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Paloma Borrajo Liton, Joshua Hirt; Autophagy and Mechanotransduction in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3257. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the role of stretch-induced autophagy and chaperone-assisted autophagy (CASA) in repair and mechanotransduction in trabecular meshwork (TM) cells.
Primary cultures of human TM cells were subjected to biaxial static (20% elongation) or cyclic mechanical (20% elongation, 1 cycle/sec) stress for 16 hours. Blockage of autophagy or CASA was achieved using siAtg5/7 or siBAG3, respectively. Scrambled siRNA was used as control. Autophagy was evaluated by monitoring the LC3-I to LC3-II conversion. Protein expression levels were quantified by WB analysis using specific antibodies against Atg5, Atg7, LC3B, BAG3, filamin A, and YAP. mRNA expression levels of BAG3, YAP, TAZ and filamin A were monitored by qPCR. Rapamycin (1 μM), bafilomycin A1 (100 nM), and 3-MA (10 mM) were used to pharmacologically modulate the autophagic pathway. Inhibition of protein synthesis was achieved by cycloheximide (25 μM).
WB analysis showed increased levels of LC3-II in TM cells subjected to either static (1.909±0.054 fold; p<0.01 fold) or cyclic mechanical stress (2.1±0.15 fold; p<0.01 fold). Downregulation of BAG3 did not alter the increase in LC3-II nor the protein levels of filamin A in response to static stretch. In contrast, the increase in LC3-II in the cyclically stretched cultures was blocked with siBAG3, siAtg5/7, or 3-MA (1.25±0.03, 1.12±0.01, 1.06±0.002 fold, respectively, compared to 2.1±0.15 fold, p<0.01). No changes were observed, however, with the mTOR-dependent activator of autophagy rapamycin. Cells exposed to cyclic mechanical stress also displayed variations in the mRNA and protein levels of several CASA components (BAG3, and FLNA), as well as those of the mechanotransducers YAP/TAZ, compared to non-stretched ones.
Our results indicate a differential activation of autophagy in TM cells in response to static stress or cyclic stress. While activation of autophagy in response to static strain was found to be BAG3-independent, activation of autophagy with cyclic mechanical stress was partially mediated by CASA, a tension-induced type of autophagy essential for mechanotransduction. Based on this, we hypothesize that autophagy is part of an integrated response triggered in TM cells in response to strain, and exerts a dual role in repair and mechanotransduction. We further hypothesize that dysregulation of this response contributes to the increased stiffness reported in the glaucomatous outflow pathway.
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