Pro370Leu mutant myocilin disturbs the endoplasm reticulum stress response and mitochondrial membrane potential in human trabecular meshwork cells.
OBJECTIVE
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.
Wang
Lina
L
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China. wanglnok@yahoo.com.cn <wanglnok@yahoo.com.cn>
Zhuo
Yehong
Y
Liu
Bingqian
B
Huang
Shengsong
S
Hou
Fei
F
Ge
Jian
J
D000328:Adult; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics* / Q000494:pharmacology; D006023:Glycoproteins / Q000235:genetics* / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007930:Leucine; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011392:Proline; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D013312:Stress, Physiological / Q000235:genetics / Q000503:physiopathology*; D014129:Trabecular Meshwork / Q000473:pathology / Q000503:physiopathology*; D014162:Transfection; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2; D005136:Eye Proteins; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D007930:Leucine; D018832:Molecular Chaperones; D011392:Proline; D012333:RNA, Messenger; D000099179:Myocilin; C000717247:HSPA5 protein, human