Pro370Leu mutant myocilin disturbs the endoplasm reticulum stressresponse and mitochondrial membrane potential in human trabecularmeshwork cells

摘要

Purpose: To investigate the impact of Pro370Leu mutant myocilin onendoplasmic reticulum (ER) stress response and mitochondria function inhuman trabecular meshwork (HTM) cells.Methods: HTM cells were transfected with wild-type Pro370Leu mutantmyocilin or pcDNA3.1 (+) expression plasmids. The effect of the mutantmyocilin on ER stress response was semiquantitatively evaluated bydetermining the expression level of 78 kDa glucose-regulated protein(GRP78) using reverse transcription-polymerase chain reaction andphosphorylation of the α subunit of eukaryotic translationinitiation factor 2 (eIF2α) using western blot analysis.Mitochondria function was determined by analyzing the changes inmitochondrial membrane potential (Δψm), measured by flowcytometry analysis using the fluorescent probe JC-1.Results: Pro370Leu mutant myocilin attenuated the induction of GRP78and the phosphorylation of eIF2α. In HTM cells expressing themutant myocilin, the reductions were evident in the level of GRP78 mRNA(65.5±2.0%), GRP78 protein (22.5±2.3%), and eIF2αphosphorylation (30.6±2.6%), compared to cells transfected with thewild-type myocilin plasmid (p less than or equal to 0.05). There was nosignificant difference between wild-type-myocilin- andpcDNA3.1(+)-transfected cells. Furthermore, Pro370Leu mutant myocilincaused a collapse of Δψm in HTM cells.Conclusions: Pro370Leu mutant myocilin down-regulates the ER stressresponse and destroys the Δψm of HTM cells. Theseobservations suggest that Pro370Leu mutant myocilin could affect ER andmitochondria function through a gain of function, increasing itsvulnerability to various cellular injuries and producing dysfunctionalHTM cells.