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 H₂O₂. Compared to control cells, H₂O₂ induced granulosa cell death in a dose- and time-dependent manner. H₂O₂ 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 H₂O₂-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 H₂O₂ were measured. The results showed that ROS was increased in a H₂O₂ dose- and time-dependent manner at the earlier time point. In addition, H₂O₂ induced an increase in Nrf2 and phosphorylation of JNK and p53. SP600125, an inhibitor of JNK, inhibits H₂O₂-induced phosphorylation of JNK and p53, and granulosa cell death. Antioxidant N-acetylcysteine (NAC) dose-dependently prevents H₂O₂-induced granulosa cell death. Furthermore, NAC also prevents phosphorylation of JNK and p53 induced by H₂O₂. Taken together, these data suggest that H₂O₂ 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.