Hyperoxic lung injury is associated with extensive disintegration of the alveolar epithelium. Numerous studies demonstrate a protective role of keratinocyte growth factor (KGF) in oxidant-induced lung injury. We have found that KGF induces the activation of the serine-threonine kinase Akt in lung epithelial which involves PI 3-kinase and results in BAD phosphorylation. Using the yeast two-hybrid system, we have isolated cDNA clones whose products interact with the cytoplasmic domain of KGFR. Two of these clones encode the kinases p90RSK and PAK4 and there are 4 novel cDNA clones. Our observation leads us to hypothesize that: 1). KGF prevents hyperoxic lung injury through its protective effects on lung epithelial cells. 2). Akt activation plays a major role in protection by KGF. 3). KGF-induced signal transduction involves interaction of specific intracellular proteins with the cytoplasmic domain of KGFR. To address our hypothesis we will:
Aim I. Investigate the mechanisms of protection from oxidant-induced injury by using inducible transgenic mice overexpressing KGF or a constitutively active form of Akt. 1) KGF expression will be induced at different times before and during hyperoxic exposure. The lungs of transgenic mice will be analyzed by: a) histology and electron microscopy, b) biochemical assays (protein leak, lipid peroxidation, redox status), c) gene expression studies (surfactant, cell death-associated genes), d) studies of lung inflammation (cytokine/chemokine expression) 2) Transgenic mice expressing a constitutively active form of Akt in an inducible fashion will be similarly analyzed.
Aim II. Characterization of intracellular proteins that interact with the KGFR. 1) One novel cDNA clone identified in multiple independent isolates and highly expressed in the lung and in epithelial cells will be characterized. 2) a) Interactions in mammalian cells will be confirmed by co-immunoprecipitation experiments, and whether the interactions b) require the tyrosine kinase activity of KGFR and c) have biologic significance will be determined by expressions of mutants in cell culture and in KGF mice using adenoviral gene transfer techniques.
Aim III. Structure-function relationships of KGFR-mediated protection from hyperoxic lung injury. 1) Specific tyrosine residues in the cytoplasmic domain of a trk-KGFR chimeric construct will be mutangenized to determine the structure-function relationships in KGFR using rat type II cells and small airways epithelial cells and a variety of molecular and biochemical approaches. 2) KGF overexpressing mice will be infected with recombinant adenovirus encoding particular KGFR mutants to determine their effects on hyperoxic injury in vivo.
Showing the most recent 10 out of 13 publications
