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Pro370Leu mutant myocilin disturbs the endoplasm reticulum stress response and mitochondrial membrane potential in human trabecular meshwork cells.

ABSTRACT: 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 alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) using western blot analysis. Mitochondria function was determined by analyzing the changes in mitochondrial membrane potential (Deltapsim), measured by flow cytometry analysis using the fluorescent probe JC-1. RESULTS: Pro370Leu mutant myocilin attenuated the induction of GRP78 and the phosphorylation of eIF2alpha. 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 eIF2alpha 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 Deltapsim in HTM cells. CONCLUSIONS: Pro370Leu mutant myocilin down-regulates the ER stress response and destroys the Deltapsim 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.

SUBMITTER: Wang L

PROVIDER: S-EPMC2669509 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Pro370Leu mutant myocilin disturbs the endoplasm reticulum stress response and mitochondrial membrane potential in human trabecular meshwork cells.

Wang Lina L Zhuo Yehong Y Liu Bingqian B Huang Shengsong S Hou Fei F Ge Jian J

Molecular vision 20070419

<h4>Purpose</h4>To investigate the impact of Pro370Leu mutant myocilin on endoplasmic reticulum (ER) stress response and mitochondria function in human trabecular meshwork (HTM) cells.<h4>Methods</h4>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 transcriptio ...[more]

PMID: 17515882

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Project description:PurposeOxidative stress is a risk factor for the onset and progression of primary open-angle glaucoma (POAG), but the exact molecular basis remains unknown. Here, we investigated the mechanisms for Pro370Leu mutant myocilin to induce mitochondrial dysfunction and subsequent reactive oxygen species (ROS) generation in trabecular meshwork (TM) cells obtained from POAG individuals.MethodsPrimary non-diseased human TM cultures were transfected with pIRES-EGFP (Mock), pIRES-wild-type (WT), or pIRES-Pro370Leu mutant myocilin. Transfection efficiency and myocilin subcellular localization were determined by polymerase chain reaction (PCR), western blot analysis, and confocal microscopy. ROS levels as well as free Ca(2+) concentrations in cytoplasm ([Ca(2+)](c)) and mitochondria ([Ca(2+)]m) were examined by 2'7'-dichlorofluorescein diacetate (H(2)-DCF-DA), Fluo-3 acetoxymethyl ester (Fluo-3/AM), and Rhod-2 acetoxymethyl ester (rhod-2/AM), respectively, using flow cytometry. Mitochondrial functions were revealed by changes in mitochondrial membrane potential (DeltaPsim) and ATP production, which were found by fluorescent probe 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1) and a luciferin/luciferase-based ATP assay, respectively.ResultsBoth WT and Pro370Leu mutant myocilin are localized in the mitochondria of TM cells as indicated using confocal microscopy and western blot analysis. Overexpression of WT myocilin decreases DeltaPsim, which is further reduced by Pro370Leu mutant myocilin. TM cells that overexpressed Pro370Leu mutant myocilin have greater cell death, higher endogenous ROS, [Ca(2+)](c), and [Ca(2+)](m) levels, and lower ATP production, and yet, these effects are not seen in the overexpression of WT myocilin.ConclusionsOur findings suggested that Pro370Leu mutant myocilin causes mitochondrial defects, which may lead to TM cell dysfunction and even cell death. Therefore, preventive measures targeting mitochondrial protection may delay the onset of glaucoma in individuals carrying the Pro370Leu myocilin mutation.

Dataset Information

Pro370Leu mutant myocilin disturbs the endoplasm reticulum stress response and mitochondrial membrane potential in human trabecular meshwork cells.

Publications

Pro370Leu mutant myocilin disturbs the endoplasm reticulum stress response and mitochondrial membrane potential in human trabecular meshwork cells.

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