The work described in this application is designed to explore the premise that the lens epithelial cell redox status may regulate gene expression. There are a number of genes whose products regulate the expression of other genes. It is proposed that some of these regulatory genes such as fos, jun and NF-kappaB are themselves controlled by redox in lens systems. These regulatory genes have been chosen since they are widely distributed, are effected by redox and binding sites for their products have been reported to be associated with certain crystallin genes. The experimental work described in this application will elucidate the extent to which oxidative stress alters the expression of these control genes and subsequently effects the expression of a reporter gene and of lens genes. The demonstration of the disruption of normal cellular gene regulation by oxidative stress would provide a new fundamental aspect to the mechanism by which oxidative stress can cause cataract and place greater emphasis on events occurring in the epithelial cells. Specifically, it is planned to determine the expression of fos, jun and NF-kappaB in the lens in resting and dividing cell lines under normal and oxidative stress conditions. The regulation by fos, jun and NF-kappaB of a reporter gene and certain lens genes containing AP-1 enhancer sites and NF-kappaB binding sites will be examined. Cellular redox set points will be determined based on GSH/GSSG, NADH/NAD and NADPH/NADP. The effect of agents which eliminate aspects of oxidative stress such as AL-3823A, desferoxamine and Tempo will also be investigated. Preliminary observations are reported indicating the feasibility of this approach.
