IL-4-induced transcription factor NFIL3/E4BP4 controls IgE class switching.

IL-4 signaling promotes IgE class switching through STAT6 activation and the induction of Ig germ-line epsilon (GLepsilon) transcription. Previously, we and others identified a transcription factor, Nfil3, as a gene induced by IL-4 stimulation in B cells. However, the precise roles of nuclear factor, IL-3-regulated (NFIL3) in IL-4 signaling are unknown. Here, we report that NFIL3 is important for IgE class switching. NFIL3-deficient mice show impaired IgE class switching, and this defect is B-cell intrinsic. The induction of GLepsilon transcripts after LPS and IL-4 stimulation is significantly reduced in NFIL3-deficient B cells. Expression of NFIL3 in NFIL3-deficient B cells restores the impairment of IgE production, and overexpression of NFIL3 in the presence of cycloheximide induces GLepsilon transcripts. Moreover, NFIL3 binds to Iepsilon promoter in vivo. Together, these results identify NFIL3 as a key regulator of IL-4-induced GLepsilon transcription in response to IL-4 and subsequent IgE class switching.

Phosphatidylinositol 3-kinase functions as a Ras effector in the signaling cascade that regulates dephosphorylation of the actin-remodeling protein cofilin after costimulation of untransformed human T lymphocytes.

The activity of cofilin, an actin-remodeling protein, is required for T lymphocyte activation with regard to formation of the immunological synapse, cytokine production, and proliferation. In unstimulated T PBL (PB-T), cofilin is present in its Ser3-phosphorylated inactive form. Costimulation of TCR/CD3 and CD28 induces dephosphorylation and, thus, activation of cofilin. In this study we characterized the signaling cascades leading to cofilin activation in untransformed human PB-T. We show that a Ras-PI3K cascade regulates dephosphorylation of cofilin in PB-T. The GTPase Ras is a central mediator of this pathway; transient expression of an activated form of H-Ras in PB-T triggered the dephosphorylation of cofilin. Inhibition of either MAPK/ERK kinase or PI3K blocked both Ras-induced and costimulation-induced cofilin dephosphorylation in PB-T, showing that the combined activities of both signaling proteins are required to activate cofilin. That Ras functions as a central regulator of cofilin dephosphorylation after costimulation through CD3 x CD28 was finally proven by transient expression of a dominant negative form of H-Ras in primary human PB-T. It clearly inhibited costimulation-induced cofilin dephosphorylation, and likewise, activation of PI3K was diminished. Our data, in addition, demonstrate that regarding the downstream effectors of Ras, a clear difference exists between untransformed human PB-T and the T lymphoma line Jurkat. Thus, in PB-T the Ras signaling cascade is able to activate PI3K, whereas in Jurkat cells this is not the case. In addition to the insights into the regulation of cofilin, this finding discloses a to date unrecognized possibility of PI3K activation in T lymphocytes.

The transcriptional co-activator p/CIP (NCoA-3) is up-regulated by STAT6 and serves as a positive regulator of transcriptional activation by STAT6.

Transcriptional activation by signal transducer and activator of transcription 6 (STAT6) has been shown to require the direct interaction not only with co-activators such as p300 and cAMP-responsive element-binding protein-binding protein (CBP) but also with nuclear co-activator 1, a member of the p160/steroid receptor co-activator family. Among the p160/steroid receptor co-activators, only p/CIP (nuclear co-activator 3) has been shown to be up-regulated by interleukin (IL)-4 in B cells through a STAT-6-dependent mechanism using Gene-Chip analysis. In this study, we have investigated the function of p/CIP in the transcriptional activation by STAT6. We found that p/CIP indirectly interacted with STAT6 via p300, and overexpression of the CBP-interacting domain of p/CIP (p/CIP(947-1084)) prevented the interaction of p/CIP with STAT6 by blocking the binding of p/CIP to p300. Whereas expression of p/CIP(947-1084) resulted in a marked reduction of STAT6-mediated transactivation, overexpression of wild type p/CIP resulted in significant enhancement of it. In addition, p/CIP(947-1084) markedly reduced CD23 expression on B cells stimulated with IL-4, whereas overexpression of wild type p/CIP enhanced it. Chromatin immunoprecipitations demonstrate that IL-4 increases the interaction of p/CIP with the murine immunoglobulin heavy chain germ line epsilon promoter in B cells. These results suggest that p/CIP positively regulates STAT6 transcriptional activation through formation of a STAT6, p300/CBP, and p/CIP complex.

Cutting edge: STAT6 serves as a positive and negative regulator of gene expression in IL-4-stimulated B lymphocytes.

STAT6 plays an important role in IL-4-mediated B cell activation and differentiation. To identify primary and secondary target genes of STAT6, gene expression profiles of IL-4-stimulated B cells from STAT6+/+ vs STAT6-/- mice were compared. Statistical analysis revealed that 106 distinct probe sets including 70 known genes were differentially expressed between the 2 genotypes. These genes include transcription factors, kinases, and other enzymes, cell surface receptors, and Ig H chains. Surprisingly, although 31 genes were expressed at higher levels in STAT6+/+ B cells, 39 genes were expressed at higher abundance in STAT6-/- B cells. This result implies both positive and negative regulatory functions of STAT6 in IL-4-mediated gene expression. Furthermore, IL-4 induces expression of the transcription factor Krox20, which is required for maximal IL-4-induced transcription.