Chemoattractant Regulation of Leukocyte Gene Expression

描述(由申请人提供)：基因表达的调节是白细胞趋化剂的一个新兴的和潜在的重要功能。越来越多的证据表明，趋化物质，包括经典的趋化因子和趋化因子，可以有效地诱导促炎细胞因子(如IL-1β)和趋化因子(如IL-8)的产生。最近的研究表明，某些趋化物质也调节免疫调节细胞因子的表达，如IL-12，从而影响获得性免疫的出现。在某些情况下，化学诱导剂已被发现抑制内毒素诱导的巨噬细胞产生IL-12。我们在最初的资助阶段进行的研究已经确定了核因子-kappaB在化学诱导剂诱导的白细胞基因表达中的重要作用。这些药物通过与异源三聚体G蛋白偶联来刺激核因子-kappaB的激活。这种独特的机制使趋化因子在诱导转录因子激活的能力上有别于细胞因子，如肿瘤坏死因子α和白介素1β。然而，尽管我们对细胞因子诱导的核因子-kappaB的激活有了更多的了解，但对趋化因子如何通过G蛋白调控转录却知之甚少。在这项申请中提出的研究将集中在趋化受体使用Galphai蛋白调节白细胞基因表达的机制上。所有七螺旋趋化受体都激活Galphai，Galphai释放Gbeta-γ激活白细胞的主要功能。在目标1中，我们将确定Gbeta-Gamma如何介导核因子-kappaB的激活。将使用正常和基因改变的小鼠的细胞来研究Gbeta伽马效应器PI-3激酶-伽马的参与。AIM 2致力于Galpha16与趋化因子受体的直接偶联和对PLCβ的激活，这是一种独特的协同激活机制，可能解释了为什么趋化因子只在造血细胞中是有效的转录调节因子。在目标3中，我们将研究Galphai在抑制IL-12基因表达中的潜在作用。利用体外重建试验，我们将比较介导抑制IL-12产生的趋化受体和那些缺乏这种能力的受体，以确定它们在G蛋白偶联特性上的差异。总而言之，这些研究旨在检验中心假设，即趋化受体通过与G蛋白的差异偶联来调节基因表达。

DESCRIPTION (provided by applicant): Regulation of gene expression is an emerging and potentially important function of leukocyte chemoattractants. Accumulating evidence indicates that chemoattractants, including classic chemotactic factors and chemokines, can effectively induce the production of proinflammatory cytokines (e.g. IL-1beta) and chemokines (e.g. IL-8). Recent studies suggest that certain chemoattractants also regulate the expression of immunomodulatory cytokines such as IL-12 and therefore influence the emergence of adaptive immunity. In some cases, chemoattractants have been found to suppress LPS-induced IL-12 production by macrophages. Our studies conducted during the initial funding period have established an important role of NF-kappaB in chemoattractant-induced leukocyte gene expression. These agents stimulate NF-kappaB activation through coupling to heterotrimeric G proteins. This unique mechanism distinguishes chemoattractants from cytokines such as TNFalpha and IL-1beta in their abilities to induce transcription factor activation. However, despite our expanding knowledge of cytokine-induced NF-kappaB activation, little is known about how chemoattractants regulate transcription through G proteins. Studies proposed in this application will focus on the mechanisms by which chemoattractant receptors uses Galphai proteins to regulate leukocyte gene expression. All heptahelical chemoattractant receptors activate Galphai, which releases Gbeta gamma for activation of major functions of leukocytes. In Aim 1, we will determine how Gbeta gamma mediates NF-kappaB activation. The involvement of a Gbeta gamma effector, PI-3 kinase-gamma, will be investigated using cells from normal and genetically altered mice. Aim 2 is devoted to studies of Galpha16 for its direct coupling with chemoattractant receptors and its permissive activation of PLCbeta, a unique mechanism of cooperative activation that may explain why chemoattractants are effective transcriptional regulators only in hematopoietic cells. In Aim 3, we will examine the potential role of Galphai in the inhibition of IL- 12 gene expression. Using in vitro reconstitution assays, we will compare chemoattractant receptors that mediate suppression of IL-12 production with those that lack this ability in order to identify the differences in their G protein-coupling properties. Collectively, these studies are designed to test the central hypothesis that chemoattractant receptors regulate gene expression through differential coupling to G proteins.