| Apoptosis, or programmed cell death, is the mechanism through which cells die and is important in the development and homeostasis of organisms. There are two major effector pathways of apoptosis utilized by the immune system: death receptor-mediated apoptosis and granule-mediated apoptosis. Many cell types, such as hepatocytes, require mitochondria involvement to complete apoptosis. The mitochondrial amplification pathway involves the Bcl-2 family of proteins, which includes both proapoptotic and antiapoptotic members. Bid is a proapoptotic member of this family that is cleaved to become an active mediator of apoptosis and plays a role in both receptor- and granule-mediated apoptosis. It has been previously demonstrated that hepatocytes are dependent on Bid cleavage to execute apoptosis. The focus of this study is to prevent rat hepatocyte apoptosis through the expression of a mutant form of Bid. To test this, we generated Bid cleavage mutants by introducing point mutations at known cleavage sites, and demonstrate protection from cleavage by caspase 8 and granzyme B.; We expressed these mutants in a rat hepatoma cell line, FaO, using recombinant adenoviruses and observed that FaO cells expressing Bid cleavage mutants were protected from death receptor-mediated apoptosis through a pathway that includes less Bid cleavage and mitochondrial membrane damage. Natural killer (NK) cell-mediated lysis of hepatocytes has been observed during viral infection, and thus we sought to determine if expression of Bid cleavage mutants would protect hepatocytes from NK lysis. Using a cytotoxicity assay, we found that expression of Bid cleavage mutants protected FaO cells from NK cell-mediated cytotoxicity. Hepatocyte apoptosis is also associated with warm ischemia/reperfusion (I/R) injury. This type of injury is common during liver surgery and can compromise liver function, and we therefore asked if mutant Bid expression would protect hepatocytes from apoptosis in this setting. We observed that rats expressing Bid cleavage mutants were protected from hepatocyte apoptosis during I/R injury and had improved liver function. Our findings demonstrate that prevention of Bid cleavage can protect hepatocytes from death receptor- and cell-mediated apoptosis, and that Bid cleavage is an attractive target for the development of novel therapeutics to control hepatocyte apoptosis and preserve liver function. |
