Wnt Activation After Inhibition Restores Trabecular Meshwork Cells Toward a Normal Phenotype

Purpose: Wnt is a spatiotemporally regulated signaling pathway whose inhibition is associated with glaucoma, elevated intraocular pressure (IOP), and cell stiffening. Whether such changes are permanent or may be reversed is unclear. Here, we determine if activation of Wnt pathway after inhibition reverses the pathologic phenotype.

Methods: Primary human trabecular meshwork (hTM) cells from nonglaucomatous donors were cultured for 12 days in the absence or presence of Wnt modulators: (i) LGK974 (Porcn inhibitor, 10 µM); (ii) LY2090314 (pGSK3β inhibitor, 250 nM); or (iii) 9 days of LGK974 followed by 3 days of LY2090314. Wnt modulation were determined by Western blotting and extracellular matrix (ECM) related genes were evaluated by quantitative PCR. Cytoskeletal morphology was determined by immunofluorescence and cell stiffness by atomic force microscopy.

Results: Wnt activation was confirmed by downregulation of pGSK3β (0.3-fold; P < 0.01), overexpression of AXIN2 (6.7-fold; P < 0.001), and LEF1 (3.8-fold; P < 0.001). Wnt inhibition resulted in dramatic changes in F-actin, which were resolved with subsequent Wnt activation. Concurrently, cell stiffness that was elevated with Wnt inhibition (11.86 kPa; P < 0.01) decreased with subsequent Wnt activation (4.195 kPa; P < 0.01) accompanied by significant overexpression of phosphorylated YAP (1.8-fold; P < 0.001) and TAZ (1.4-fold; P < 0.001). Additionally, Wnt activation after inhibition significantly repressed ECM genes (SPARC and CTGF, P < 0.01), cross-linking genes (LOX and TGM2, P < 0.05), inhibitors of matrix metalloproteinases (TIMP1 and PAI1, P < 0.001), and overexpressed MMP 1/9/14 (P < 0.01).

Conclusions: These data strongly demonstrate that, in normal hTM cells, activation of the Wnt pathway reverses the pathological phenotype caused by Wnt inhibition and may thus be a viable therapeutic for lowering IOP.

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S7063 LY2090314 LY2090314 is a potent GSK-3 inhibitor for GSK-3α/β with IC50 of 1.5 nM/0.9 nM; may improve the efficacy of platinum-based chemotherapy regimens. LY2090314 is highly selective towards GSK3 as demonstrated by its fold selectivity relative to a large panel of kinases.

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