This is a continuation application for a long-term project on effects of trace metal ions on gene expression in human cells. During the last period, we began to investigate the effects of toxic metal ions on signal transduction pathways used for global regulation of gene expression, cell proliferation and survival. The tumor promoter arsenite (As+3) was found to be a potent activator of two mitogen activated protein kinase (MAPK) cascades that stimulate the activity of transcription factor AP-1. This effect of As+3 is due to inhibition of a dual-specificity protein phosphatase, JNK phosphatase, whose normal function is to keep the MAPKs, JNK and p38 in a low activity state. As induction of AP-1 activity is closely linked to tumor promotion, the JNK phosphatase is probably an important mediator of As+3 cocarcinogenesis. It may also be involved in As+3 induced inflammatory disease. To examine the physiological role of the JNK phosphatase we will characterize and molecularly identify it using a combination of biochemical and molecular biological approaches. Dominant-negative mutants will be transfected into cultured cell lines to inhibit endogenous JNK phosphatase activity and thus assess its function in cell physiology. We will also examine the susceptibility of mouse strains and cell lines deficient in JNK or JNK phosphatase to As+3 induced cocarcinogenesis and toxicity. As+3 and other trace metals were also proposed to act through non-specific induction of oxidant stress. We therefore started to investigate the regulation of transcription factor NF-KB, which was proposed to be a major sensor of oxidant stress. We recently purified and cloned a key component in the pathway leading to NF-KB activation, the protein kinase responsible for phosphorylation and eventual degradation of the inhibitors of NF-KB, the IKBs. We now propose to study the molecular mechanism by which oxidants lead to activation of this IKB kinase (IKK). As NF-KB plays a key role in inflammation, understanding the regulation of IKK activity by oxidants, such as ozone, will provide a molecular basis for oxidant induced inflammatory disease.
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