The goal of this proposal is to determine the pathobiochemical mechanisms responsible for acute lung injury. HYPOTHESIS: the overall hypothesis to be tested is that reactive oxygen species (ROS) play a critical role in many types of acute lung injury and that xanthine oxidoreductase (XDH/XO) is a highly regulated and critically important enzyme for the generation of ROS in these settings.
SPECIFIC AIMS : The first specific aim is to determine the mechanisms by which pro-inflammatory cytokines and hypoxia direct XDH/XO molecular and cellular regulation. The second specific aim is to directly determine the role of XDH/XO in models of acute lung injury associated with inflammation and hypoxia. RESEARCH PLAN: Our strategy for the first specific aim is to define and fully characterize the cis-acting elements within the XDH/XO 5' regulatory region that mediate the gene activation induced by cytokines and hypoxia and the trans-acting factors that bind to the critical elements. We will complete a functional analysis of the XDH/XO 5' regulatory region using step deletion constructs cloned into a luciferase reporter vector. This will be followed by the definition of protein-DNA interactions via gel shift assays and DNAse footprinting. Supershift assays and nuclear protein purification will be used to identify putative transacting factors. Our strategy for the second specific aim is to selectively manipulate the level of XDH/XO gene expression in mice and to evaluate effects of this manipulation in clinically relevant models of lung injury (Viral Pneumonia, reperfusion injury and sepsis). To accomplish this we will study the effects of: a) loss of function of XDH/XO by generating (XDH/XO (-/-) mice by creating targeted deletions using homologous recombination and B) gain of function of XDH/XO by generating transgenic mice overexpressing the enzyme. SIGNIFICANCE: This work will provide a better understanding of the importance of XDH/XO in clinically relevant models of acute lung injury. The results will be of wide interest to investigators studying gene activation by inflammation and hypoxia. The creation of XDH/XO (-/-) and transgenic mice will snot only assist with elucidating the role of XDH/HO in lung injury, but also its roles in a host of other diseases and in normal physiology.
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