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

描述（由申请人提供）：人类哮喘，特别是严重哮喘的病理生物学仍然知之甚少，关于Th2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，在缺乏高水平Th2的情况下，15脂氧合酶（LO）1、其产物15羟二糖四烯酸（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Th2炎症和抑制¿2肾上腺素受体（AR）反应的核心。基于我们的初步数据，我们提出3旨在更好地了解15 HETE-PE/15 LO1联合PEBP1在体外和离体人气道上皮细胞中扩增IL-13/IL-4Ra信号的作用，以及该过程是否进一步影响¿2受体信号传导。在Aim 1中，我们将确定长期暴露于IL-13（以及15lo1的上调）是否会导致上皮细胞中MEK-ERK通路长期激活，而PI3K通路下调，以及IL-4Ra多态性是否会影响这些过程。我们将讨论15 LO1/15 HETE-PE在这一过程中所起的作用，以及这种上调对下游基因表达的影响，无论是在急性（低15 LO1）状态，还是在慢性（高15 LO1）状态。我们假设活跃的15 LO1/15 HETE-PE通路的存在将大大增强IL-13诱导的基因表达。基因阵列将在急性和慢性刺激、存在和不存在敲低以及药物抑制下进行评估，以确定15 LO1/15 HETE-PE途径的总体影响。在目标2中，我们将探讨15 LO1/15 HETE-PE途径与pERK途径相互作用以调节基因表达的机制。本研究将专注于15 LO1/15 HETE-PE与PEBP1的相互作用，进一步确定相互作用的亚细胞位置、物理相互作用的机制以及PEBP1磷酸化发生的作用和机制。在Aim 3中，我们将确定15 HETE-PE与PEBP1的相互作用是否竞争性地抑制GRK2与PEBP1的结合，从而在体外和体内增强2¿AR的脱敏性。我们相信，在哮喘和正常受试者的原代人细胞中评估这些过程，将揭示一个新的和令人兴奋的途径，该途径似乎严格调节pERK，“Th2”型反应，可能还有哮喘上皮细胞中的¿2AR途径。我们相信，更好地了解这些相互作用将为新的治疗方法开辟许多机会。

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.