Cataract is a significant health and economic problem worldwide. Oxidative damage has been implicated as a causative factor in cataract formation and iron-catalyzed free radical reactions are responsible for virtually all oxidative tissue damage. In addition to catalyzing oxidative damage and serving as an essential component of key enzymes, new roles for iron in cellular physiology are emerging. For example, we have made the novel observation that iron regulates glutamate production in a number of cell types, including the lens. Furthermore, others have shown that iron regulates the availability of the transcription factor, hypoxia inducible factor (HIF-1 a) which exerts significant effects on iron metabolism by controlling transcription of key iron regulatory proteins. All cells carefully control iron uptake, utilization and storage. Most intracellular iron is safely stored in ferritin or incorporated into iron-dependent enzymes. However, there is a pool of iron in cells that is chelatable and is called the labile iron pool (LIP). The LIP may be the key regulator of iron metabolism in cells and our experiments are designed to determine how it is regulated and its role in the lens. Therefore we have hypothesized that alterations in cellular iron metabolism and storage result in significant changes in the size of the LIP with concomitant physiological changes including glutamate and glutathione production and secretion as well as the activity of HIF-1. The following specific aims will be addressed: SA #1. Hypothesis: Age-related alterations in ferritin H:L chain makeup, as seen in lens epithelial cells of varying age, and modification of ferritin structure as found in ferritin from fiber have profound effects on ferritin function. These include the ability of ferritin to sequester iron as well as long term storage and release of iron from these modified ferritin molecules. SA #2. Hypothesis: Changes in intracellular iron distribution significantly impact glutamate synthesis and secretion resulting in alterations in cystine uptake and glutathione synthesis. SA #3. Hypothesis: Glutamate vesicles, glutamate/cystine antiporter and ferroportin have specific localization within the lens which is related to their function. SA #4. Hypothesis: Changes in oxygen levels and intracellular iron distribution alter the activity of the transcriptional regulator HIF-1. SA #5. Hypothesis: Alterations in the activity of HIF-1 modify iron metabolism with downstream effects on glutamate and glutathione production. ? ? ? ?
