Oxidative stress-induced apoptosis in granulosa cells involves JNK, p53 and Puma

Abstract

Reactive oxygen species (ROS) play important roles in follicular development and survival. Granulosa cell death is associated with increased ROS, but the mechanism of granulosa cell death induced by ROS is not clear. In order to define the molecular link between ROS and granulosa cell death, COV434, human granulosa tumor cells, were treated with H ₂O ₂. Compared to control cells, H ₂O ₂ induced granulosa cell death in a dose- and time-dependent manner. H ₂O ₂ induced an increase in Bax, Bak and Puma, and a decrease in anti-apoptotic molecules such as Bcl-2, Bcl-xL and Mcl-1. Both knockdown of Puma and overexpression of Bcl-xL could inhibit H ₂O ₂-induced granulosa cell death. These results suggest that suppression of Puma and overexpression of anti-apoptotic Bcl-2 family members could improve granulosa cell survival. To explore the mechanisms responsible for these findings, ROS in granulosa cells treatment with H ₂O ₂ were measured. The results showed that ROS was increased in a H ₂O ₂ dose- and time-dependent manner at the earlier time point. In addition, H ₂O ₂ induced an increase in Nrf2 and phosphorylation of JNK and p53. SP600125, an inhibitor of JNK, inhibits H ₂O ₂-induced phosphorylation of JNK and p53, and granulosa cell death. Antioxidant N-acetylcysteine (NAC) dose-dependently prevents H ₂O ₂-induced granulosa cell death. Furthermore, NAC also prevents phosphorylation of JNK and p53 induced by H ₂O ₂. Taken together, these data suggest that H ₂O ₂ regulates cell death in granulosa cells via the ROS-JNK-p53 pathway. These findings provide an improved understanding of the mechanisms underlying granulosa cell apoptosis, which could potentially be useful for future clinical applications.