Although phosphatidylserine (PS) externalization is a well-established early marker of apoptosis and a signal for macrophage-dependent recognition, the functional molecular links between these processes and oxidative stress produced during apoptotic execution are unclear. Our preliminary data suggest a previously unknown signaling role for oxidized PS during Fas-induced apoptosis in Jurkat cells. Since Fas-triggered apoptosis modulates turnover of pulmonary epithelial cells (PUEC) in both healthy and diseased tissue, we propose studying the mechanisms involved in selective PS oxidation and the subsequent PS-dependent signaling events during Fas-mediated apoptosis in PUEC. Our central hypothesis is that PS oxidation is an essential component in the pathway leading to PS externalization during Fas-induced apoptosis in PUEC as well as in efficient recognition and phagocytosis of apoptotic cells by macrophages. As a corollary, inhibition of PS oxidation during Fas-induced apoptosis by antioxidants may affect subsequent externalization/recognition and phagocytosis of apoptotic cells.
Our Specific Aims designed to test this hypothesis will: 1) establish a mechanistic link between Fas-induced PS peroxidation and PS-dependent signaling in the PUEC plasma membrane (PS externalization and recognition by macrophages), relationship of PS oxidation to other components of Fas-triggered apoptosis, and extent to which the inhibition of PS oxidation by antioxidants blocks PS-dependent signaling without affecting other major Fas-triggered pathways of apoptosis; 2) establish the catalytic role and mechanisms of cyt c in PS oxidation in plasma membrane; 3) determine the role of PS peroxidation in PS externalization; and 4) reveal the mechanisms for enhanced macrophage recognition and phagocytosis of PUEC expressing oxidized PS. Developmental remodeling of the lung and various acute and chronic lung diseases are characterized by marked apoptosis. Inflammation is limited by effective recognition and phagocytosis of apoptotic cells by macrophages whose critical function is mediated by externalized PS on the surface of apoptotic cells. Our studies will define a new role for site-specific oxidative stress during apoptosis in PUEC, identify oxidized PS as a key signaling molecule that affects macrophage recognition of apoptotic cells and identify antioxidant interventions that can differentially prevent or preserve PS oxidation and downstream signaling events. This will aid in developing new strategies for directed modulation of inflammation in lung disease.
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