The effects and regulatory mechanism of RIP3 on RGC-5 necroptosis following elevated hydrostatic pressure

• Published in: Acta biochimica et biophysica Sinica 2017-02, Vol.49 (2), p.128-137
• Main Authors: Shang, Lei, Ding, Wei, Li, Na, Liao, Lvshuang, Chen, Dan, Huang, Jufang, Xiong, Kun
• Format: Article
• Language: English
• Subjects: Animals; Antioxidants - pharmacology; Apoptosis - physiology; Blotting, Western; blot分析; Butylated Hydroxyanisole - pharmacology; Caspase 8 - metabolism; Cell Line; Flow Cytometry; Glycogen Phosphorylase - metabolism; Hydrostatic Pressure; Malondialdehyde - metabolism; Mice; Necrosis; Reactive Oxygen Species - antagonists & inhibitors; Reactive Oxygen Species - metabolism; Receptor-Interacting Protein Serine-Threonine Kinases - genetics; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; Retinal Ganglion Cells - drug effects; Retinal Ganglion Cells - metabolism; RNA Interference; Up-Regulation; 压力升高; 流式细胞仪分析; 细胞死亡; 裂解产物; 视网膜神经节细胞; 调控机制; 静水
• ISSN: 1672-9145; 1745-7270
• DOI: 10.1093/abbs/gmw130

Necroptosis is a type of regulated cell death that has been implicated in various diseases. Receptor-interacting protein 3 （RIP3）, a member of the RIP family, is an important mediator of the necroptotic pathway. Cleavage of RIP3 at Asp328 by caspase-8 abolishes the kinase activity of RIP3, which is critical for necroptosis. Moreover, RIP3 is significantly upregulated during the early stages of acute high intra-ocular pressure and oxygen glucose deprivation. In this study, the effects of RIP3 during elevated hydrostatic pressure （EHP） were investigated and the possible mechanism through which caspase-8 regulated RIP3 cleavage was explored. Flow cytometry ana- lysis revealed that the number of EHP-induced necrotic retinal ganglion cell 5 （RGC-5） cells was reduced after RIP3-knockdown. Furthermore, malondialdehyde （MDA） levels and glycogen phos- phorylase （PYGL） activity in normal RGC-5 cells were much higher than those in RIP3-knockdown cells after EHP. EHP-induced RGC-5 necrosis was significantly reduced after treatment with buty- lated hydroxyanisole （BHA）, a reactive oxygen species （ROS） scavenger. MDA levels and PYGL activity were lower in normal RGC-5 cells than those in cells with caspase-8 inhibition after EHP. Western blot analysis demonstrated that the RIP3 cleavage product was upregulated in cells with caspase-8 inhibition. Additionally, flow cytometry analysis revealed that the number of EHP- induced necrotic RGC-5 cells was increased after caspase-8 inhibition. Our results suggested that RGC-5 necroptosis following EHP was mediated by RIP3 through induction of PYGL activity and subsequent ROS accumulation, Thus, caspase-8 may participate in the regulation of RGC-5 necroptosis via RIP3 cleavage.