Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

Abstract

Purpose : We previously reported autophagy activation in primary-cultured human trabecular meshwork (TM) cells upon mechanical stress. Here we investigate its mechanosensor and downstream signaling pathway, as well as its role in IOP homeostasis.

Methods : Human TM cells were subjected to cyclic mechanical stretch (CMS, 8% elongation, 1 cycle/sec) for up to 24 h. Autophagy activation was monitored by measuring LC3-II and p62 levels or LC3 puncta formation using western blot or confocal microscopy. Primary cilia (PC) were disrupted by chloral hydrate (CH, 4 mM for 3 day). Ca²⁺ channels were inhibited with 10 µM of amiloride, amlodipine and HC067047. Hedgehog signaling was inhibited by cyclopamine (10 µM). TGFβ and AKT signaling were inhibited chemically (10 µM of LY2109761 or SB431542 for TGFβ and 4 µg/ml of SC66 for AKT) or genetically (siRNAs targeting SMAD2/3 and AKT1). The effect of deciliation on outflow facility were measured in pig eyes by using iPerfusion system.

Results : Primary cilia disruption with CH abolished autophagy induction, as evaluated by LC3-II and p62 levels (0.23 ±0.18 and 0.63± 0.31fold, p<0.05, n=5 and 3, respectively) in CMS cells. Inhibiting Ca²⁺ channels or hedgehog signaling had no effect on it. In contrast, SMAD2/3 knock-down reduced the CMS-induced increase in LC3-II level by approximately 3 folds (p<0.01, n=3). However, similar effect was not observed by the inhibition of TGFβ receptors. Intriguingly, AKT inhibition increased LC3-II levels, and the levels were significantly lowered by 1.5~2 folds (p<0.01, n=3) together with SMAD2/3 knock-down in both NS and CMS cells. Furthermore, knock-down of SMAD2/3 or AKT1 reciprocally decreases AKT1 phosphorylation at S473 or SMAD2/3 protein level. In addition, deciliated cells showed higher levels of pAKT1/AKT in both NS (2.36 ± 0.57 folds, p<0.01, n=6) and CMS (3.42 ± 2.14 folds, p<0.05, n=6). Finally, removal of PC disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge in pig eye (CNT VS deciliated; 2.55 ± 0.14 VS 1.31 ± 0.44 folds, p<0.05, n=3).

Conclusions : Our results strongly indicate that PC act as a mechanosensor for CMS-induced autophagy, and a novel cross-regulatory talk between AKT1 and SMAD2/3 signaling is critical components for its mechanism of action, which play a role in regulating IOP homeostasis.

This is a 2021 ARVO Annual Meeting abstract.