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Peter Pechan, Jeffery Ardinger, Hillard Rubin, Michael Lukason, Elizabeth Barry, Seng Cheng, Abraham Scaria; Role of Myocilin Mutants and Wnt Signaling in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1973.
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© ARVO (1962-2015); The Authors (2016-present)
Non-secreted autosomal dominant mutants of Myocilin (MYOC) accumulate intracellularly and can cause an increase in intraocular pressure (IOP), retinal ganglion cell death, and optic nerve head (ONH) damage through mechanisms that are as yet unclear. In this study, we examine whether the MYOC mutants P370L or Y437H have an effect on Wnt signaling (previously implicated in glaucoma) in trabecular meshwork (TM) cells and thus contribute to the development of glaucoma.
Wnt signaling in immortalized (using SV40 T-Ag) human TM cell line (hTM-T) and HEK293T cells was evaluated using the TOPFlash luciferase assays and by Western blotting (for beta-catenin). For the TOPFlash assays, HEK 293T or hTM-T cells were co-transfected with the TOPFlash reporter plasmid and plasmids expressing wild type MYOC (wtMYOC), MYOC mutants P370L, Y437H, the Wnt agonist R-spondin 3 (RSPO3) or MYOC shRNA. Samples were treated with 400 ng/mL of recombinant human or mouse wnt3a protein and incubated for an additional 24 hours. Western blot analysis was then performed on cell lysates and conditioned medium collected at 48 hours post transfection using a mouse anti-human MYOC antibody.
Using the TOPFlash assay, we showed that the MYOC mutants P370L and Y437H, but not wtMYOC, significantly blocked Wnt signaling in 293T and in hTM-T cells. Western blot analysis demonstrated that a shRNA targeting MYOC, but not control shRNA, reduced both extracellular and intracellular wtMYOC levels in the 293T and hTM-T cells co-transfected with wtMYOC and P370L plasmids. Blockage of Wnt signaling by MYOC mutants could be restored by treatment with RSPO3 or by gene silencing of MYOC using a shRNA targeting MYOC transcripts.
Our results indicate that MYOC mutants inhibit Wnt signaling, and targeting MYOC gene expression may represent a potential therapy for some forms of glaucoma.
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