Figure 4.

C/EBPβ is required, but CREB and STAT-6 are dispensable, for the stimulatory effect of adenosine (ado) on arginase-1 expression in IL-4-stimulated macrophages. A) Adenosine or NECA augments IL-4-induced arginase-1 promoter activity in macrophages. To measure arginase-1 promoter activity, RAW 264.7 cells were transiently transfected with a promoter-luciferase reporter construct containing −3810/−31 fragment of the arginase-1 promoter in a pGL3-basic vector. Cells were treated with IL-4 and adenosine or NECA for 8 h. Luciferase reporter activities were normalized to protein content. Results are representative of ≥3 experiments; n = 5/experiment. **P < 0.01 vs. control; ^#P < 0.05, ^##P < 0.01 vs. IL-4. B) Adenosine synergizes with IL-4 to up-regulate C/EBP-driven promoter activity in RAW 264.7 macrophages. RAW 264.7 macrophages were transfected with a luciferase reporter vector driven by C/EBP (pC/EBP-luc). Cells were treated with IL-4 and adenosine (ado; 100 μM) for 4 h; cells were then lysed, and luciferase activity was determined. Luciferase reporter activities were normalized to protein concentration. Results are representative of ≥3 experiments; n = 4/experiment. **P < 0.01 vs. IL-4 or ado. C) Adenosine fails to increase arginase activity in IL-4-activated C/EBPβ-deficient macrophages. C/EBPβ-WT and -KO macrophages were activated with IL-4 or IL-4 plus adenosine, and arginase activity was determined from the cell lysate after 8 h of stimulation. Results are representative of ≥3 experiments; n = 6/experiment. ***P < 0.001 vs. control (con); ^###P < 0.001 vs. IL-4 in C/EBPβ WT. D) Stimulatory effect of adenosine on arginase activity is CREB independent in IL-4-activated macrophages. Top panel: RAW 264.7 cells were transduced with Lentivirus particles containing either CREB-specific shRNA or nontargeting (NT) shRNA. CREB-silenced or NT-transduced cells were selected in the presence of puromycin. CREB protein levels were evaluated by Western blotting using protein extract isolated from NT shRNA- and CREB shRNA-transduced RAW 264.7 macrophages. Note that silencing of CREB expression was inefficient in clone 32. Blots are representative of ≥4 experiments. Bottom panel: CREB-silenced (clone 33/4 is shown) or NT-transduced macrophages were stimulated with IL-4 and adenosine, and arginase activity was measured from the cell extracts prepared at the end of the incubation period. Results are representative of ≥3 experiments; n = 6/experiment. ***P < 0.001 vs. con; ^###P < 0.001 vs. IL-4. E) Adenosine diminishes IL-4-induced STAT-6 activation. Peritoneal macrophages were challenged with 5 ng/ml IL-4 in the presence or absence of adenosine for the indicated times, and STAT-6 phosphorylation, which is indicative of activation, was determined from cell extracts taken at the end of the incubation period, using Western blotting with antibodies raised against the active phosphorylated STAT-6. β-Actin was employed as internal control. Blots are representative of 3 separate experiments. Results are shown as means ± se.