Novel Functions of the Ah Receptor

点击翻译按钮获取中文摘要

EXCEED THE SPACE PROVIDED. The biological functions of the aryl hydrocarbon receptor (AhR) are normallyattributed to its functioning as a ligand-activated transcription factor. However, recent studies suggest that the AhR may have ligand- independent functions. Studies with murine hepatoma cells engineered to have different AhR contents show a direct correlation between AhR content and susceptibility to apoptosis induced by ceramide, but not other agents. Recent studies have shown that ceramide causes lysosome disruption, cleavage of cytosolic Bid, release of cytochrome c, and activation of the 'mitochondria!'apoptotic pathway in AhR-containing cells. The effects of ceramide could be duplicated by the targeted disruption of lysosomes with photodynamic therapy (PDT). Because PDT and ceramide disrupt the lysosomes of AhR-deficient cells, but do not cause Bid cleavage we hypothesize that 1) lysosome disruption triggers the 'mitochondria!' apoptotic pathway via lysosomal protease cleavage of Bid, and 2) the AhR modulates processes involved in Bid cleavage. In this application we propose to use Bid null and AhR null cells to determine if activation of the 'mitochondria!' apoptotic pathway by ceramide/PDTis dependent upon 1) Bid and 2) AhR content, respectively. 3) We will determine if the inability of our AhR-deficient cell lines to cleave Bid reflects the absence of a lysosomal Bid cleavage activity, or the presence of cytosolic protein(s) that inhibit this activity. 4) We will attempt to identify the lysosomal activity responsible for Bid cleavage by conventional purification techniques, selective pharmacological inhibition, or a proteomic approach. 5) Transfection of mutated forms of the AhR into AhR-deficient cells will be used to determine the regions/functions (e.g., DNA and ligand-binding, heterodimerization, transactivation, etc.) of the AhR important in the modulation of Bid cleavage/ceramide- induced apoptosis. Lastly, 6) a series of 'oxidative stressors' will be tested to determine if oxidant-induced activation of the 'mitochondria!' apoptotic pathway is a downstream consequence of lysosome disruption and Bid cleavage. These studies will provide a mechanism linking lysosome disruption and apoptosis, and characterize a novel AhR function. The concept that lysosome disruption can trigger apoptosis, in addition to necrosis, is relevant to the mechanism of action of many toxicants, and health-related areas such as neuro and pulmonary toxicology, chemo-and phototherapy, stroke research, and reperfusion induced injury. PERFORMANCE SITE ========================================Section End===========================================