Oxidant induced cellular damage is an important factor in the pathogenesis of injury to the developing lung. Such injury is exacerbated in the premature infant due to a gestational-age dependent deficiency in antioxidant defenses. The long-term objective of these studies is to define the molecular mechanisms of antioxidant gene expression in the developing human lung.
The specific aims of this proposal are to define the cellular and molecular determinants of pulmonary ceruloplasmin gene expression. Ceruloplasmin is a blue-copper oxidase with multiple roles in host antioxidant defense including free-radical scavenging, prevention of lipid-peroxidation, cellular compartmentalization of copper and provision of copper to antioxidant and repair enzymes such as Cu/Zn superoxide dismutase and lysyl oxidase. Ceruloplasmin is abundantly expressed in the mesenchymal cells of the peripheral airways of the developing rodent and human lung and in alveolar and interstitial macrophages during hyperoxia, inflammation and lung injury. Detailed molecular studies of the ceruloplasmin gene indicate that ceruloplasmin expression in the developing lung is determined in part by a unique, previously uncharacterized cis- acting element in the 5' flanking region of the gene. This sequence specifically binds a nuclear protein present in extracts from the developing lung but not the adult lung or liver. We now propose to extend these observations by comparing the conservation of this cis-acting sequence in the rat ceruloplasmin gene to the sequence of the corresponding region in the human and murine genes. We shall then purify and clone this nuclear binding protein and determine the primary structure of this putative transcription factor by nucleotide sequencing of isolated clones. The cellular site of expression will then be identified by in-situ hybridization in developing lung tissue. Finally we will generate transgenic mice using characterized upstream regions of the ceruloplasmin gene and human growth hormone and E. Coli lacZ reporter genes. These studies will permit a detailed definition of the cis and trans-acting elements determining ceruloplasmin gene expression in the developing lung and will generate several unique mouse models for the study of cell-cell interactions in lung development. These data may allow the development of new strategies for manipulating antioxidant gene expression to prevent or ameliorate lung injury in the premature human infant.
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