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Haiyan Li, Haven Roberts, Daniel W Stamer, Preethi S Ganapathy, Samuel Herberg; TGFβ2 modulates YAP/TAZ activity in human trabecular meshwork cells through ERK and ROCK signaling pathways. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1657.
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Primary open angle glaucoma (POAG) is associated with increased trabecular meshwork (TM) stiffness and elevated levels of transforming growth factor beta2 (TGFβ2) in the aqueous humor. YAP/TAZ are important mechanotransducers and have been implicated in the pathogenesis of POAG. However, the molecular underpinnings of YAP/TAZ activity modulation in TM cells under glaucomatous conditions are not well understood. The purpose of this study is to elucidate how TGFβ2 and substratum stiffness regulate YAP/TAZ in human TM (HTM) cells using viscoelastic biomimetic hydrogels.
Primary HTM/glaucomatous HTM (GTM) cells were isolated from surgical discard corneal rims/donor POAG globe and validated using accepted protocols. HTM/GTM cells were plated on soft photocrosslinked hydrogels (collagen I, elastin-like polypeptide and hyaluronic acid) or stiff glass coverslips/tissue culture polystyrene and stimulated with 2.5 ng/ml TGFβ2 ± ERK inhibitor (U0126; 10 µM)/ROCK inhibitor (Y27632; 10 µM). YAP/TAZ were knocked down in HTM cells using siRNA. Vinculin, YAP/TAZ, transglutaminase 2 (TGM2), TGFβ2, fibronectin (FN), alpha-smooth muscle actin (α-SMA) and phospho-myosin light chain (p-MLC) expression were determined by qRT-PCR, immunoblotting and immunocytochemistry analyses.
HTM cells displayed increased vinculin puncta density, cell spreading and YAP/TAZ nuclear localization/activation when cultured on stiff substratum vs. soft hydrogels. On hydrogels, TGFβ2 exposure induced a more rounded HTM cell shape with decreased nuclear aspect ratio (p<0.001). GTM cells and TGFβ2-induced HTM cells showed decreased p-YAP (cytoplasmic; inactive) and increased nuclear YAP/TAZ (active) together with increased downstream TGM2 vs. normal HTM cells. U0126 completely abolished TGFβ2-induced YAP/TAZ expression and nuclear translocation as well as TGM2 activation. Y27632 slightly rescued TGFβ2-induced YAP/TAZ activation. YAP/TAZ depletion using siRNA resulted in decreased expression of TGM2, TGFβ2, FN, α-SMA and p-MLC (p<0.001), all associated with cell contractility and tissue stiffening.
Our data suggest that substratum stiffness and TGFβ2 modulate YAP/TAZ activity in HTM cells under simulated POAG conditions to varying degrees through ERK and ROCK signaling pathways. YAP/TAZ play critical roles in regulating HTM cell contractility associated with HTM stiffness and POAG pathogenesis.
This is a 2021 ARVO Annual Meeting abstract.
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