Thus, the effect of STAT2

Thus, the effect of STAT2 over-expression was first examined on the suppression of the IL-4 signaling in terms of STAT6 localization in Ramos B cells. In the STAT2 over-expressing cell system, IFN-α not only increased cytoplasmic accumulation of the endogenous and transfected pY-STAT2, but also upregulated cytoplasmic levels of the IL-4-activated pY-STAT6 compared with the mock-transfected system (Fig. 7A: The CE/NE ratio of pY-STAT6/STAT6 increased

from 4.2 to 10.9). Next, we analyzed the effect of STAT6 over-expression on the inhibitory action of IL-4 on IFN-α signaling. We found that the cytoplasmic retention of pY-STAT2 induced by IL-4 treatment was promoted corresponding to the increment of pY-STAT6 cytoplasmic levels, resulting in a further reduction in nuclear pY-STAT2 levels (Fig. 7B: The CE/NE ratio of pY-STAT2/STAT2 increased from 3.2 to 13.7). The effects of STAT over-expression were then investigated on the target gene expression in Ramos B cells. Upon STAT2 over-expression, IL-4-induced CD23 mRNA levels were severely reduced, and the suppression by IFN-α proceeded faster check details than in mock cells, reducing the lag

time for inhibition from 4 to 2 h (Fig. 8A: The graph scale in the box was enlarged in the right panel). A similar phenomenon was observed in STAT6 over-expressing cells; IRF7 mRNA levels induced why by IFN-α were substantially downregulated, and the suppressive effect of IL-4 on the IFN-α-induced IRF7 gene expression obtained by 8 h was more prominent as compared with the mock-transfected

cells (Fig. 8B). The data demonstrate that increase in cytoplasmic STAT2 or STAT6 levels caused a concomitant retention of STAT6 or STAT2, respectively, which in turn promoted the inhibitory effects of IFN-α and IL-4 on CD23 and IRF7 gene expression, respectively. The increased co-retention of STAT6 and STAT2 observed in cells over-expressing either STAT2 or STAT6 is likely to occur through the molecular interaction and complex formation between activated STATs induced by cytokine treatment. We have utilized the CD23 gene expression system in Ramos B cells to investigate the regulation mechanism of IL-4 signaling pathways by IFN-α. While IFN-α was shown to suppress the IL-4-induced IL-4R expression in primary immune cells 21, it had no effect on IL-4R levels throughout 12 h-period sufficient for the regulation of CD23 expression in Ramos cells (data not shown). Yet, IFN-α perturbed IL-4 signaling leading to CD23 gene activation in these cells as shown by a significant decrease in IL-4-induced nuclear pY-STAT6 levels and the subsequent STAT6 binding to the CD23 promoter, leading to the effective downregulation of the IL-4-induced CD23 expression at both protein and mRNA levels (Figs. 1 and 2).

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