Analysis of the life cycle of stat6. Continuous cycling of STAT6 is required for IL-4 signaling.

Signal transducer and activator of transcription (Stat)6 is a transcription factor important for the development of Th2 cells and regulation of gene expression by IL-4 and IL-13. It is known that Stat6 is rapidly activated in response to IL-4; however, the fate of activated Stat6 is less clear. We examined the fate of activated Stat6 and found that during continuous exposure to IL-4, Stat6 activity was sustained for 72 h and that the maintenance of a constant level of activated Stat6 did not require new protein synthesis. In contrast, when cells were pulsed with IL-4 and then incubated in the absence of IL-4, the half-life of Stat6 phosphorylation and DNA binding activity was less than 1 h. Stat6 did not accumulate in the nucleus, and protein degradation did not play a major role in the disappearance of activated Stat6. Inhibition of kinase activity by staurosporine or the JAK inhibitor, AG490, revealed that maintenance of Stat6 activation in the continuous presence of IL-4 required ongoing phosphorylation of latent cytoplasmic Stat6 molecules. Cells treated with an inhibitor of nuclear export, leptomycin B, were unable to maintain Stat6 activation. Thus, the maintenance of Stat6 activation requires a constant cycle of activation, deactivation, nuclear export, and reactivation.

DNA binding activity of cytoplasmic phosphorylated Stat6 is masked by an interaction with a detergent-sensitive factor.

Signal transducer and activator of transcription (Stat) 6 is vital to interleukin (IL)-4 and IL-13 responses and the generation of Th2 immunity. We investigated the cellular location of phosphorylated Stat6 and Stat6 DNA binding activity in A201.1 murine B cells and primary splenocytes. Phosphorylated Stat6 was present in cytoplasmic and nuclear extracts from IL-4-treated cells. Confocal microscopy confirmed the presence of phosphorylated Stat6 in the cytoplasm of IL-4-treated cells. In contrast, Stat6 DNA binding activity was present in nuclear extracts, but not in cytoplasmic extracts. Thus, cytoplasmic extracts from IL-4-stimulated cells were devoid of Stat6 DNA binding activity despite the presence of phosphorylated Stat6. Addition of cytoplasmic extracts to nuclear extracts did not inhibit Stat6 DNA binding present in the nuclear extracts. Detergent treatment restored Stat6 DNA binding activity in cytoplasmic extracts of IL-4-stimulated cells. Thus, DNA binding activity of cytoplasmic phosphorylated Stat6 is masked by a factor dissociable by detergent treatment.

V75R576 IL-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function.

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4R alpha in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4R alpha allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4R alpha with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4R alpha allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4R alpha haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4R alpha requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.