expression of CXCR4 on HPC mobilized into the blood by G-CSF is reduced relative to HPC in the bone marrow. Given its importance in regulating HPC trafficking in the bone marrow, these data suggest that disruption of SDF-1/CXCR4 signaling is a key step in G-CSF induced HPC mobilization. In addition to G-CSF, there are many other cytokines that can induce HPC mobilization. Though the cellular targets and biological activities of mobilizing cytokines are diverse, it is possible that disruption of SDF-1/CXCR4 signaling is a common shared mechanisms of HPC mobilization. To test this hypothesis, we characterized SDF-1 and CXCR4 expression in mice treated with flt-3 ligand or stem cell factor (SCF), two cytokines that induce HPC mobilization in both mice and humans. C57BL/6 mice (n=6–8, each) were treated with human flt-3 ligand (10 mg per day x 7 days) or pegylated-rat SCF (200 mg/kg/day x 7 days). As previously reported, both cytokines induced robust HPC mobilization [number of CFU-C per ml of blood ± SEM: 17,000 ± 795 (SCF); 〉40,000 (flt-3 ligand); 138 ± 63 (saline)]. At the time of peak mobilization, mice were sacrificed and SDF-1 protein in the bone marrow measured by ELISA. Both flt-3 ligand- and SCF-treated mice showed significant reduction in SDF-1 protein compared with untreated controls [percent reduction ± SEM: 64% ± 12% (flt-3 ligand), 79% ± 10% (SCF)]. We next determined whether SDF-1 expression in the bone marrow during flt-3 ligand and SCF induced mobilization is primarily regulated at an mRNA level, similar to G-CSF. Isolated femurs were directly flushed with Trizol and SDF-1 mRNA was measured using a real time RT-PCR assay. The amount SDF-1 mRNA with respect to beta-actin mRNA was reduced in both flt-3 ligand- and SCF-treated mice compared with the untreated controls [percent reduction ± SEM: 79% ± 8% (flt-3 ligand), 60% ± 24% (SCF)]. Finally, we determined whether functional CXCR4 expression of mobilized HPC decreased during flt-3 and SCF induced mobilization. A transwell migration assay was used to measure the percentage of BM and blood HPC that migrated in response to SDF-1. As reported previously, the percentage of blood HPC that migrated to SDF-1 was significantly decreased compared with BM HPC in G-CSF treated mice [percent migrated ± SEM: 9.6 ± 3.0% (blood) and 16.2 ± 1.2% (BM)]. A similar trend was observed in flt-3 ligand treated [5% ± 4% (blood) and 32 ± 9% (BM)] and SCF treated mice [2.0 ± 2% (blood) and 21 ± 8% (BM)]. Collectively, these data show that during flt-3 ligand and SCF induced HPC mobilization SDF-1/CXCR4 signaling is disrupted. Similar to G-CSF, both of these agents primarily regulate SDF-1 expression in the BM at an mRNA level. In addition, functional expression of CXCR4 on mobilized HPC is decreased. These data suggest the hypothesis that disruption of SDF-1/CXCR4 signaling may be a common mechanism by which all hematopoietic cytokines induce HPC mobilization.

Description: Accumulating evidence suggests that osteoblast-lineage cells play a key role in supporting hematopoiesis, providing signals that maintain the normal function of hematopoietic stem cells (HSC). We and others previously showed that treatment with G-CSF reduces the number and activity of osteoblasts in the bone marrow. Here we confirm these findings using transgenic mice expressing GFP in osteoblast lineage cells under control of a 2.3 kb fragment of the col1a1 promoter. Analysis of these mice confirmed that G-CSF treatment reduced the number of osteoblasts 2.9-fold compared to control mice (n=4, p

Description: There is strong evidence that CXCL12 (stromal-derived factor-1)/CXCR4 signaling is a key regulator of hematopoietic stem and progenitor cell (HSPC) trafficking in the bone marrow. CXCL12 protein and mRNA expression in the bone marrow are markedly reduced with G-CSF treatment. We and others recently showed that G-CSF treatment results in a marked loss of mature endosteal and trabecular osteoblasts. Since osteoblasts are a major source of CXCL12, this observation provides a potential mechanism by which G-CSF downregulates CXCL12 expression in the bone marrow. To test this hypothesis, we performed RNA in situ studies for CXCL12. These studies show that CXCL12 is highly expressed in endosteal osteoblasts as well as in scattered cells in the bone marrow in untreated mice. In mice treated with G-CSF, there is a near complete loss of CXCL12 signal along the endosteum. Together, these data suggest a model in which G-CSF induced suppression of osteoblasts leads to a decrease in CXCL12 expression in the bone marrow, disrupting CXCR4 signaling and ultimately leading to HSPC mobilization. This model raises several important questions. Many different hematopoietic cytokines can induce HSPC mobilization. Is loss of CXCL12 expression by osteoblasts a common mechanism by which cytokines induce HSPC mobilization? To address this question, we studied HSPC mobilization by Flt3 ligand (Flt3L) and stem cell factor (SCF), two potent mobilizing hematopoietic cytokines. Treatment with Flt3L or SCF resulted in a significant decrease in bone marrow CXCL12 protein and mRNA expression. Moreover, the decrease in CXCL12 expression was accompanied by a loss in trabecular osteoblasts [number osteoblasts per mm bone ± SEM: 5.1 ± 0.1 (control), 3.1 ± 0.4 (Flt3L), 1.8 ± 0.8 (SCF); n=2, p