Abstract
Purpose :
The Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) signaling pathway is a pivotal cytosolic DNA sensor system in innate immunity, known to contribute to retinal degeneration. BRD4, a member of the Bromodomain and Extraterminal Domain (BET) proteins functioning as epigenetic readers, plays a crucial role as a proinflammatory factor. Our previous RNA-seq analysis indicates that OS induces an upregulation of BRD4 in ARPE-19 cells. Here, our aim is to investigate whether or not OS-induced BRD4 promotes STING expression and inflammation.
Methods :
To induce the OS-induced retinal degeneration mouse model, C57BL/6J mice (5-6 weeks old) were intraperitoneally injected with 35 mg/kg of sodium iodate (SI). Control mice received PBS. Retinas were collected at specified time points or 3 days after SI injection.To assess intracellular reactive oxygen species (ROS) and cell viability, BRD4 knockout or control ARPE-19 cell lines were constructed. FLAG or FLAG-STING was transfected into ARPE cells. After 24 hours, cells were treated with or without H2O2 (1.8 mM or 0.6 mM) for 2 hours and then allowed to recover for 12 hours before analysis. The antioxidant N-Acetylcysteine (NAC) (1 mM) was added 2 hours before H2O2 treatment and maintained throughout the experiment. Western blot and real-time quantitative analyses determined the expression of proinflammatory cytokines.
Results :
Increased BRD4 was detected in SI-injected mouse retinas and increased in a time-dependent manner. BRD4 knockdown decreased STING expression and prevented OS-induced ARPE cell death, which was reversed by STING overexpression. In line with recent report that STING loss reduces ROS and expression of the ROS-related genes, overexpression of STING promoted ROS generation and enhanced expression of the genes involved in ROS homeostasis. Finally, STING induced expression of inflammatory factors, whereas treatment of the cells with NAC represses STING-induced upregulation of IL6 and IFNβ.
Conclusions :
Our results indicate that OS-induced BRD4 activates STING and hence ROS production and promotes inflammatory response. Our findings suggest that BRD4 may be a potential drug target for retinal degeneration and inflammation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.