Fig. 1. SDF-1 activates Erk synergistically with IL-3 in hematopoietic cells. (A) After overnight starvation from cytokine in serum-free ASF104 medium, 32Dcl3 cells were left unstimulated or stimulated with IL-3 (1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for indicated times. Cell lysates were prepared, resolved by SDS-PAGE, and immunoblotted with anti-phospho-Erk antibody (α-P-Erk), as indicated. The membrane was stripped and reproved with anti-Erk (α-Erk) and anti-α-tubulin (α-α-tubulin) antibodies, as indicated. (B) Fold increases in phosphorylation of Erk were determined by densitometric analysis and shown.

Fig. 2. SDF-1 and IL-3 prominently activate Rac and Ras, respectively, in non-synergistic manners. After overnight starvation, 32Dcl3 cells were left unstimulated or stimulated with IL-3 (1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 5 min. Cell lysates were prepared and activated small GTPases were purified from the lysates using GST-Raf-RBD (A), GST-Pak2-RBD (C), or GST-RalGDS-RBD (E) fusion proteins, as described under Materials and methods. The eluates from the precipitates or total cell lysates (upper or lower panels, respectively) were resolved by SDS-PAGE and subjected to Western blot analysis with antibody against Ras (A), Rac (C), or Rap1 (E). Fold increases in activities of Ras (B), Rac (D) and Rap1 (F) were determined by densitometric analysis and shown.

Fig. 3. SDF-1 synergistically enhances IL-3-induced activation of Raf-1 and phosphorylation of MEK. After overnight starvation, 32Dcl3 cells were left unstimulated or stimulated with IL-3 (1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 5 min. (A) Cell lysates were prepared and immunoprecipitated (IP) with anti-Raf-1, as indicated. The immunoprecipitates were subjected to the in vitro kinase assays using GST-MEK1 fusion protein as substrate. The reaction products were subjected to Western blot analysis with anti-phospho-MEK antibody (α-P-MEK), followed by reprobing with anti-GST (α-GST) and anti-Raf-1 (α-Raf-1) antibodies, as indicated. (B) Fold increases in activities of Raf-1 were determined by densitometric analysis and shown. (C) The total cell lysates were subjected to Western blot analysis with anti-phospho-MEK antibody (α-P-MEK), followed by reprobing with anti-MEK antibody (α-MEK), as indicated. (D) Fold increases in MEK phosphorylation were determined by densitometric analysis and plotted.

Fig. 4. Involvement of Rac and Ras in synergistic activation of the Erk/Elk-1 signaling pathway by SDF-1 and IL-3. (A) 32Dcl3 cells were transfected with 20 μg of pcDNA-Ha-Ras17N (RasN), pSR-myc-Rac17N (RacN), or an empty vector (Vector), as indicated, along with 20 μg of S3H-Erk2. After overnight starvation, transfected cells were left unstimulated or stimulated with IL-3 (1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 5 min. The cells were lysed and transfected HA-Erk was immunoprecipitated by anti-HA antibody. Immunoprecipitates were resolved by SDS-PAGE and blotted with anti-phospho-Erk (α-P-Erk) antibody. The membrane was stripped and reprobed with anti-HA (α-HA) antibody to estimate the expression levels of transfected HA-Erk. (B) The relative intensities of phospho-Erk bands were determined by densitometry. (C) 32Dcl3 cells were transfected with 10 μg of pcDNA-Ha-Ras17N (Ras17N), pSR-myc-Rac17N (RacN), or an empty vector (Vector), as indicated, along with 1 μg of pFA-Elk-1, 10 μg of pFR-Luc, and 0.1 μg of pRL-SV40. After 1-day starvation, the cells were left unstimulated or stimulated with IL-3 (1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 5 h and then harvested for the dual luciferase assay. The luciferase activity was normalized by the Renilla luciferase activity and expressed as fold increase from the unstimulated vector control. The data represent averages±S.D. of three independent experiments.

Fig. 5. Possible involvement of the Pak kinases in synergistic activation of the Erk/Elk-1 signaling pathway by SDF-1 and IL-3. 32Dcl3 cells were transfected with indicated amounts of pCMV6M-Pak-1-K299R (Pak-KR) (A) or pCMV6M-Pak-1-T423E (Pak-TE) (B) along with 1 μg of pFA-Elk-1, 10 μg of pFR-Luc, and 0.1 μg of pRL-SV40. After overnight starvation, the cells were left unstimulated or stimulated with IL-3 (1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 5 h and then harvested for the dual luciferase assay. (C) After overnight starvation, 32Dcl3 cells were left unstimulated or stimulated with IL-3 (1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 5 min. The cell lysates were prepared and subjected to Western blot analysis with anti-phospho-Raf-1-S338 (α-P-RafS338), followed by reprobing with anti-Raf-1 (α-P-Raf-1) antibody.

Fig. 6. IL-3 and SDF-1 synergistically increase the number of viable cells and induce chemotactic response in Erk-dependent manners. (A) BAF3 cells were cultured with indicated concentrations of IL-3 and SDF-1 for 72 h. The numbers of viable cells were measured by the XTT proliferation assay as described under Materials and methods. The data represent averages±S.D. of triplicate determinations. (B) BAF3 cells were cultured with IL-3 (0.01 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 48 h and subjected to the apoptosis assay with annexin V staining as described under Materials and methods. The data represent averages±S.D. of three separate experiments. (C) BAF3 cells were cultured with IL-3 (0.1 ng/ml) and SDF-1 (50 ng/ml), as indicated, for 18 h. Cells were further cultured for 2 h in the presence of BrdU. Cells that had incorporated BrdU were analyzed by flow cytometry as described under Materials and methods. The data represent averages±S.D. of three separate experiments. (D) BAF3 cells were cultured with indicated concentrations of IL-3 and SDF-1 with or without 50 μM of PD98059 for 72 h. The numbers of viable cells were measured by the XTT proliferation assay. (E) BAF3 cells were added to the upper chamber while the lower chamber contained medium with IL-3 and SDF-1 at indicated concentrations. After 4-h incubation at 37 °C, the number of cells that had migrated to the lower chamber was determined. The data are expressed as the percentage of the total input cells that had migrated into the lower chamber. The data represent averages±S.D. of triplicate determinations. (F) BAF3 cells were preincubated with or without 50 μM PD98059 for 30 min, as indicated, before addition to the upper chamber for the chemotaxis assay as described above.

Fig. 7. A model for the signaling mechanisms for synergistic activation of the Erk pathway by SDF-1 and IL-3.