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M. Joe, S. I. Tomarev; Expression of Myocilin Mutants Sensitizes Cells to Oxidative Stress-Induced Apoptosis: Implications for Glaucoma Pathogenesis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3224.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the pathogenic mechanisms of myocilin-induced glaucoma, which are still largely unknown
Stably transfected HEK293 cell lines expressing wild-type or mutant (Y437H and I477N) myocilin under an inducible promoter were produced. These cell lines were treated with different concentrations (100 to 500 µM) of hydrogen peroxide for 24 hours. Unfolded protein response was analyzed by Western blotting using antibodies against corresponding markers. Reactive oxygen species were measured after treatment of cells with 5 µM 2'7'-dichlorodihydrofluorescein diacetate. Iridocorneal angle tissues from wild-type mice or transgenic mice expressing Y437H mutant myocilin were analyzed by Western blotting and fluorescence immunohistochemistry.
Expression of two mutant myocilins led to different levels of endoplasmic reticulum (ER) stress and increased apoptosis after treatment of cells with 100 µM hydrogen peroxide. The Y437H mutant myocilin cell line showed the highest sensitivity to the oxidant treatment. Several antioxidant genes were downregulated in the Y437H mutant myocilin cell line, but not in other cell lines. The Y437H mutant myocilin cell line also produced more reactive oxygen species than other cell lines examined. Consistent with the data obtained in cultured cells, the ER stress marker 78 kDa glucose-regulated protein was up-regulated, while antioxidant proteins paraoxonase 2 and glutathione peroxidase 3 were down-regulated in the eye angle tissue of aged transgenic mice expressing the Y437H myocilin mutant. In addition, the pro-apoptotic factor CCAAT/enhancer-binding protein-homologous protein was up-regulated in the aged transgenic mouse angle tissue.
Our results suggest that expression of mutated myocilins may have a sensitization effect which can lead to a severe phenotype in combination with oxidative stress. Different myocilin mutants may confer various sensitivities to oxidative stress depending upon the mutation.
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