c‐Maf increases apoptosis in peripheral CD8 cells by transactivating Caspase 6

In addition to transactivation of interleukin‐4 (IL‐4), cellular muscular aponeurotic fibrosarcoma (c‐Maf) enhances CD4 cell apoptosis by limiting Bcl‐2 expression. The CD8 cells also express c‐Maf and peripheral CD8 cell numbers are reduced in c‐Maf transgenic mice, suggesting that c‐Maf may influence CD8 cell survival in a manner similar to CD4 cells. Here we confirm that, similar to CD4 cells, c‐Maf enhances CD8 cell susceptibility to apoptosis induced by multiple stimuli, independent of IL‐4. However, unlike CD4 cells, c‐Maf enhancement of apoptosis is independent of Bcl‐2, suggesting that c‐Maf uses other mechanisms to regulate CD8 cell apoptosis. Real‐time reverse transcription–polymerase chain reaction reveals that the pro‐apoptotic gene Caspase 6 is upregulated in c‐Maf transgenic CD8 cells, suggesting that Caspase 6 is a novel c‐Maf target gene. Luciferase reporter assays and site‐directed mutagenesis reveal a functional c‐Maf recognition element (MARE) within the first intron of Caspase 6. Binding of c‐Maf to the MARE site is detectable by chromatin immunoprecipitation using non‐transgenic T‐cell lysates, so c‐Maf can interact with the Caspase 6 MARE site in normal T cells. Furthermore, caspase 6 activity is increased among CD8 cells from c‐Maf transgenic mice following T‐cell receptor engagement. As expected, activity of the downstream caspases 3 and 7 is also increased. Consistent with the ability of caspase 6 to participate in positive feedback loops, cytochrome c release and caspase 8 activation are also increased. Together these results indicated that c‐Maf increases CD8 cell sensitivity to apoptotic stimuli, at least in part, by direct transactivation of Caspase 6, providing increased substrate for Caspase 6‐dependent apoptosis pathways.