The central hypothesis of our project has been that short peptides can be used as potent, specific inhibitors which can be targeted to desire organelles and cell types in order to precisely elucidate cellular pathways. We have shown that the nuclear function of calmodulin in type II cells is critical for murine lung development. The highly flattened type I cells provide a vast cellular surface for O2/CO2 exchange. Lung alveoli are critical for the maintenance of oxygen homeostasis. Type I cells are extremely sensitive to injury from environmental challenges and they do not divide. Following lung damage, type II cells terminally differentiate to repopulate the alveolar epithelium with new type I cells. The research in this project will use novel transgenic approaches to study, in vivo, the role of CaM kinase II- and CalN-regulated pathways involved in lung development and type II cell response to lung injury. CaM kinase II and CalN are the major enzyme systems which mediate Ca/2+/CaM regulation of cellular activities such as secretion, ion fluxes, DNA replication and RNA transcription. Studies are performed in transgenic mice in which the SP-C (surfactant protein C) promoter is used to direct expression of synthetic dominant-negative genes which neutralize the function of CaM Kinase II and CalN in lung type II epithelial cells. These synthetic genes encode peptide concatemers which bind to the active site in the catalytic subunit and cause inhibition of their targeted enzymes in a cell-specific manner. The targeting of these inhibitory peptide concatemers to the lung epithelium will lead to the generation of unique phenotypes in which Ca/2+- activated signal transduction systems are altered. This project will evaluate the role of CaM kinase II and CaIN in lung development and physiological responses to oxidative stress, ozone and silica-induced injury. CaM kinase II and CalN may be required for proper lung function such as fluid and surfactant secretion. It is anticipated that unique mouse lines will be developed that have altered lung compliance, mucous congestion and lung fibrosis. These pathophysiological conditions will result in decreased gas exchange in the alveolus which will cause hypoxemia, plasma pH imbalance and physical disability.?
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