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Joshua Hirt, Paloma Borrajo Liton; New Role of Autophagy Genes in Trabecular Meshwork Mechanotransduction. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3484.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies reported the activation of autophagy in TM cells in response to high pressure and elongation. Here, we investigate the role and the mechanistic pathway triggering stretch-induced autophagy.
Human TM cells were subjected to biaxial cyclic mechanical stress (20% elongation, 1 cycle/sec) for up to 16 hours. Blockage of autophagy or chaperon assisted-selective autophagy (CASA) was achieved using siRNA targeting Atg5 and Atg7 (siAtg5/7) or BAG3 (siBAG3), respectively. Scrambled siRNA was used as control (siNC). Autophagy was evaluated by monitoring the LC3-I to LC3-II conversion by WB and via confocal imaging using the tfLC3 assay. Protein expression levels of autophagy related proteins were quantified in nuclear and cytosolic fractions. Whole-transcriptome expression profiling and alternative splicing analyses were performed using Affymetrix Clariom D arrays and analyzed with Transcriptome Analysis Console and Partek software.
Microarrays analysis revealed 240 genes and 131 genes differentially expressed (> 2-fold, p<0.05, n=3) in TM cells with inhibited autophagy or CASA, respectively. Among the genes commonly found under both conditions (total = 22) we highlight intraflagellar transport 27 (IFT27), a core component of the intraflagellar transport complex implicated in mechanosensation. Other genes showing common differential expression with siAtg5/7 and siBAG3 include the actin-binding proteins tropomyosin 4, phosphatase and actin regulator, and phospholipase C. Very interestingly, we observed for the first time the nuclear expression of the autophagosome marker LC3, as well as Atg7, p62 and BAG3 in TM cells. No endosomal or lysosomal markers were detected in the nuclear fractions. Moreover, the levels of nuclear LC3, in particular LC3-II significantly increased in response to mechanical stretch in an Atg5-indepependent manner.
We have conducte the first whole-transcriptome expression profiling of TM cells with blocked autophagy. Our results indicate a role of stretch-induced autophagy in actin cytoskeleton and primary cilia function. Intriguingly, our data demonstrates for the first time the stretch-induced nuclear translocation of LC3. We hypothesize a role of LC3 in regulating cell signaling in TM mechanotransduction. We further hypothesize that dysregulation of this response contributes to the loss of TM homeostasis in the glaucomatous outflow pathway.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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