Effects of GATA-1 on IL-6 signaling.

(A) Induction of TIS11 and jun B genes by recombinant human (rh) interleukin 6 (IL-6) in M1-V1, M1-G1, and M1-G2. Total cellular RNA was extracted from each clone before and after the stimulation with rhIL-6 (20 ng/mL) for 30 minutes and subjected to Northern blot analysis. The filters were hybridized with ³²P-labeled probes for TIS11, jun B, and CHO-B. (B) Changes in tyrosine phosphorylation of STAT3 in response to rhIL-6 in M1-V1, M1-G1, and M1-G2. The cells of each clone were serum starved for 12 hours and then stimulated with rhIL-6 (20 ng/mL) for 15 minutes. Total cell lysates were isolated before and after the stimulation with rhIL-6, immunoprecipitated with anti-STAT3 polyclonal antibody, and subjected to SDS-PAGE. The blots were probed with anti-phosphotyrosine monoclonal antibody. Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system. Then, the filters were stripped and reprobed with anti-STAT3 antibody. (C) Effects of GATA-1 on DNA-binding activities of STAT3. M1-V1 and M1-G1 were serum deprived for 12 hours then unstimulated or stimulated with rhIL-6 (20 ng/mL) for 15 minutes, and nuclear extracts were isolated. The nuclear extract was incubated in binding buffer containing 2 μg of poly(dI-dC) and labeled probe (30 000 cpm) for 20 minutes at 4°C. The reaction mixture was electrophoresed, dried, and subjected to autoradiography. In competition assays, nuclear extracts were preincubated with a 200-fold molar excess of unlabeled competitor oligonucleotide before the binding reaction. In supershift assays, the nuclear proteins were preincubated with 1 μg of rabbit anti-STAT3 polyclonal monoclonal at 4°C for 30 minutes, and then the binding reaction was performed. (D) Effects of GATA-1 on transcriptional activities of STAT3. NIH3T3 cells were transfected with 1 μg of 4 × APRE-Luc, 10 ng of pRL-CMV-Rluc, and various amounts of pcDNA3-GATA-1 or pCAGGS-neo-dn-STAT3 by calcium phosphate coprecipitation method. After 12 hours, the cells were serum starved for 24 hours and then stimulated with rhIL-6 (20 ng/mL) for 5 hours. The cells were lysed in lysis buffer, followed by the measurement of the firefly and the renilla luciferase activities. The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the renilla luciferase activities. The results are shown as the mean ± SD of triplicate experiments.