Abstract
Purpose :
To investigate the effects of dysregulated autophagy in TM cell and tissue physiology.
Methods :
Three independent primary cultures of human TM were transfected with siRNA targeting the autophagy genes Atg5 and Atg7. Scrambled siRNA was used as control. Whole-transcriptome expression profiling was performed from total RNA using Affymetrix Clariom D arrays. Gene and functional analyses were conducted using Partek Flow and Metacore software. Gene array data was confirmed by qPCR, ELISA and/or WB. Treatment of hTM cells with TGFβ1 and TGFβ2 was performed during indicated times (0, 24, 48 hours) at increasing concentrations (0-10 ng/mL). Autophagy was assesed by WB, confocal imaging using the tfLC3 assay, and by electron microcopy. ERK, p38 and JNK pathways were blocked with PD98059, SB203580, and SP600125 inhibitors, respectively. The Smad2/3 signaling pathway was silenced via siRNA transfection by lipofectamine. Intracellular and extracellular ECM content (collagen I, fibronectin) were quantified by WB and ELISA. ECM degradation was monitored by flow cytometry using DQ-ECM substrates.
Results :
Gene expression profile analysis revealed the upregulated expression (> 2-fold, p<0.05, n=3) of TGFβ2 and the pseudoreceptor BAMBI in siAtg5/7-transfected hTM cells compared to siNC controls. Functional analysis identified regulation of endothelial to mesenchymal transition (TGFβ-mediated), as one of the cellular pathways most preferentially affected in human TM cells with blocked autophagy function. Treatment of human TM cells with TGFβ triggered activation of autophagy in a concentration-dependent manner, reaching a peak at 48 h post-treatment with 10 ng/mL of the cytokines (TGFβ1: 158% ± 19%, TGFβ2: 142% ± 11%, p<0.05, n=4). Activation of autophagy by TGFβ was significantly reduced in siSmad2/3-transfected cells (81% ± 7% and 75% ± 10% reduction, TGFβ1 and TGFβ2, respectively, p<0.05, n=4). p38, ERK, and JNK inhibitors did not cause any significant effect. Higher rates of ECM degradation were observed in the presence of activators of autophagy. Autophagy inhibitors, in contrast, negatively affected ECM degradation elevating ECM content
Conclusions :
Our results show for the first-time a cross-talk between autophagy and TGFβ signaling in human TM cells. We hypothesize autophagy to play a critical role in counteracting TGFβ-induced fibrogenesis and endothelial to mesenchymal transition in TM cells.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.