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

Investigative Ophthalmology & Visual Science
Kamesh DhamodaranVijayKrishna Raghunathan

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. 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. 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...Continue Reading

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Citations

Aug 25, 2020·Investigative Ophthalmology & Visual Science·Felix YemanyiVijay Krishna Raghunathan
Mar 12, 2021·The American Journal of Pathology·Chenna Kesavulu SugaliWeiming Mao
Mar 18, 2021·The Application of Clinical Genetics·Altaf A Kondkar
Aug 17, 2020·Progress in Retinal and Eye Research·Ted S AcottMary J Kelley

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Methods Mentioned

BETA
AFM
nuclear translocation
atomic force microscopy
protein assay
PCR
GTPases
deamination
acylation

Software Mentioned

ImageJ

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