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化学引诱物的一种新兴的和潜在的重要功能。越来越多的证据表明，趋化因子，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-1 β）和趋化因子（如IL-8）的产生。最近的研究表明，某些化学引诱物也调节免疫调节细胞因子如IL-12的表达，从而影响适应性免疫的出现。在某些情况下，已经发现化学引诱剂抑制LPS诱导的巨噬细胞产生IL-12。我们在初始资助期间进行的研究已经确定了NF-kappaB在化学引诱剂诱导的白细胞基因表达中的重要作用。这些试剂通过偶联异源三聚体G蛋白刺激NF-κ B活化。这种独特的机制将化学引诱物与细胞因子如TNF α和IL-1 β在诱导转录因子活化的能力上区分开来。然而，尽管我们扩大了对苦参碱诱导的NF-κ B活化的了解，但对化学引诱物如何通过G蛋白调节转录知之甚少。本申请中提出的研究将集中在化学引诱物受体使用Galphai蛋白调节白细胞基因表达的机制上。所有七螺旋化学引诱物受体激活Galphai，Galphai释放G β γ以激活白细胞的主要功能。在目标1中，我们将确定G β γ如何介导NF-κ B激活。将使用正常和遗传改变小鼠的细胞研究G β γ效应子PI-3激酶-γ的参与。目的2致力于研究Galpha 16与趋化因子受体的直接偶联及其对PLC β的允许激活，这是一种独特的协同激活机制，可以解释为什么趋化因子仅在造血细胞中是有效的转录调节因子。在目的3中，我们将研究Galphai在抑制IL- 12基因表达中的潜在作用。使用体外重建试验，我们将比较介导抑制IL-12产生的趋化因子受体与缺乏这种能力的趋化因子受体，以确定其G蛋白偶联特性的差异。总的来说，这些研究旨在验证化学引诱物受体通过与G蛋白的差异偶联来调节基因表达的中心假设。

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.