Amplification of IL-4Ralpha signaling pathways in human airways through 15 LO1

描述（由申请人提供）：人类哮喘，特别是重度哮喘的病理生物学仍然知之甚少，关于Th 2炎症的作用和重要性存在相当大的争议。我们的初步数据表明，15脂肪氧合酶（LO）1、其产物15羟基二十碳四烯酸（HETE）及其与磷脂酰乙醇胺（PE）结合蛋白（BP）1的相互作用可能是促进和放大Th 2炎症和抑制的核心在缺乏高水平Th 2的情况下，2肾上腺素受体（AR）反应。基于我们的初步数据，我们提出3的目的是更好地理解15 HETE-PE/15 LO 1与PEBP 1组合在体外和离体人气道上皮细胞中IL-13/IL-4 Ra信号传导放大中的作用，以及该过程是否进一步影响2受体信号传导。在目标1中，我们将确定长期暴露于IL-13（和15 LO 1的上调）是否会导致MEK-ERK通路被长期激活的上皮细胞，而PI 3 K通路被下调，以及IL-4 Ra多态性是否会影响这些过程。我们将讨论15 LO 1/15 HETE-PE在这一过程中的作用，以及这种上调对下游基因表达的影响，无论是在急性（低15 LO 1）状态，还是在慢性（高15 LO 1）状态。我们假设活性15 LO 1/15 HETE-PE途径的存在将极大地增强IL-13诱导的基因表达。将在急性和慢性刺激下，在存在和不存在敲低以及药理学抑制的情况下评价基因阵列，以确定15 LO 1/15 HETE-PE途径的总体影响。在目标2中，我们将讨论15 LO 1/15 HETE-PE通路与pERK通路相互作用以调节基因表达的机制。该目标将专门关注15 LO 1/15 HETE-PE与PEBP 1的相互作用，进一步定义相互作用的亚细胞位置、物理相互作用的机制以及PEBP 1磷酸化发生的作用和机制。在目标3中，我们将确定15 HETE-PE与PEBP 1的相互作用是否竞争性抑制GRK 2与PEBP 1的结合，从而导致人哮喘患者体内和体外2 <$AR的脱敏增强。我们相信，在哮喘和正常受试者的原代人类细胞中评估这些过程，将揭示一种新的令人兴奋的途径，该途径似乎密切调节pERK，“Th 2”型反应，也许还有哮喘上皮细胞中的<$2AR途径。我们相信，更好地理解这些相互作用将为新的和新颖的治疗方法开辟许多机会。 公共卫生相关性：哮喘是一种非常常见的疾病，人们对其了解甚少。虽然Th 2细胞因子（IL-4/-13）被认为是重要的，但是使用阻断该途径的试剂的初始研究令人失望。尽管如此，在哮喘患者的气道中有证据表明，这一途径的部分是高度活跃的。这项拨款申请将建立在新的和高度新颖的相互作用，在人类哮喘气道细胞之间的15脂氧合酶途径和磷脂酰乙醇胺结合蛋白-1。这些相互作用可以在缺乏高水平IL-14/-13的情况下放大和维持“Th 2”型炎症，以及改变对b2激动剂药物的反应，这是哮喘治疗的支柱。最后，这些相互作用为药物开发的创新分子方法开辟了新的机会。

DESCRIPTION (provided by applicant): The pathobiology of human asthma and severe asthma in particular, remains poorly understood, with considerable controversy as to the role and importance of Th2 inflammation. Our preliminary data suggest that 15 lipoxygenase (LO)1, its product 15 hydroxyeicosatetraenoic acid (HETE) and their interactions with phosphatidyl-ethanolamine (PE) binding protein (BP)1 may be central to promoting and amplifying Th2 inflammation and dampening ¿2 adrenoreceptor (AR) responses in the absence of high levels of Th2. Based on our preliminary data, we propose 3 aims to better understand the role that 15 HETE-PE/15 LO1, in combination with PEBP1, plays in amplification of IL-13/IL-4Ra signaling in human airway epithelial cells in vitro and ex vivo, and whether the process further impacts ¿2 receptor signaling. In Aim 1, we will determine whether prolonged exposure to IL-13 (and upregulation of 15 LO1) leads to epithelial cells in which MEK-ERK pathways are chronically activated, while PI3K pathways are downregulated and whether IL-4Ra polymorphisms impact these processes. We will address the role that 15 LO1/15 HETE-PE plays in this process and the impact this upregulation has on downstream gene expression, both in an acute (low 15 LO1) state, as well as in a chronic (high 15 LO1) state. We hypothesize that the presence of an active 15 LO1/15 HETE-PE pathway will greatly enhance IL-13 induced gene expression. Gene arrays will be evaluated under both acute and chronic stimulation, and in the presence and absence of knockdown, as well as pharmacologic inhibition to determine the overall impact of the 15 LO1/15 HETE-PE pathway. In Aim 2, we will address the mechanisms by which the 15 LO1/15 HETE-PE pathway interacts with the pERK pathway to modulate gene expression. This aim will focus exclusively on the interactions of 15 LO1/15 HETE-PE with PEBP1, further defining the subcellular location of the interaction, the mechanism of the physical interaction and the role for and mechanism by which phosphorylation of PEBP1 occurs. In Aim 3, we will determine whether the interaction of 15 HETE-PE with PEBP1 competitively inhibits the binding of GRK2 to PEBP1, leading to enhanced desensitization of 2¿AR in vitro and in vivo in human asthmatics. We believe that evaluating these processes in primary human cells, from asthmatic and normal subjects, will unravel a new and exciting pathway which appears to tightly regulate pERK, "Th2"-type responses and perhaps the ¿2AR pathway in asthmatic epithelial cells. We believe a better understanding of these interactions will open up many opportunities for new and novel therapeutic approaches. PUBLIC HEALTH RELEVANCE: Asthma is an extremely common disease which remains poorly understood. Although Th2 cytokines (IL-4/-13) have been believed to be important, initial studies using agents which block this pathway have been disappointing. Despite that, there is evidence in the airways of asthmatic people that portions of this pathway are highly active. This grant application will build on new and highly novel interactions identified in human asthmatic airway cells between the 15 lipoxygenase pathway and phosphatidylethanolamine binding protein-1. These interactions could serve to amplify and sustain a "Th2"-type of inflammation in the absence of high levels of IL-14/-13, as well as alter responses to b2 agonist drugs, mainstays of asthma therapy. Finally, these interactions open up new opportunities for innovative molecular approaches to drug development.