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

描述(申请人提供)：人类哮喘，尤其是严重哮喘的病理生物学仍然知之甚少，对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及其产物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Th2炎症和抑制[2]肾上腺素受体(AR)反应的中心。基于我们的初步数据，我们提出3个目的是为了更好地了解15 HETE-PE/15 LO1与PEBP1联合在体外和体外对人呼吸道上皮细胞IL-13/IL-4ra信号的放大作用，以及这一过程是否进一步影响2受体信号转导。在目标1中，我们将确定长期暴露于IL-13(和15个LO1上调)是否会导致上皮细胞中MEK-ERK途径被慢性激活，而PI3K途径被下调，以及IL-4ra基因多态是否影响这些过程。我们将讨论15LO1/15HETE-PE在这一过程中所扮演的角色，以及这种上调对下游基因表达的影响，无论是在急性(低15LO1)状态下，还是在慢性(高15LO1)状态下。我们推测，活跃的15LO1/15HETE-PE通路的存在将极大地增强IL-13诱导的基因表达。基因阵列将在急性和慢性刺激下进行评估，并在存在和不存在击倒以及药物抑制的情况下进行评估，以确定15LO1/15HETE-PE途径的整体影响。在目标2中，我们将阐述15LO1/15HETE-PE途径与PERK途径相互作用调节基因表达的机制。这一目标将专门集中在15 LO1/15 HETE-PE与PEBP1的相互作用上，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发生磷酸化的作用和机制。在目标3中，我们将确定15HETE-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的脱敏作用。我们相信，评估来自哮喘和正常受试者的原代人类细胞的这些过程，将揭开一条新的、令人兴奋的途径，似乎严格调控哮喘上皮细胞中的PERK、“Th2”型反应，以及可能的2AR途径。我们相信，对这些相互作用的更好理解将为新的和新的治疗方法打开许多机会。 与公共卫生相关：哮喘是一种极其常见的疾病，人们对此仍知之甚少。尽管Th2细胞因子(IL-4/-13)一直被认为是重要的，但使用阻断这一途径的药物的初步研究一直令人失望。尽管如此，哮喘患者的呼吸道中有证据表明，这一途径的一部分非常活跃。这项拨款申请将建立在人类哮喘呼吸道细胞中发现的15脂氧合酶途径和磷脂酰乙醇胺结合蛋白-1之间的新的和高度新颖的相互作用基础上。在缺乏高水平IL-14/-13的情况下，这些相互作用可能有助于放大和维持一种“Th2”型炎症，以及改变对b2激动剂药物的反应，而b2激动剂是哮喘治疗的主要药物。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.