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.
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.
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