Cooperative Mechanisms in IL-4-Induced Gene Expression

描述(由申请人提供)：细胞因子IL-4是参与体液免疫反应发展和调节的唯一最重要的细胞因子。IL-4促进初始的CD4+T细胞分化为辅助性T细胞2型(Th2)，引导B细胞向IgE和IgG4(小鼠的IgG1)的同型转换，并调节细胞的生长和死亡。因此，IL-4在调节炎症反应、自身免疫性疾病、对感染的免疫反应以及包括哮喘在内的过敏反应中起着关键作用。IL-4在一定程度上通过激活不同细胞类型中的特定基因集来调节这种不同的免疫功能。IL-4通过诱导Janus激酶(JAK)介导的转录因子Stat6的激活来诱导基因表达。重要的是，单靠Stat6与DNA的结合不足以激活转录，需要与其他因子协同作用。最近的证据表明，其他信号通路，包括IRS-2/PI3-Kinase和PKC通路，可能对Stat6依赖的基因表达起重要作用，但其他通路如何与Stat6合作尚不清楚。这个项目的长期目标是了解IL-4信号如何调节基因表达，从而调节不同的生物学效应。本研究的目的是阐明JAK/STAT6通路与其他信号通路和细胞因子协同作用以激活基因表达以响应IL-4的机制。这项研究的中心假设是，IL-4反应基因的表达不仅受到JAK介导的Stat6酪氨酸磷酸化的调控，还受到丝氨酸激酶信号通路的调控，这些信号通路聚集在Stat6和其他所需的转录辅助因子上，促进它们之间的协同作用。这一假说得到了我们最近发现的IL-4诱导Stat6丝氨酸磷酸化的支持。为了验证中心假说，这项提议将追求三个特定的目标：1)确定Star6丝氨酸磷酸化的位置，丝氨酸磷酸化的动力学和稳定性，以及IL-4R介导的信号事件在Stat6丝氨酸磷酸化中的作用；2)确定IL-4诱导的Stat6丝氨酸磷酸化在调节Stat6依赖的基因表达中的作用；以及3)建立Stat6与其他必要的辅助因子在调节IL-4依赖的基因表达中协同作用的机制。STAT6的丝氨酸磷酸化位点将通过肽图谱和质谱学来确定。它们在调节Stat6表达和功能中的作用将通过逆转录病毒介导的转导在Stat6缺陷的B细胞中进行分析。建议的研究将集中在小鼠B细胞中未重排的IgG1免疫球蛋白基因的调控，作为IL-4诱导基因表达的模型。我们目前对IL-4信号和STAT6如何调节淋巴细胞和其他类型细胞中基因表达的了解有限，这些拟议的研究将极大地促进我们的理解。更好地了解IL-4介导的信号如何协同调节基因表达，也将有助于开发新的方法来调节免疫反应和受IL-4调控或影响的疾病过程。

DESCRIPTION (provided by applicant): The cytokine IL-4 is the single most important cytokine involved in the development and regulation of humoral immune responses. IL-4 drives the differentiation of naive CD4+ T cells into T helper type 2 (Th2) cells, directs isotype switching in B cells to IgE and IgG4 (IgG1 in mice), and regulates cell growth and death. As such, IL-4 has a pivotal role in regulating inflammatory responses, autoimmune diseases, immune responses to infection, and allergy, including asthma. IL-4 regulates this diverse array of immune functions in part through the activation of specific sets of genes in different cell types. IL-4 induces gene expression by inducing Janus kinase(Jak)-mediated activation of the transcription factor Stat6. Importantly, binding of Stat6 alone to DNA is not sufficient to activate transcription and cooperation of Stat6 with other factors is necessary. Recent evidence suggests that other signaling pathways, including the IRS-2/PI 3-kinase and PKC pathways, may be important for Stat6-dependent gene expression, but how other pathways cooperate with Stat6 is entirely unknown. The long-range goal of this project is to understand how IL-4 signaling regulates gene expression to mediate diverse biological effects. The objective of this application is to elucidate the mechanisms by which the Jak/Stat6 pathway cooperates with other signaling pathways and cellular factors to activate gene expression in response to IL-4. The central hypothesis for the proposed research is that IL-4-responsive gene expression is regulated not only by Jak-mediated tyrosine phosphorylation of Stat6 but also by serine kinase signaling pathways that converge on Stat6 and other required transcription cofactors to promote their cooperative interaction. This hypothesis is supported by our recent demonstration that IL-4 induces serine phosphorylation of Stat6. To test the central hypothesis, this proposal will pursue three specific aims: 1) determine the sites of Star6 serine phosphorylation, the kinetics and stability of serine phosphorylation and the role of IL-4R-mediated signaling events in Stat6 serine phosphorylation; 2) determine the role of IL-4-induced Stat6 serine phosphorylation in the regulation of Stat6-dependent gene expression; and 3) establish the mechanisms by which Stat6 cooperates with other necessary cofactors in the regulation of IL-4-dependent gene expression. Serine phosphorylation sites in Stat6 will be determined by peptide mapping and mass spectrometry. Their role in regulating Stat6 expression and function will be analyzed in Stat6-deficient B cells by retrovirus-mediated transduction. The proposed studies will focus on the regulation of the unrearranged IgG1 immunoglobulin gene in mouse B cells as a model for IL-4-induced gene expression. The proposed studies will significantly advance our current limited understanding of how IL-4 signaling and Stat6 regulate gene expression in lymphocytes as well as in other cell types. A better understanding of how IL-4-mediated signals cooperate to regulate gene expression will also allow development of novel approaches for modulating immune responses and disease processes regulated or influenced by IL-4.