This project, which builds on our recent observations of regulation of Fas death receptor, takes us to a new area of research, the analysis of liver cells, where Fas plays a critical role mediating liver disorders. Death receptors have key roles in numerous cell death/proliferation decisions. More specifically, the Fas death receptor plays a pivotal role in liver tissue to determine the outcomes of challenges induced by cell stress due to infection and toxins. The Fas system is an important cell elimination system that has several established functions, including the elimination of autoreactive lymphocytes, infected and defective cells and cells damaged by chemotherapy and irradiation. Without the Fas system, for example, in Fas -/- cells, many clinical chemotherapies and ionizing radiation are no longer effective in eradicating cells. Given the common expression of Fas, the common inability to eliminate some cells that express Fas, we suspect Fas to be under tight control. While Fas activation serves as a clinically beneficial mediator of chemotherapy, inadvertent Fas signaling can perpetuate liver damage and death. In this project we searched for potential binding regulators of Fas and identified a tumor suppressor protein promyelocytic leukemia protein (PML), which is associated with enhanced apoptosis. We chose to test the dominant-negative PMLRARa because its phenotype is easier to visualize. We found that PMLRARa (and PML) binds to Fas and potently blocks apoptosis in cells. When express in mouse liver, PMLRARa effectively protects mice from death induced by a lethal dose of agonistic anti-Fas antibody. The B-box domain of PML is necessary for binding to Fas, suggesting that PML and PMLRARa use this site to bind Fas. A model of opposing effects of PML and PMLRARa on regulation of transforming growth factor 2 receptor (TGF2R) has been established and we suspect that Fas operates in a similar model. Our hypothesis is that PML positively regulates Fas signaling, which plays a critical role in death and proliferation decisions. We will use PML dominant-negative mutant PMLRARa as a probe to characterize the blocking effects on Fas. We anticipate that PML will express have Fas-promoting effects and will explain the widely known apoptosis enhancement effect of PML. Our hypothesis is that PML is a binding regulator of Fas and this interaction can be modulated to preserve liver tissue function. The long-term goal of this project is to understand apoptosis regulation of liver cells to reverse liver pathology driven by the Fas receptors.
The Fas system is an important cell elimination system that has several established functions, including the elimination of autoreactive lymphocytes and infected and defective cells and cells damaged by chemotherapy and irradiation. We have identified promyelocytic leukemia protein as a binding and potential key regulator of Fas signaling. This project will characterize the binding with Fas and demonstrate how Fas is regulated in liver cells and in mice;thus by showing the site of regulation of Fas, we will be able to identify an effective therapeutic approach for liver disorders driven by Fas signaling.
