Article Figures & Data
Figures
- FIGURE 1.
Macrophage differentiation of M1 cells transduced with pMX-IRES-EGFP-STAT5A1*6 (M1/STAT5A1*6). A, Quantitation of cell differentiation and expression of GFP 4 days after retroviral gene transduction (2 days after cell sorting) on flow cytometry in M1/WT-STAT5A and M1/STAT5A1*6 cells. Upper panels, The x-axis indicates FSC, and the y-axis indicates SSC. Lower panels, The x-axis indicates fluorescence intensity as a log scale ranging from 100 to 104, and the y-axis indicates the number of cells. Parental M1 cells were used as a control. B, Morphological analysis of STAT5A1*6-induced differentiation of M1 cells 10 days after gene transduction. Parental M1, M1/WT-STAT5A, and M1/STAT5A1*6 cells were centrifuged onto glass slides and stained with May-Grünwald-Giemsa. Photographs were taken at ×400 magnification. C, Flow cytometric analysis in parental M1, M1/WT-STAT5A, and M1/STAT5A1*6 cells 10 days after gene transduction. Upper panels, Quantitation of cell differentiation on flow cytometry. Differentiated M1 cells were detected in region R2. Increased size and vacuolization of differentiated cells are reflected by increases in FSC (x-axis) and SSC (y-axis), respectively. Lower panels, Quantitation of expression of GFP on flow cytometry. The x-axis indicates fluorescence intensity as a log scale ranging from 100 to 104. The y-axis indicates the number of the cells. D, CD14 expression of M1/WT-STAT5A and M1/STAT5A1*6 cells. M1/WT-STAT5A (blank area) and M1/STAT5A1*6 (shaded area) cells were preincubated with an isotype control rat IgG1 and stained with PE-conjugated rat anti-mouse CD14 Ab. The x-axis indicates fluorescence intensity as a log scale ranging from 100 to 104. The y-axis indicates the number of the cells. E, Quantitation of M1 cell differentiation on FACS in continuously growing clones, MD1, MD2 and MD3, derived from M1/STAT5A1*6 cells. The x-axis indicates FSC, and the y-axis indicates SSC.
- FIGURE 2.
Characterization of the STAT5A1*6 expressed in MD cells. A, Tyrosine phosphorylation of STAT5A in parental M1, M1/WT-STAT5A, and MD cells. Cells were lysed, and STAT5A proteins were immunoprecipitated with the anti-STAT5A Ab. The immunoprecipitated proteins were separated by a SDS-polyacrylamide gradient gel and analyzed by Western blot with the anti-phosphotyrosine Ab (upper panel), followed by probing with the anti-STAT5A Ab (lower panel). B, DNA binding activities of STAT5A in nuclear extracts derived from parental M1, M1/WT-STAT5A, and MD1–3 cells. The DNA-protein complexes were confirmed to contain STAT5A by supershift analysis using anti-STAT5A Ab. An arrowhead and an arrow indicate positions of the STAT5A bands and supershifted complexes, respectively.
- FIGURE 3.
Analysis of STAT3 in MD1–3 cells. A, Tyrosine phospholylation of STAT3 in parental M1, M1/WT-STAT5A, and MD cells. Cells were treated as described for Fig. 2A. An anti-STAT3 Ab was used here instead of anti-STAT5A Ab. B, Failure to detect heterodimers of STAT3 and STAT5A1*6. Bulk population of MD cells was lysed and STAT3 and STAT5A proteins were immunoprecipitated with anti-STAT3 (lanes 1 and 3) and anti-STAT5A (lanes 2 and 4) Abs, respectively. The immunoprecipitated proteins were subjected to SDS-PAGE analysis using a gradient gel and analyzed by Western blot with anti-STAT3 (lanes 1 and 2) and anti-STAT5A Abs (lanes 3 and 4).
- FIGURE 4.
The supernatant of MD cells contains a sufficient amount of IL-6 to induce macrophage differentiation of parental M1 cells via activation of STAT3. A, Parental M1 cells were cultured in the medium containing half-volume of the supernatant of the MD1 cells with (right panel) or without (left panel) an anti-IL-6 neutralizing Ab (10 μg/ml). Flow cytometric analysis was performed to quantitate the morphological changes in these cells 3 days after the treatment. Differentiated M1 cells were detected in region R2. Increased size and vacuolization of differentiated cells are reflected by increases in FSC (x-axis) and SSC (y-axis), respectively. MD2 and MD3 cells gave similar results (data not shown). B, Tyrosine phosphorylation of STAT3 in parental M1 (lane 1) and M1 cells incubated in the supernatant of MD cells for 30 min with (lane 3) or without (lane 2) the anti-IL-6 neutralizing Ab (10 μg/ml). Cells were treated as described for Fig. 3A. C, Expression of IL-6 mRNA in parental M1, MD, and Ba/F3 cells cultured in medium containing IL-3 (1 ng/ml) as a positive control. A total of 2 μg poly(A)+ RNA was subjected to Northern blot analysis. A full-length cDNA for mIL-6 was used as a probe.
- FIGURE 5.
A1 expression prevented M1 cells from IL-6-induced terminal differentiation. A, A1 gene expression was detected by Northern blot analysis in MD cells (lane 2) but not in parental M1 cells (lane 1). B, Morphological analysis of M1 cells expressing A1 treated with IL-6 (10 ng/ml).
- FIGURE 6.
Inhibition of macrophage differentiation in M1/STAT5A1*6 cells with an anti-mIL-6 neutralizing Ab. M1 cells were retrovirally transduced with pMX-STAT5A1*6-IRES-EGFP, and EGFP-positive cells were sorted on a cell sorter 2 days after gene transduction. pMX-STAT5A-IRES-EGFP served as a control vector. M1/STAT5A1*6 cells were then separated and cultured under different conditions: half were cultured in the medium with an anti-IL-6 Ab (10 μg/ml), and the other half were cultured with a control Ab (10 μg/ml). Quantitation of M1 cell differentiation and expression of EGFP were analyzed on FACS.
- FIGURE 7.
Transactivation of the wild-type IL-6 promoter and its mutants by STAT5A. A, The structures of the wild-type and various mutants of the IL-6 promoter. The wild-type IL-6 promoter sequence, k0 (−1161/+59), contains AP-1 (−283/−277), NF-IL-6 (−145/−158), and NF-κB (−73/−64) binding sites. The deletion mutants were designed as k4 (−310/+59), k18 (−180/+59), and k9 (−122/+59). The mk9 and mk0 was produced by introducing a mutation in the NF-κB binding site of the k9 and k0 to reduce the NF-κB binding activity, as described in Materials and Methods. B, The sequence for luciferase was connected up with the wild-type IL-6 promoter (k0) or the various mutants of the IL-6 promoter (k4, k18, k9, mk9, and mk0) in reporter plasmids. Luciferase activities were examined in the lysates of Ba/F3 cells cotransfected with each of these reporter plasmids and either of the mock vector (pMX), the expression vector for the wild-type STAT5A (pMX/STAT5A-WT), or that for the STAT5A1*6 (pMX/STAT5A1*6), as described in Materials and Methods. The results shown are the averages ± SD of three independent experiments. C, Transactivation of the wild-type IL-6 promoter (k0) by the wild-type STAT5A and STAT5A1*6 in the presence of IL-3. Luciferase activities were examined in the lysates of Ba/F3 cells cotransfected with the k0 reporter plasmids and either of the mock vector (pMX), the expression vector for the wild-type STAT5A (pMX/STAT5A-WT), or that for the STAT5A1*6 (pMX/STAT5A1*6), as described in Materials and Methods. Cells were stimulated with 1 ng/ml of mIL-3 for the last 6 h before cell lysates were prepared. The results shown are the averages ± SD of three independent experiments.
- FIGURE 8.
Enhanced DNA binding activities of NF-κB p65 by STAT5A activation. DNA binding activities of NF-κB p65 were examined by EMSA using nuclear extracts derived from COS cells expressing the Epo receptor and either the wild-type STAT5A (WT), the STAT5A1*6 (1*6), or the control vector pMX (−), with or without Epo stimulation. The DNA-protein complexes were confirmed to contain NF-κB p65 in a supershift analysis with an anti-NF-κB p65 Ab. The arrowhead and arrow indicate positions of the DNA-NF-κB p65 complexes and supershifted complexes, respectively.
- FIGURE 9.
Enhanced DNA binding activities of NF-κB p65 and c-Rel and modulation of DNA-protein complexes of NF-κB p65 by STAT5A activation in MD cells. A, Nuclear extracts of M1/WT-STAT5A (lanes 1–3) and MD cells (lanes 4–6) were prepared and subjected to EMSA with 32P-labeled oligonucleotide probe containing an NF-κB binding sequence. Competition assays were performed by adding 5-fold molar excess of cold competitor (lanes 2 and 5) or nonspecific oligonucleotides (lanes 3 and 6) in the reaction mixture. The arrowhead indicates the specific DNA-protein complexes, and arrows indicate STAT5A1*6-induced supershifted complexes. B, DNA binding activities of NF-κB p65 were examined by EMSA using nuclear extracts derived from M1/WT-STAT5A (lanes 1–3) and MD cells (lanes 4–6). The filled arrowhead and arrow indicate positions of the DNA-NF-κB p65 complexes in M1/WT-STAT5A cells and MD cells, respectively. The open arrowheads indicate positions of the supershifted bands. C, DNA binding activities of c-Rel were examined by EMSA using nuclear extracts derived from MD cells. The open arrow and the open arrowhead indicate positions of the DNA-c-Rel complexes and supershifted bands, respectively. The filled arrowhead indicates positions of the DNA-NF-κB p65 complexes.
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