Prostaglandin E2 (PGE2) is an important cyclooxygenase metabolite in the lung where it regulates smooth muscle tone as well as immune/inflammatory responses. Understanding of which PGE2 receptor subtypes mediate these responses is incomplete, an the integration of isolated cellular effects into the multicellular pathophysiology of immunologic/allergic diseases is even less well understood. Increases in intracellular cAMP have been implicated in the PGE2 mediated anti-inflammatory effect. The recent cloning of four distinct prostaglandin E receptor subtypes designated, EP1, EP2, EP3 and EP4, combined with pharmacologic studies, will allow a more complete understanding of the signal transduction pathways participating in these immuno-regulatory actions. The main hypothesis of this project is that EP receptor subtypes couple to different effector systems and display differential regulatory properties; thus expression of different EP receptor subtypes on individual inflammatory cell types determines the physiologic response to PGE2 evoked in each target cell. Based upon our preliminary data and studies in the literature, we hypothesize that the EP2 receptor is a major determinant of the anti-inflammatory effects of PGE2. To test these related hypotheses the following specific aims are propose;
In specific aim 1, we will determine the pattern of expression of EP receptor subtypes on relevant human and murine immune/inflammatory cells including monocytes, eosinophils, alveolar macrophages, murine T-helper (Th)1 and Th2 lymphocyte clones and mast cells. Since the abundance of EP receptors usually is quite low, sensitive reverse transcription-polymerase chain reaction (RT-PCR) and RNAse protection assays will be employed. Cells positive for EP receptor gene expression will be analyzed for ligand binding, second messenger generation and functional responses using subtype-selective ligands.
In specific aim 2, we will address the importance of EP2 and receptor subtypes through studies of models of airway allergic or viral inflammation and eosinophil accumulation, and systemic anaphylaxis. We have isolated the gene encoding the murine EP2 receptor as well as the murine EP2 receptor cDNA.
In specific aim 3, if based upon the findings in specific aims 1 and 2, we establish that the EP2 receptor is indeed localized on target cells in such a way as to support its role in PGE2 evoked anti- inflammatory response, we will generate EP2 receptor-deficient mice by standard gene targeting methodology, starting with homologous recombination in embryonic stem cells. This EP2 deficient mouse will be used as a tool for the study of EP2 receptor function. These studies will elucidate the role of EP2 receptor function in inflammatory lung disease and possibly identify this receptor subtype as a therapeutic target for the development of novel ligands for the treatment of these disorders.
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