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

Abstract

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.

Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating β-catenin and YAP/TAZ Signaling Pathways

“…Finally, because 10% XCDM stiffened hTM cells in correlation with inactivating the Wnt pathway, and antagonism of this pathway had previously been implicated in hTM cell stiffening, 50 we explored whether activation of this pathway could rescue hTM cells from 10% XCDM-induced stiffening. Intriguingly, canonical Wnt pathway activation (via inhibition of GSK3β) abrogated 10% XCDM-dependent stiffening of hTM cells, consistent with a recent study from our group, 80 where canonical Wnt pathway activation reversed the pro-fibrotic effects (for instance, cell stiffening) of Wnt pathway inhibition in hTM cells seeded on glass/plastic substrates.…”

“… 71 , 77 Correspondingly, in hTM cells cultured on 10% XCDM, at an early time point (24 hours), we observed reduced expression of critical target genes and proteins of Wnt/β-catenin pathway like Axin2, CD44, total fibronectin, and fibronectin EDA isoform. Whereas Wnt antagonism has been shown to be implicated in POAG, elevated intraocular pressure, 49 and cell stiffening, 50 activation of this pathway may have a therapeutic benefit by inhibiting transforming growth factor beta 2 (TGFβ2) signaling pathway, 69 a well-established profibrotic pathway in POAG, 78 , 79 reducing the expression of profibrotic ECM proteins, 70 , 80 and/or regulating cell-cell contacts via K-cadherin expression. 51 Regarding the latter, cadherins’ role in maintaining cell-cell adhesion is partly to allow for their mechanosensitive/mechanotransduction function, 81 – 83 together with formation of gap junctions to enhance cell-cell communication.…”

“…Intersecting the Wnt/β-catenin signaling pathway are YAP and TAZ, 38 – 44 whose mechanotransduction function conventionally involves their subsequent nuclear translocation, following activation of the actin cytoskeleton or actomyosin machinery. 34 , 52 , 80 Several studies have reported inhibitory crosstalk between canonical Wnt/β-catenin and Hippo/YAP/TAZ pathways in non-ocular cells/tissues, in response to chemical ligands. In these previous studies, YAP/TAZ pathway inhibited the Wnt pathway via the following mechanisms: (i) direct binding of Hippo-induced inactivated YAP or TAZ to either β-catenin or disheveled in the cytoplasm, 38 , 39 (ii) prevention of β-catenin’ nuclear translocation, 38 (iii) direct binding between YAP and β-catenin in the nucleus, 43 and/or (iv) RhoA/ROCK-mediated activation of YAP/TAZ to promote expression of Wnt antagonists.…”

The Canonical Wnt Signaling Pathway Inhibits the Glucocorticoid Receptor Signaling Pathway in the Trabecular Meshwork

“…Using this mouse model, a great reduction of ocular hypertension with concomitant Wnt3a expression was observed, suggesting activation of canonical Wnt signaling could be a potential therapeutic strategy for GC-induced ocular hypertension prevention and/or treatment. Recently, Dhamodaran et al 55 demonstrated in vitro that another GSK3b inhibitor softens primary TM cells as well as inhibits the expression of ECM, cross-linkers of ECM, and inhibitors of matrix metalloproteinases. Although the changes in genes involved in ECM remodeling were not determined in that study, their roles in ocular hypertension prevention were inferred in that study.…”

Cannabidiol and the Canonical WNT/β-Catenin Pathway in Glaucoma

“…Moreover, the cotreatment with a small-molecule WNT pathway activator can downregulate sFRP1-induced OHT in mouse eyes. The activation of WNT/β-catenin pathway in the TM using lithium chloride decreases the production of some ECM and matricellular proteins [ 125 , 126 ]. WNT/β-catenin signaling and K-cadherin expression are major for the control of IOP, and the downregulation of this pathway leads to IOP elevation in glaucoma [ 127 ].…”