Induction of SM-20 in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death.

Sympathetic neurons deprived of nerve growth factor (NGF) release cytochrome c into the cytosol and undergo caspase-dependent cell death through a process that requires de novo gene expression. Expression of the SM-20 gene increases after NGF withdrawal, and ectopic SM-20 expression induces cell death in NGF-maintained neurons. To further evaluate the mechanism by which SM-20 promotes cell death, we developed a PC12-derived cell line in which SM-20 expression can be induced by addition of doxycycline to the culture medium. Induction of SM-20 in either undifferentiated or NGF-differentiated cells resulted in cell death. Cell death was accompanied by an increase in caspase activity and was inhibited by the caspase inhibitor zVAD-FMK. Analysis of cytochrome c in cytosolic and mitochondria-enriched subcellular fractions revealed that induction of SM-20 led to the accumulation of cytochrome c in the cytosol. Surprisingly, SM-20 expression also resulted in a selective increase in the total amount of cytochrome c protein. Thus, induction of SM-20 expression appears to affect both the amount and subcellular localization of cytochrome c in PC12 cells. These results suggest that SM-20 promotes caspase-dependent cell death through a mechanism involving cytochrome c.