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Tatsuo Itakura, M. Elizabeth Fini; Myocilin Mutations Activate the IL-1 Stress Response. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4641.
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Our larger collaborative team has reported that activation of an ELAM-1/IL-1/NF-kappaB stress pathway in the trabecular meshwork (TM) is a marker for high tension glaucomas of diverse etiology and protects against oxidative stress (Wang et al., Nature Medicine, 2001). Myocilin (MYOC) mutations are causatively linked to high tension forms of primary open angle glaucoma (POAG). In this study, we investigated a possible connection between stress pathway activation and myocilin glaucoma.
We constructed MYOC expression vectors with mutations at sites that cause POAG (Q368X and Y437H). The double tagged (Myc and FLAG) constructs were made in two different kinds of vectors; one for standard transfection (pCMV6), and one for lentivirus infection (pSLIK). Mutants and wild type MYOCs were individually expressed in cells of the human trabecular meshwork line TM-1 (a gift from Dr. Donna Peters). Subsequent changes in gene expression were quantified by qRT-PCR and western blotting.
Transfection of TM-1 cells with mutant MYOC genes caused a high degree of cell death as compared to wild-type MYOC when introduced at high DNA concentrations. Over-expressed wild-type MYOC was secreted into cell culture media whereas mutants MYOCs remained within cells. These findings are confirmatory of published reports. Wild-type MYOC significantly decreased expression of the IL-1-regulated gene IL-6, as well as another glaucoma associated gene TGF-beta2, to approximately half of the control levels. Expression of IL-1beta, as well as the TGF-beta-regulated gene alpha-smooth muscle actin (SMA), was also slightly decreased. In contrast, mutant MYOCs significantly increased expression of IL-1beta, SMA, and the oxidative stress gene FOXO1 by 1.5 to 2-fold. The level of expression of these genes tended to correlate with the degree of glaucoma phenotype (Q368X <Y437H).
These findings connect an additional high tension form of glaucoma - myocilin glaucoma - to our previous findings on an ELAM-1/IL-1/NF-kappaB-regulated stress pathway diagnostic of high tension glaucomas. This provides further evidence that a common mechanism is operative in the pathophysiology of high tension glaucomas.
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