Molecular mechanisms that regulate eosinophil cytokine production

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(Applicant's Abstract) The mechanisms by which peripheral blood eosinophils (PBEos) differentiate into airway based, effector cells responsible for the pathophysiology of asthma are poorly understood. A candidate mediator for this process is granulocyte macrophage colony stimulating factor (GM-CSF), a potent cytokine produced by eosinophils. GM-CSF is commonly elevated in the BAL fluid of symptomatic asthmatics. PBEos which are activated in vitro secrete immunologically detectable GM-CSF and express GM-CSF mRNA. PBEos also express cell surface GM-CSF receptors, suggesting GM-CSF functions as a critical autocrine growth and survival factor both in vitro and in vivo. Despite the likely functional significance of GM-CSF, very little is known about the molecular mechanism(s) which controls its production and release by eosinophils. Recently we have shown that GM-CSF mRNA stability was significantly enhanced in PBEos treated with tumor necrosis factor alpha (TNF) and fibronectin or in BAL derived eosinophils from allergen challenged volunteers. Using a yeast 3 hybrid screen, we identified YB-1, a known nucleic acid binding protein as a GM-CSF mRNA binding protein. Recombinant YB-1 specifically bound in vitro to the AU-rich, 3' UTR instability determinants of GM-CSF mRNA. When transfected into peripheral blood eosinophils, YB-1 enhanced in vitro survival by 3-5 fold, which was completely blocked by anti-GM-CSF antibodies. Finally, in preliminary studies, transfected YB-1 stabilized GM-CSF mRNA in PBEos. Therefore, we hypothesize that YB-1 mediates the post-transcriptional regulation of GM-CSF mRNA in activated eosinophils. Thus, the aims of this project are to 1). Characterize how YB-1 increases GM-CSF mRNA in PBEos, 2).Characterize the signaling cascades induced by TNFalpha, and fibronectin which enable YB-1 to interact with and regulate GM-CSF mRNA, 3). Determine if YB-1 is the sole effector in this system or interacts with additional protein components to regulate GM-CSF mRNA, 4). Determine which domain(s) of YB-1 is/are required for GM-CSF post-transcriptional gene regulation. In aggregate these studies will clarify the molecular mechanisms underlying GM-CSF mRNA regulation in activated eosinophils, and as such, provide additional, novel therapeutic targets for the prevention and treatment of asthma.