CXCR7 Silencing Attenuates Cell Adaptive Response to Stromal Cell Derived Factor 1α after Hypoxia

<div><p>Previous studies have shown that chemotactic factor stromal-cell derived factor 1α (SDF1α) promotes cell recovery from hypoxic injury via its main receptor C-X-C chemokine receptor type (CXCR) 4. However, the role of its new receptor CXCR7 on cell repair against hypoxia and cell response to SDF1α remains largely unknown. In this study, neurons induced from hippocampal progenitor cells were pre-conditioned in hypoxia for 4h and subsequently monitored to investigate the function of SDF1α on cell repair after hypoxia. Neurons were assessed for their cell morphology, actin filament polymerization and migration capability. SDF1α protein levels increased significantly 1 h after hypoxia compared to control (<em>P</em><0.01), and it reached a peak at 24 h after hypoxia. Moreover, addition of SDF1α promoted neurite outgrowth and actin filament polymerization both in normoxic and hypoxic cells compared to untreated cells. Cell migration showed a time-dependent increase with SDF1α stimulation in both groups, and hypoxic cells illustrated a significant augment at 0.5 h, 1 h and 12 h after SDF1α application compared to normoxic cells (<em>P</em><0.01). CXCR7 expression also increased with time dependence after hypoxia and demonstrated a two-fold upregulation compared to control at 24 h after hypoxia. With CXCR7 silencing, axon elongation and actin filament polymerization induced by SDF1α were inhibited sharply both in normoxic and hypoxic cells. CXCR7 silencing also leads to reduced hypoxic cell migration at 0.5 h, 1 h, 12 h, 24 h and 36 h after SDF1α application (<em>P</em><0.01), but it failed to reduce normoxic cell migration induced by SDF1α at 0.5 h, 1 h and 12 h (<em>P</em>>0.05). 24 h SDF1α stimulation led to higher ERK1/2 phosphorylation compared to control, and ERK1/2 phosphorylation increased more in hypoxic cells than that in normoxic cells. This study suggested that CXCR7 plays an important role on cell repair processing induced by SDF1α, and CXCR7 silencing attenuates cell adaptive response to acute SDF1α stimulation (≤12 h) after hypoxia.</p> </div

Effects of CXCR7 ShRNA on cell morphology, actin filament polymerization and migration capability induced by SDF1α in normoxic cells.

<p>A, CXCR7 expression was increased with 24 h stimulation of SDF1α. Adhered hippocampal cells were cultured in normoxia for seven days. Then cells were treated with SDF1α (50 ng/ml) for 24 h or left untreated (control). Cell lysates were obtained to detect CXCR7 expression by western blot. B, CXCR7 protein expression was determined after CXCR7 silencing. Three ShRNA (CXCR7 ShRNA1, CXCR7 ShRNA2, CXCR7 ShRNA3) lentiviral expression vectors with green fluorescent protein (GFP) were transfected into hippocampal cells targeting CXCR7 mRNA on the 7^th day of culture. Three days later, cell lysates of transfected and untreated cells (control) were obtained to determine CXCR7 protein expression by western blot. The lentivirus-GFP that expressed GFP only was used as a blank control (data was not shown). Here, we used CXCR7 ShRNA1 as an effective inhibitor to do subsequent experiments. C, D, Cell morphology was observed and the ratio of the longest axon length to the soma perimeter was calculated by image analysis system. Cells were cultured under normoxia with or without CXCR7 ShRNA transfection followed by SDF1α (50 ng/ml) for 24 or 36 hours, or left untreated. Neuronal cells were counted under a light microscope. Six predetermined areas in each independent well (From a total of six wells) at each time point were selected and photographed. Then the longest axon length and the soma perimeter were counted and calculated manually. Bar = 10 µm. *<i>P</i><0.01, vs untreated. E, Actin filament polymerization was observed by staining with phalloidin and imaging with confocal microscopy. Neural cells were transfected with CXCR7 ShRNA with following SDF1α (50 ng/ml) application for 24 h or not. Arrows indicate the distribution changes of actin filament polymerization. Bar = 10 µm. F, Number of migrated cells was counted by transwell chamber analysis. Some of cells were transfected with CXCR7 ShRNA, and some were left untreated. Then cells were placed in the upper chamber and SDF1α (50 ng/ml) was added to the lower chamber for 0.5, 1, 12, 24, and 36 hours respectively for migration analysis. *<i>P</i><0.01, vs untreated.</p

Expression of SDF1α in hippocampal cells after hypoxia.

<p>Adhered hippocampal cells were exposed to hypoxia (3%O₂, 5% CO₂ and 92% N₂) for 4 h or left untreated on the seventh day of culturing. Supernatants and cell lysates were obtained and measured at 0.5, 1, 12, 24, and 36 h after hypoxia for ELISA and western blot respectively. Untreated cells were used as control. A, Concentrations of supernatant SDF1α in the medium were determined by ELISA. *<i>P</i><0.01, vs control. B, Expression of SDF1α in the cells was measured by western blot analysis. GAPDH was used as reference.</p

Expression of CXCR7 in hippocampal cells after hypoxia.

<p>Adhered hippocampal cells were exposed to hypoxia or normoxia (control), and then CXCR7 expression was determined by immunofluorescence and western blot at 0.5, 1, 12, 24, and 36 h after hypoxia. Six independent experiments were applied for both cell count and western blot. A, Hippocampal cells were digested from the culture dish and measured for CXCR7 expression by immunofluorescence. *<i>P</i><0.01, vs control. B–C, Cell lysates were obtained to perform western blot assay for CXCR7 expression. Data showed strong up-regulation of CXCR7 after hypoxia. *<i>P</i><0.01, vs control.</p

Effects of CXCR7 ShRNA on cell morphology and migration capability induced by SDF1α in hypoxic cells.

<p>A, Cell morphology was observed after hypoxia with pre-treatment of CXCR7 ShRNA or not. Some of cells were transfected with CXCR7 ShRNA, or left untreated. Then all of cells were treated with hypoxia followed by stimulation of SDF1α (50 ng/ml) for 24 h. Neuronal cell morphology was observed and photographed under a fluorescence microscope. Arrows indicate the changes of dendrite length and neurite outgrowth formation. Bar = 30 µm. B, Number of migrated cells was counted by transwell chamber analysis. Cells with or without CXCR7 silencing were treated with hypoxia, and then were placed in the upper chamber with SDF1α (50 ng/ml) in the lower chamber for 0.5, 1, 12, 24, and 36 hours respectively. *<i>P</i><0.01, vs hypoxia.</p

Effect of ATPsyn-β recombinant plasmid on adriamycin sensitivity in HL-60/ADM cells.

<p>A. 48-β and MRP1 were determined by western blot. Results are representative of three repeats. β-actin is used as loading control. B. Inhibition percentages of cell proliferation in various concentrations of adriamycin-treated HL-60/ADM cells after ATPsyn-β-pcDNA3.1(+) transfection (1.5625, 3.125, 6.25, 12.5, 25, 50, 100 µM, for 48 h) determined by MTT assay. The values or Columns represent mean±SD of triplicate experiments. C. Morphologic change in HL-60/ADM cells transfected with ATPsyn-β-pcDNA3.1 (+). 48 h after transfection, HL-60/ADM cells were treated with 10 µM adriamycin, and cultured for 48 hours. Then cells were stained with Wright-Giemsa and photographed (×1000). D. Morphological features for apoptosis in control pcDNA3.1 (+) or ATPsyn-β pcDNA3.1 (+)-treated HL-60/ADM cells were revealed by Hoechst 33258 staining (×200, up panel; ×1000, below panel). After transfection, cells were treated with 10 µM adriamycin for 48 hours. For each Hoechst experiment at least 200 cells in 5 random scope fields were counted for apoptotic rate. The mean percentage of apoptotic cells treated with ATPsyn-β-pcDNA3.1 (+) was 33.3%, which was higher than the percentage of apoptotic cells transfected with empty control pcDNA3.1(+) (23.7%). Experiments in this figure were repeated twice and similar results were obtained. E. Percentages of apoptotic cells in adriamycin-treated HL-60/ADM cells after plasmid transfection based on annexin V-APC expression assays. HL-60/ADM cells with indicated treatment (PBS buffer, pcDNA3.1(+) empty vector, ATPsyn-β pcDNA3.1(+)) were incubated with different dose of adriamycin(1.5625, 3.125, 6.25, 12.5, 25 µM) for 36 h. (i) Representative flow cytometry results(adriamycin = 25 µM). (ii) Data are presented as mean ± SD for three independent experiments. *P<0.05 vs pcDNA3.1 (+) empty vector treated cells.</p

Correlation of mitochondrial ATPsyn-β expression and IC₅₀ of AML primary samples to adriamycin in vitro.

<p>ATPsyn-β mRNA (A) and protein expression (B) showed an inverse correlation with IC₅₀ of primary cells to adriamycin from remission patients. ATPsyn-β mRNA (C) and protein expression (D) showed an inverse correlation with adriamycin IC₅₀ of primary cells from relapsed/refractory AML patients.</p

Clinical characteristic of AML patients.

<p>Clinical characteristic of AML patients.</p

Analysis of mitochondrial ATPsyn-β in HL-60 and HL-60/ADM cell lines.

<p>A. Loss of mitochondrial ATPsyn-β mRNA and gain of MRP1 mRNA in HL-60/ADM cells. Columns, means of three experiments; bars, SD. B. Western blot detection of mitochondrial ATPsyn-β and MRP1 protein. β-actin is used as loading control. Data represent one of three repeats. C. Flow cytometric histogram of mitochondrial ATPsyn-β expression from HL-60/ADM cells (red line, 6.1%) and HL-60 cells (purple color, 36.3%).</p

Down-regulation of mitochondrial ATPsyn-β in CD34+ cells from relapsed/refractory AML patients.

<p>A. Loss of ATPsyn-β mRNA in relapsed/refractory AML samples. The dotted line indicates means of primary samples; bars, SD;n = 20. B. Mitochondrial ATPsyn-β protein expression (n = 5). No. 16, 17, 19, relapsed/refractory samples; No. 5, 8, presentation samples from remission samples.</p