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W. Mao, R. J. Wordinger, A. F. Clark; The Existence of a Canonical Wnt Signaling Pathway in Gtm-3 Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4884.
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Increased intraocular pressure (IOP) is the primary risk factor of glaucoma, and we have previously shown that the canonical Wnt signaling pathway plays a role in regulating IOP. In order to better understand Wnt signaling in the trabecular meshwork (TM), we investigated whether a similar canonical Wnt signaling pathway exists in GTM-3 cells, a transformed glaucomatous TM cell line widely used in glaucoma research.
The Wnt signaling pathway in GTM-3 cells was examined using a TCF dual-luciferase reporter gene assay, and we determined the effects of Wnt signaling on the endogenous ß-catenin levels with Western immunoblots. For the dual luciferase assay, reporter vectors containing TCF cis-elements were transfected into GTM-3 cells, which were then treated with recombinant Wnt3a, Wnt5a with or without SFRP-1 at different concentrations. For Western blots, nuclear and cytoplasmic proteins were extracted from GTM-3 cells treated with or without Wnt3a and/or SFRP-1 for 4 hours. ß-catenin, Lamin A/C and GAPDH (nuclear and cytoplasmic protein loading controls, respectively) were probed with corresponding antibodies.
The dual-luciferase assay showed that Wnt3a dose-dependently induced luciferase expression in GTM-3 cells. The normalized relative luminescence unit (RLU) in GTM-3 cells increased 10 fold with 100ng/ml Wnt3a and 40 fold with 500ng/ml Wnt3a (both p<0.01). In contrast, Wnt5a only regulated luciferase expression with low efficacy. Treating GTM-3 cells with SFRP-1, an inhibitor of Wnt pathway, dose-dependently blocked the effect of Wnt3a with complete inhibition at 10ug/ml (p<0.01). Wnt3a caused the accumulation of endogenous ß-catenin in the nucleus and cytoplasm in GTM-3 cells, and this accumulation was also inhibited by SFRP-1.
GTM-3 cells have a canonical Wnt signaling pathway. These cells will be useful in further examining Wnt/β-catenin signaling and identifying Wnt regulated genes in the TM.
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