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Abstract #17498 Published in IGR 9-2
Pro370Leu mutant myocilin disturbs the endoplasm reticulum stress response and mitochondrial membrane potential in human trabecular meshwork cells
Wang L; Zhuo Y; Liu B; Huang S; Hou F; Ge JMolecular Vision 2007; 13: 618-625
PURPOSE: To investigate the impact of Pro370Leu mutant myocilin on endoplasmic reticulum (ER) stress response and mitochondria function in human trabecular meshwork (HTM) cells. METHODS: HTM cells were transfected with wild-type Pro370Leu mutant myocilin or pcDNA3.1 (+) expression plasmids. The effect of the mutant myocilin on ER stress response was semiquantitatively evaluated by determining the expression level of 78 kDa glucose-regulated protein (GRP78) using reverse transcription-polymerase chain reaction and phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) using western blot analysis. Mitochondria function was determined by analyzing the changes in mitochondrial membrane potential (Δ(psi)m), measured by flow cytometry analysis using the fluorescent probe JC-1. RESULTS: Pro370Leu mutant myocilin attenuated the induction of GRP78 and the phosphorylation of eIF2α. In HTM cells expressing the mutant myocilin, the reductions were evident in the level of GRP78 mRNA (65.5 ± 2.0%), GRP78 protein (22.5 ± 2.3%), and eIF2α phosphorylation (30.6 ± 2.6%), compared to cells transfected with the wild-type myocilin plasmid (p less than or equal to 0.05). There was no significant difference between wild-type-myocilin- and pcDNA3.1(+)-transfected cells. Furthermore, Pro370Leu mutant myocilin caused a collapse of Δ(psi)m in HTM cells. CONCLUSIONS: Pro370Leu mutant myocilin down-regulates the ER stress response and destroys the Δ(psi)m of HTM cells. These observations suggest that Pro370Leu mutant myocilin could affect ER and mitochondria function through a gain of function, increasing its vulnerability to various cellular injuries and producing dysfunctional HTM cells.
Dr. J. Ge, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou 510060, China. gejian@mail.sysu.edu.cn
Classification:
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)
3.7 Biochemistry (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)