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. 2006 Mar 1;107(5):1847-56.
doi: 10.1182/blood-2005-04-1612. Epub 2005 Nov 10.

G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth

Quan-Sheng Zhu  1 , Ling Xia, Gordon B Mills, Clifford A Lowell, Ivo P Touw, Seth J Corey
Affiliations

G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth

Quan-Sheng Zhu et al. Blood. .

Abstract

Granulocyte colony-stimulating factor (G-CSF) drives the production, survival, differentiation, and inflammatory functions of granulocytes. Reactive oxygen species (ROSs) provide a major thrust of the inflammatory response, though excessive ROSs may be deleterious. G-CSF stimulation showed a time- and dose-dependent increase in ROS production, correlating with activation of Lyn and Akt. Inhibition of Lyn, PI3-kinase, and Akt abrogated G-CSF-induced ROS production. This was also blocked by DPI, a specific inhibitor of NADPH oxidase. Following G-CSF stimulation, neutrophils from Lyn-/- mice produced less ROSs than wild-type littermates. G-CSF induced both serine phosphorylation and membrane translocation of p47phox, a subunit of NADPH oxidase. Because patients with a truncated G-CSF receptor have a high risk of developing acute myeloid leukemia (AML), we hypothesized that dysregulation of ROSs contributes to leukemogenesis. Cells expressing the truncated G-CSF receptor produced more ROSs than those with the full-length receptor. G-CSF-induced ROS production was enhanced in bone marrow-derived neutrophils expressing G-CSFRdelta715, a truncated receptor. The antioxidant N-acetyl-L-cysteine diminished G-CSF-induced ROS production and cell proliferation by inhibiting Akt activation. These data suggest that the G-CSF-induced Lyn-PI3K-Akt pathway drives ROS production. One beneficial effect of therapeutic targeting of Lyn-PI3K-kinase-Akt cascade is abrogating ROS production.

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Figures

Figure 1.
Figure 1.
G-CSF-induced intracellular ROS production. Ba/F3 cells stably transfected with the wild-type G-CSF receptor were treated with variable G-CSF concentrations or for variable time periods. ROS production was measured in cells labeled with DHR. ROS levels induced by G-CSF normalized to that of untreated cells were determined by monitoring the increased fluorescence in the cells. (A) Dose-dependent increase in intracellular ROS production (mean ± SD). (B) Time course for increased intracellular ROS production (mean ± SD). (C) G-CSF-induced neutrophil differentiation. (Left) 32DGR cells grown in medium with IL-3 appear as myeloblasts. (Right) 32GR cells grown in medium with 100 ng/mL G-CSF for 10 days display a population of granulocytes (Giemsa staining; × 200 magnification). Photographs were obtained using a Nikon Microphot-FX microscope (Nikon, Tokyo, Japan) and a Nikon plan/apo 20×/0.75 numeric aperture objective lens. Images were captured using a Sony 3CCD color video camera, model DXC-990 (Sony, Tokyo, Japan) and were processed using Acquisitions software (Media Cybernetics, Silver Spring, MD). (D) G-CSF-induced intracellular ROS production in 32DGR cells. Purple indicates unstimulated cells; green, G-CSF for 30 minutes; red, 100 μM H2O2 for 30 minutes.
Figure 2.
Figure 2.
Time course for G-CSF-induced activation of Lyn, Akt, and ERK1/2. Ba/F3GR cells were stimulated with or without 100 ng/mL G-CSF for indicated time periods, then whole-cell lysates were prepared. (A) Lyn activation. Immunoblotting (IB) was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn, and anti-phospho-Lyn Y507, which detects the nonactivated state of Lyn. The blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (B) Akt activation. Immunoblotting was performed using anti-phospho-Akt S473 antibody and reprobed with anti-Akt antibody. (C) ERK1/2 activation. Immunoblotting was performed using anti-phospho-ERK1/2 2T202/Y204 antibody and reprobed with antiactin antibody. Comparable results were observed in 4 independent experiments.
Figure 3.
Figure 3.
Effect of kinase inhibitors on G-CSF-induced activation of Akt or ERK1/2. (A) Inhibition of downstream substrates of Akt, mTOR, and GSK3 by a specific Akt inhibitor A838450. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then stimulated with or without 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-mTOR S2448 or anti-phospho-GSK3 S21/9 antibodies. Blots were stripped and reprobed with anti-mTOR or anti-GSK3 antibody to demonstrate comparable protein loading. (B) Akt inhibition. Ba/F3GR cells were pretreated for 1 hour with the indicated concentrations of Akt inhibitor A838450 or DMSO (the diluent control), then left unstimulated or stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-AktS473 and anti-phospho-ERK1/2 T202/Y204 antibody. Blot was stripped and reprobed with anti-Akt or antiactin antibody to demonstrate comparable protein loading. (C) ERK1/2 inhibition. Ba/F3GR cells were pretreated with the indicated concentrations of MEK inhibitor or DMSO (diluent control) for 1 hour and then stimulated with 100 ng/mL G-CSF. Lysates were prepared, and immunoblotting was performed using anti-phospho-ERK1/2 T202/Y204 antibody or anti-phospho-Akt S473. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable protein loading.
Figure 4.
Figure 4.
Inhibition of G-CSF-induced intracellular ROS production. Ba/F3GR cells were pretreated with the Src kinase inhibitor PP1, PI3-kinase inhibitor LY294002, Akt inhibitor A838450, or ERK1/2 inhibitor MEKI for 1 hour and then stimulated with 100 ng/mL G-CSF for 30 minutes (green) or not (purple). The increased levels of intracellular ROSs were determined by flow cytometry. (A) Inhibition of ROS production by kinase inhibitors. Results from a representative experiment. (B) Effects of kinase inhibitors on ROS production due to G-CSF (mean ± SD). (C) Effect of siRNA knock-down of Akt on ROS production. Ba/F3GR cells were treated for 24 hours with 200 nM siRNA to murine Akt1, scrambled sequence, or mock nucleofection. Cell lysates were analyzed for Akt and actin protein expression. Cells were also stimulated with 100 ng/mL G-CSF for 60 minutes or not, stained with DHR, and then analyzed for ROS production by flow cytometry. Data represented the mean ± SD. (Bottom) Western blot of nucleofected Ba/F3GR cells. Lane 1, untransfected cells; lane 2, mock nucleofection of cells; lane 3, nucleofection with Akt1 siRNA; lane 4, scrambled siRNA. (D) Neutrophils from bone marrow of wild-type and Lyn-/- mice. The results showed that more than 95% of cells are neutrophils. Images obtained as in Figure 1C. (E) G-CSF-induced ROS production was impaired in neutrophils from Lyn-/- mice. Neutrophils from bone marrow of wild-type and Lyn-/- mice were loaded with 5 μM DHR with (green) or without (purple) 100 ng/mL G-CSF for 60 minutes. One representative is shown in the left panel; quantitative results (mean ± SD) were shown in the right panel.
Figure 5.
Figure 5.
G-CSF induced activation of p47phox. (A) Serine phosphorylation of p47phox induced by G-CSF. Ba/F3GR cells were treated with or without 100 ng/mL G-CSF for indicated time periods, and lysates were prepared. Proteins were immunoprecipitated with anti-p47phox antibody and blotted with either anti-phosphoserine antibody or anti-p47phox antibody. The latter demonstrates comparable protein loading. (B) Inhibition of serine phosphorylation of p47phox by kinase inhibitors. Ba/F3GR cells were untreated or pretreated with kinase inhibitors for 1 hour, followed by no or 100 ng/mL G-CSF for 60 minutes, then the lysates were prepared. Proteins were immunoprecipitated with anti-p47phox antibody and blotted with either anti-phosphoserine antibody or anti-p47phox antibody. (C) Membrane translocation of p47phox. Ba/F3GR cells were untreated or treated with 100 ng/mL G-CSF for 10 minutes, and then cells were lysed and subjected to ultracentrifugation. Membrane and cytosolic fractions were purified and then subjected to electrophoresis and immunoblotting with anti-p47phox antibody. Lane 1, whole-cell lysate from Ba/F3GR cells; lanes 2-3, whole-cell lysates after sonication; lane 4, cytosolic fraction of Ba/F3GR cells unstimulated by G-CSF; lane 5, cytosolic fraction of Ba/F3GR cells stimulated by G-CSF; lane 6, membrane fraction of Ba/F3GR cells unstimulated by G-CSF; lane 7, membrane fraction of Ba/F3GR cells stimulated by G-CSF. (D) Abrogation of membrane translocation of p47phox following treatment with Src or Akt kinase inhibitors. Ba/F3GR cells were pretreated with DMSO (-) or Src kinase inhibitor PP1 (P), the PI3K inhibitor Ly294 002 (L), the Akt inhibitor A838450 (A), or ERK1/2 inhibitor MEKI (M) for 1 hour, followed by stimulation with 100 ng/mL G-CSF for 10 minutes. Cytosolic and membrane fractions were prepared and analyzed for trafficking of p47phox. Comparable results were obtained in 3 independent experiments. (E) DPI, a specific inhibitor of NADPH oxidase, blocked G-CSF-induced ROS production. Ba/F3GR cells were pretreated with 0.1 μMor1 μM DPI for 1 hour, loaded with 5 μM DHR for 10 minutes, and then stimulated with 100 ng/mL G-CSF for 30 minutes. ROS production was determined by flow cytometry. Purple indicates untreated cells without G-CSF; green, untreated cells + G-CSF; pink, 0.1 μM DPI + G-CSF; blue, 1 μM of DPI + G-CSF.
Figure 6.
Figure 6.
Enhanced ROS production in cells expressing a truncated G-CSF receptor. (A) Increased ROS production in cells expressing the truncated G-CSF receptor. Stable transfectants of Ba/F3 cells, expressing comparable levels of either wild-type or truncated G-CSF receptor, were labeled with dihydrorhodamine123 and unstimulated or stimulated with 100 ng/mL G-CSF for indicated periods of time. The relative ROS level induced by G-CSF was determined by monitoring the increased fluorescence (means ± SD) of rhodamine123 in the cells. (B) Truncated G-CSF receptor knock-in mice showed enhanced ROS production. Neutrophils from bone marrow of wild-type and GRΔ715 mice were loaded with DHR followed by stimulation with 100 ng/mL G-CSF for 60 minutes (green) or not (purple). One representative is shown in the left panel; quantitative results (means ± SD of 3 independent experiments) are shown in the right panel. (C) G-CSF-induced sustained activation of Lyn in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Src Y416 antibody, which detects the activated state of Lyn. Blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes. (D) G-CSF-induced activation of Akt in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-Akt S473 antibody, then the blot was stripped and reprobed with anti-Akt antibody to demonstrate comparable levels of protein loaded in respective lanes. (E) G-CSF-induced activation of ERK1/2 in Ba/F3GRprox. Immunoblotting was performed using anti-phospho-ERK1/2 T202T204 antibody, then the blot was stripped and reprobed with antiactin antibody to demonstrate comparable levels of protein loaded in respective lanes.
Figure 7.
Figure 7.
N-acetyl-L-cysteine on the ROS production and cell proliferation. (A) Effect of NAC on the activation of Akt and ERK1/2. Ba/F3GR and Ba/F3GRprox cells were pretreated without or with 1 mM or 10 mM NAC, respectively, for 1 hour, followed by stimulation with or without100 ng/mL G-CSF for 10 minutes. Cell lysates were prepared and blotted with anti-phosphor-Akt S473 or anti-phospho-ERK1/2 antibody and reprobed with Akt or ERK1 antibody. (B) G-CSF-induced ROS production was diminished by the administration of NAC. Ba/F3GR and Ba/F3GRprox cells were pretreated with or without1 mM or 10 mM NAC, respectively, for 1 hour, then loaded with 5 μM DHR123 and stimulated with or without 100 ng/mL G-CSF for 30 minutes. The relative ROS level induced by G-CSF was determined by monitoring the increased fluorescence in the cells (mean ± SD). Blue bars indicate BaF3GR; red bars, BaF3GRprox. (C) NAC inhibited the proliferation of Ba/F3GR and GRprox. Ba/F3GR and Ba/F3GRprox cells growing in IL-3 were washed extensively with PBS, 105/mL cells for each sample was plated in G-CSF containing medium, the cells were untreated or treated with 1 mM or 10 mM NAC, and the cells were counted at 0, 24, 48, and 72 hours by trypan blue exclusive assay. The results represent 3 independent experiments. (D) Effect of NAC on cell-cycle progression. Ba/F3GR and Ba/F3GRprox cells were treated with 10 mM NAC for 48 hours in the presence of 100 ng/mL G-CSF. The cells were subjected to cell-cycle analysis by flow cytometry (Modfit LT). (E) Effect of H2O2 on cell growth. Trypan blue counting of Ba/F3GR cells grown in IL-3 containing medium wit or without varying doses of H2O2 was performed daily.
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