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SDF-1/CXCL12 is a potent chemokine required for the homing and engraftment of hematopoietic stem and progenitor cells. Previous Data from our group has shown that in an SDF-1/CXCL12 transgenic mouse model, Lineage− Sca-1+ c-Kit+ (LSK) bone marrow cells have reduced mitochondrial membrane potential versus wild-type, These results suggested that SDF-1/CXCL12 may function to keep mitochondrial respiration low in immature blood cells in the bone marrow. Low mitochondrial metabolism helps to maintain low levels of reactive oxygen species (ROS), which can influence differentiation. To test whether SDF-1/CXCL12 regulates mitochondrial metabolism, we employed the human leukemia cell line HL-60, that expresses high levels of the SDF-1/CXCL12 receptor, CXCR4, as a model of hematopoietic progenitor cells in vitro. We treated HL-60 cells with SDF-1/CXCL12 for 2 and 24 hours. Oxygen consumption rates (OCR), mitochondrial-associated ATP production, mitochondrial mass, and mitochondrial membrane potential of HL-60 cells were significantly reduced at 2 hours and increased at 24 hours as compared to untreated control cells. These biphasic effects of SDF-1/CXCL12 were reproduced with lineage negative primary mouse bone marrow cells, suggesting a novel function of SDF-1/CXCL12 in modulating mitochondrial respiration by regulating mitochondrial oxidative phosphorylation, ATP production and mitochondrial content.
Stromal cell-derived factor 1α (SDF-1), also known as CXCL12, is an important member of the CXC family of chemokines. SDF-1/CXCL12 is expressed in a wide array of different tissues and cell types, including immune cells, endothelial cells, stromal cells, fibroblasts, and cancer cells . The gene encoding SDF-1/CXCL12 is located at 10q11.1 and has 6 exons encoding 68 amino acids. It has a molecular weight of 8 kDa, and its promoter contains binding sites for transcription factors such as SP1 . Signal transduction induced by SDF-1/CXCl12 is mediated through the chemokine receptor CXCR4 [3–6]. Knockout of SDF-1/CXCL12 is perinatal lethal and mice lacking SDF-1/CXCL12 have severe defects in gastrointestinal vascularization, cerebral development, and hematopoietic defects [7–9]. CXCR4 knockout studies reveal a strikingly similar phenotype to that of SDF-1/CXCL12 knockout mice, suggesting that the SDF-1/CXCL12 and CXCR4 signaling axis is non-promiscuous .
SDF-1/CXCL12 is a potent chemotactic factor for hematopoietic stem (HSCs) and progenitor (HPCs) cells [11,12]. It plays an essential role in the maintenance of HSCs, including homing, engraftment and repopulating activity, as well as HSC quiescence and retention in the bone marrow [13–17]. It has been shown to enhance the survival of HSCs and HPCs, an effect increased in synergy with other cytokines [5,18–20]. Treatment of mouse bone marrow cells and human cord blood HPCs with soluble SDF-1/CXCL12 enhanced their replating efficiency, and bone marrow cells from mice expressing a human SDF-1/CXCL12 transgene exhibited increased replating capacity of single macrophage-and multipotent progenitor- derived colonies .
SDF-1/CXCL12 appears to be a key regulator of HSCs in the bone marrow microenvironment . The niche provides signals regulating HSC functions, such as self-renewal and long term repopulating capability, as well as the ability to undergo multiline age differentiation. Several groups have shown in genetic studies that mesenchymal progenitor, endothelial, and stromal cell populations play a critical role in the maintenance of HSCs in the niche and depending on which niche cells HSCs interact with, helps to define the specific “sub-niche” in which HSCs may reside [23–30]. Deletion of SDF-1/CXCL12 from different types of niche cells leads to the reduction in HSC numbers, competitive repopulation, and increases in splenic HSCs, all of which indicate an essential role for SDF-1/CXCL12 in HSC function in the bone marrow microenvironment [23,25,27,29,30].
Despite work from several groups describing the role of SDF-1/CXCL12 in the maintenance of HSCs and HPCs in the various niches in the bone marrow [23,25,27,29,30], there is a paucity of information on the mechanism by which SDF-1/CXCL12 functions at the molecular level for immature blood cell function in the bone marrow. Regulation and restriction of mitochondrial metabolism has been shown to be critical in maintaining the quiescent state of HSCs in the bone marrow by preventing mitochondrial produced reactive oxygen species (ROS), which can promote differentiation and HSC attrition and potential dysfunction [31–36]. Recent work from our group has shown that SDF-1/CXCL12 can modulate mitochondrial activity and mitochondrial mass in murine bone marrow cells expressing a human SDF-1/CXCL12 transgene . We therefore hypothesized that SDF-1/CXCL12 regulates mitochondrial respiration in early hematopoietic cells.
Basal oxygen consumption rates (OCR) and mitochondrial-associated ATP production were obtained using the Seahorse Bioscience XF96 Extracellular Flux Analyzer from Seahorse Bioscience, and measurements were performed according to the manufacturer’s instructions and as described previously [37–39]. Mitochondrial-associated ATP production is the difference between the basal OCR and oligomycin-A repressed OCR .
HL-60 cells (ATCC CCL-240) were obtained from the American Type Culture Collection (Manassas, VA) and maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) with 20% FBS. HL-60 cells were incubated in IMDM +20% FBS with and without 50 ng/ml SDF-1 (R&D, Minneapolis, MN) for two and 24 hours, respectively. This concentration of SDF-1 has been shown to elicit optimal responses in numerous of our assays [12,18,19,21,41]. C57Bl/6 strain mice were used to isolate lineage negative bone marrow cells. The Indiana University Committee on Use and Care of Animals approved the mouse studies. Mouse lineage negative cells were isolated using the Miltenyi Biotech (San Diego, CA) mouse Lineage Cell Depletion Kit. After lineage depletion, lineage negative cells were incubated in IMDM +10% FBS and stimulated with or without 50 ng/ml SDF-1 (R&D, Minneapolis, MN) for two and 24 hours, respectively.
Anti-human CXCR4 APC conjugated antibody (Clone 12G5) and anti-human CXCR7 FITC conjugated antibody (Clone 358426) was from R&D, Minneapolis, Minnesota. Mitotracker Green FM and Mitotracker Red CMXRos were from Molecular Probes, Eugene, Oregon. Flow cytometry was performed with a FACS Calibre flow cytometer (BD Biosciences, Franklin Lakes, NJ). Flow cytometry data were analyzed using FlowJo (Ashland, Oregon). Oligomycin-A was purchased from Sigma-Aldrich (St. Louis, MO) and AMD3100 was a kind gift from AnorMed (Langley, BC, Canada).
Data were statistically analyzed and plotted using GraphPad Prism 6 (San Diego, CA). Differences were assessed with a 2-tailed Student t-test or one-way ANOVA with Tukey’s post-hoc correction. P values ≤0.05 were considered significant.
To investigate the potential role that SDF-1/CXCL12 plays in controlling mitochondrial respiration, we employed the human promyelocitic leukemia cell line, HL-60, as a beginning of modeling human immature subsets of hematopoietic cells and then proceeded to confirm the results in lineage− primary mouse bone marrow cells.
We maintained HL-60 cells in culture with IMDM +20% fetal bovine serum under standard cell culture conditions, then cultured them for two and 24 hours, respectively, with or without 50 ng/ml of SDF-1/CXCL12 to assess rapid and more delayed effects. We assessed oxygen consumption rates (OCR), a measure of mitochondrial respiration, for each group on a Seahorse Bioscience XF96 Extracellular Flux Analyzer (Seahorse XF96). Cells treated for two hours with SDF-1/CXCL12 had a significant reduction in OCR of 24.8±1.0% (mean ± SEM) as compared to two-hour unstimulated control cells (Figure 1Ai). In contrast, HL-60 cells treated for 24 hours with SDF-1/CXCL12 had a significantly increased OCR of 96.7±13.3% over unstimulated 24-hour control cells (Figure 1Aii). This suggests that SDF-1/CXCL12 plays a potential role modulating mitochondrial respiration in a bi-phasic manner as indicated by changes in mitochondrial OCR.
We next analyzed mitochondrial-associated ATP production by using the Seahorse XF96 (Figure 1Bi and 1Bii). Mitochondrial-associated ATP production is the difference between the OCR and oligomycin-A (0.6 μM) repressed OCR . Mitochondrial-associated ATP production was significantly reduced in the two-hour SDF-1/CXCL12 treated group compared to the unstimulated control cells (−29.2 ± 2.9%), while the 24-hour treated group was significantly increased versus control (+120.1 ± 20.3%). These results show that as OCR is reciprocally decreased or increased over time by SDF-1/CXCL12 treatment of HL-60 cells, there are closely related changes in ATP production suggesting that these processes are tightly coupled and regulated in a biphasic manner in vitro.
Next we sought to confirm that the changes in mitochondrial respiration and ATP production in HL-60 cells are mediated through the SDF-1/CXCL12-CXCR4 axis. We first measured surface expression of human CXCR4 and CXCR7, both receptors for SDF-1/CXCL12, using fluorochrome conjugated antibodies. (Figure 2A). Once we determined that the HL-60 cells expressed CXCR4 with minimal or no expression of CXCR7, we used the CXCR4 antagonist AMD3100  to attempt to block the SDF-1/CXCL12-CXCR4 mediated changes in mitochondrial respiration. HL-60 cells were pretreated for 30 minutes with 100 ng/ml of AMD3100 and then stimulated for 2 and 24 hours, respectively, with 50 ng/ml of SDF-1/CXCL12. HL-60 cells treated with SDF-1/CXCL12 for two hours have significantly reduced OCR as compared to untreated control cells, while both the AMD3100 alone group and the SDF-1/CXCL12 + AMD3100 group were not significantly different from untreated control (Figure 2Bi). Cells treated with SDF-1/CXCL12 for 24 hours had significantly increased OCR as compared to untreated control and cells pretreated with AMD3100 alone or pretreated with AMD3100 and then stimulated with SDF-1/CXCL12 for 24 hours had significantly reduced OCR as compared to untreated control cells (Figure 2Bii). These results show that blocking CXCR4 with AMD3100 oblates the SDF-1/CXCL12 mediated biphasic changes in OCR, suggesting that these metabolic effects of SDF-1/CXCL12 are mediated through the CXCR4 receptor.
To further investigate the mechanism by which SDF-1/CXCL12 mediates changes in mitochondrial respiration, we investigated changes in mitochondrial mass and mitochondrial membrane potential (Δψm). We hypothesized that changes in mitochondrial respiration and ATP production should be accompanied by concomitant changes in mitochondrial mass and Δψm. To assess these changes we used the mitochondrial specific dyes MitoTracker Green FM (50nM) and MitoTracker Red CMXROS (50 nM). MitoTracker Green FM is a mitochondrial specific dye that is mitochondrial membrane potential independent and is an indicator of mitochondrial mass, while Mitotracker Red CMXRos is membrane potential dependent and used as an indicator of Δψm (a measure of mitochondrial electron transport chain function). HL-60 cells were cultured for two and 24 hour, respectively, with 50 ng/ml of SDF-1/CXCL12. After each time point, cells were harvested and stained with MitoTracker Green FM or MitoTracker Red CMXRos and mitochondrial mass and Δψm was measured by flow cytometry. Both the mitochondrial mass and Δψm for the two-hour SDF-1/CXCL12 stimulated group was significantly decreased versus unstimulated control (Figure 3Ai and 3Bi). There was a trend toward increased mitochondrial mass at 24 hours (Figure 3Aii). The Δψm of the 24-hour SDF-1/CXCL12 stimulated group was significantly increased versus control (Figure 3Bii). Much like the OCR and ATP production, SDF-1/CXCL12 is also producing biphasic changes in mitochondrial mass and Δψm, suggesting that it may be regulating several different aspect of mitochondrial respiration through regulation of electron transport, oxidative phosphorylation, and mitochondrial biogenesis.
To determine if SDF-1/CXCL12 effects on HL-60 cells could be mimicked in primary cells, we isolated lineage negative (Lin−) bone marrow cells (enriched for immature blood cells) from C57Bl/6 mice and cultured them with or without 50 ng/ml SDF-1/CXCL12 for two and 24 hours, respectively. Cells were then collected after each time point and OCR rates of each group were measured on the Seahorse Bioscience XF96 Extracellular Flux Analyzer. As already noted for the human HL-60 cells (Figure 1), the OCR for the two-hour SDF-1/CXCL12 stimulated group was significantly reduced versus unstimulated two-hour control (Figure 4Ai). The OCR of the 24-hour SDF-1/CXCL12 stimulated group, however, was significantly increased (Figure 4Aii). These results are similar to the changes in OCR of the SDF-1/CXCL12 treated HL-60 cells. Next we analyzed mitochondrial-associated ATP production using the Seahorse Bioscience XF96 Extracellular Flux Analyzer (Figure 4Bi and 4Bii). Treatment of Lin− cells with SDF-1/CXCL12 produced a significant decrease in the mitochondrial-associated ATP production versus two-hour unstimulated control and treatment of Lin− cells with SDF-1/CXCL12 for 24 hours produced a significant increase in the mitochondrial-associated ATP production as compared to control cells. These results demonstrate that ex vivo treatment of primary mouse immature blood cells in Lin- bone marrow cells with SDF-1/CXCL12 regulates mitochondrial respiration in a biphasic manner similar to that seen in HL-60 cells.
Regulation of mitochondrial respiration is essential to HSC and HPC maintenance and proper function [31–36]. Disregulation of mitochondrial function can lead to various disorders including leukemia or bone marrow failure. Many groups have shown an essential role for SDF-1/CXCL12 in HSC maintenance, but its role in mitochondrial regulation had yet to be established. Our findings in HL-60 and primary Lin− bone marrow cells suggest that SDF-1/CXCL12 regulates mitochondrial OCR, ATP production as well as Δψm and mitochondrial mass in a biphasic fashion and that these effects are mediated through CXCR4. These results suggest that SDF-1/CXCL12 plays a potential role in regulating mitochondrial respiration in immature blood cell types. Mitochondrial regulation in a biphasic manner also suggests that time-dependent changes in SDF-1/CXCL12, as well as changes in the expression of SDF-1CXCL12 by various niche cells, may differentially effect hematopoietic cells in the bone marrow and potentially influence HSC maintenance and differentiation. It is clear that mitochondrial metabolism of HSCs and HPCs is important [33–37] and these results may have important implications for the regulation of mitochondrial metabolism of HSCs and HPCs in the bone marrow microenvironment. By continuing to study the role of SDF-1/CXCL12 in mitochondrial regulation in more defined HSC and HPC populations, studies may elucidate the role that it plays in regulating HSC and HPC function by various SDF-1/CXCL12 expressing niche cells.
These Studies were supported by: U.S. Public Health Service Grants from the National Institutes of Health: R01 HL67384, R01 HL56416, R01 HL112669, P01 DK90948, and U54 DK106846 to H.E.B. S.M-G was supported as a Pre-Doctoral Fellow by NIH T32 DK07519 to H.E.B.
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