FIGURE 4.

The role of CREB for IL-22 expression as detected in T/A-stimulated Jurkat T cells. A, Jurkat T cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm). After 4 h, nuclear pCREB/pATF1 and HDAC1 content was evaluated by Western blot analysis. One representative of five independently performed experiments is shown. B, Jurkat T cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm). After 4 h, cells were harvested followed by ChIP analysis as described under “Experimental Procedures.” One representative of three independently performed experiments is shown. C, Jurkat T cells were either kept as unstimulated control or stimulated with the indicated concentrations of forskolin. After 4 h, IL-22 mRNA was assessed by real time PCR. IL-22 mRNA was normalized to that of GAPDH and is shown as mean fold-induction compared with unstimulated control ± S.D. (n = 5); **, p < 0.01 compared with unstimulated control; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. D, Jurkat T cells were transfected for 5 h with either pGL3-Prom3 or pGL3-Prom3 displaying a mutated CRE (−194/−190 nt) (PM) or a mutated NF-ATS4 (−95/−91 nt) (PM) or with pGL3-Prom3 displaying a CRE/NF-ATS4 double mutation (DM) together with Renilla luciferase as described under “Experimental Procedures.” After 15 h of rest, cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm) for another 8 h. Thereafter, cells were harvested and luciferase assays were performed. Means of luciferase activity are shown as fold-induction compared with unstimulated control (transfected with the same plasmid) ± S.D. (n = 3); **, p < 0.01 compared with unstimulated control (transfected with the same plasmid); ##, p < 0.01 compared with unmutated pGL3-Prom3 stimulated with T/A; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for T/A, forskolin).