MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch

Abstract

The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30mmHg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.

Keywords: Autophagy; Chaperon-assisted autophagy; Glaucoma; MTOR pathway; Mechanical stress; Trabecular meshwork.

Figure 1

LC3-I to LC3-II turnover in human TM cells subjected to static mechanical stress. (A) Protein expression levels of LC3-I and LC3-II in primary cultures of human TM cells subjected to static mechanical stress (20% elongation) for 30 min, 1 hour or 16 hours, evaluated by WB analysis. TUBB/β-tubulin was used as loading control. (B) Normalized fold-changes of LC3B-II in stretched cultures compared to non-stretched cultures calculated from densitometric analysis of the blots. (C) Expression levels of LC3-I and LC3-II in TM cells mechanically stressed (static, 20% elongation) treated with CQ (30 nM), added during the last hour of the stretching. Data are mean ± SD, n=3, **p < 0.001, *** p < 0.0001, one-way ANOVA with Bonferroni post hoc test.

Figure 2

Autophagy inhibition in mechanically stressed TM cells. (A) Protein expression levels of LC3-I and LC3-II in TM cells subjected to static mechanical stress (20% elongation, 1hour) in the presence of 3-MA (10 mM) added to the culture media one hour prior to stretching, evaluated by WB analysis. TUBB was used as loading control. Blots are representative from four independent experiments. (B) Normalized relative protein levels of LC3B-II calculated from densitometric analysis of the blots. Data are the means ± SD, n = 4, ***p < 0.001, t-test.

Figure 3

Expression levels of proteins participating in the induction of autophagy or autophagic flux in mechanically stretched TM cells. (A) Representative immunoblots using the indicated specific antibodies. (B) Normalized relative protein levels calculated from densitometric analysis of the blots. Data are the means ± SD, n = 4, *p<0.05, **p < 0.001, Two-way ANOVA followed by Bonferroni's post hoc test.

Figure 4

Autophagic flux in mechanically stressed TM cells evaluated by tfLC3: Primary cultures of TM cells were infected with AdtfLC3 (m.o.i = 10 pfu/cell). At 2 d.p.i., cells were subjected to mechanical stress (20% elongation, 1 hour). (A) Representative confocal images showing the presence of yellow and red puncta in non-stretched and stretched cells. Bottom panels represents the top panel pictures processed with the Find Edges tools in Image J to facilitate manual counting. (B) Number of yellow and red puncta per cell. (C) Autophagic flux measurement as a ratio of red/yellow puncta per cell. (D) Size of yellow and red puncta. Data are means ± SD of three independent experiments, ten cells/experiment, n = 30, *p<0.05, **p < 0.00, *** p < 0.0001, t-test.

Figure 5

Ultrastructural appearance of TM cells exposed to static mechanical stress. (A) Non-stretched cells. (B) Stretched cells. (C) Stretched cells at higher magnification. Note the intracellular presence of autophagic structures (B, representative examples marked as *) in the cells subjected to mechanical stress identified as initial vacuoles (C, AVi) and degradative vacuoles (C, AVd). Pictures are representative of three independent experiments.

Figure 6

MTOR pathway in mechanically stressed TM cells. (A) Protein levels of phosphorylated and non-phosphorylated downstream target of MTOR, RPS6KB. (B) Normalized relative protein levels of pRPS6KB calculated from densitometric analysis of the blots. Data are the means ± SD, n = 3, **p < 0.01, t-test. NS: non-stretched; S: stretched.

Figure 7

Chaperon-assisted autophagy in TM cells under static mechanical stress: Protein levels of the CASA components FLNA and BAG3, as well as the autophagic marker LC3-II in (A) HTM cells subjected to static stretch (20% elongation, 16 hours) in the presence of BafA1 (100 nM) or Chx (25 μM) added to the culture media 30 minutes prior to stretching; (B) NTM cells transfected with siBAG3. TUBB was used as loading control. Blots are representative from three independent experiments.

Figure 8

Induction of Autophagy in High Pressure-Perfused Eyes. Enucleated porcine eyes were perfused for one hour at either normal physiological pressure (8 mmHg equivalent to 15 mmHg in vivo), or at high pressure (fellow eye, 30 mmHg). (A) Protein expression levels of LC3-I and LC3-II. (B) Normalized relative protein levels of LC3B-II calculated from densitometric analysis of the blots. (C) Electron micrographs showing the presence of autophagic figures (arrowheads) in the cells of the corneoscleral meshwork of eyes perfused at high pressure.