Abstract
Purpose :
Our previous work demonstrated that primary cilia (PC)-dependent cyclic mechanical stress (CMS)-induced autophagy is crucial for maintaining intraocular pressure (IOP) homeostasis. This activation was found to be regulated by reciprocal AKT and SMAD2/3 signaling. Here, we investigate in more detail the upstream signal that orchestrates PC-mediated autophagy activation in TM cells under CMS.
Methods :
Primary-cultured human TM cells underwent cyclic mechanical stretch (CMS, 8% elongation, 1 cycle/sec) for up to 24 h. Autophagy activation was assessed by monitoring LC3-II and p62 levels, or LC3 puncta formation of mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3), a marker for autophagy flux. Immunocytochemistry and Cilia Q software were employed to determine the localization of PI3K isoforms on PC. Chemical (PIK75, 0.02~2 µM) and genetic inhibition (siRNAs targeting each PI3K) were used to inhibit PI3Ks. Phosphoinositides, phosphorylated forms of phosphatidylinositol (PI), were detected using GFP-PHAKT1 (specifically for PI(3,4)P2 or PIP3) or antibodies against PI3P.
Results :
Human TM cells express class IA PI3Ks (PIK3CA, B, and C), class II (PIK3C2A), and Class III (PIK3C3/VPS34). Among them, PIK3CA is localized in primary cilia, with decreased colocalization upon CMS (NS: 54.49 ± 19.15% vs CMS: 41.01 ± 28.62%, p<0.01, n=55 and 38). PIK3CA knockdown alone did not impact CMS-induced autophagy activation. However, triple knockdown of PIK3CA, B, and D significantly reduced the increased LC3 II levels upon CMS (siCNT vs siTriple: 1.34±0.13 vs 1.07 ±0.18, n=6, p<0.05). Furthermore, PIK75, a class I PI3Ks inhibitor, concentration-dependently inhibited autophagy flux. CMS increased the number of GFP-PHAKT1-positive endosomes in TM cells (NS: 39.00 ± 24.81 vs CMS: 93.10 ± 42.82, p<0.05, n=13 and 20). However, CMS-induced autophagy in TM cells remained unaffected by PIK3C2A-mediated PI3P.
Conclusions :
Our findings reveal, for the first time, that class IA PI3K is the upstream signaling pathway regulating PC-mediated autophagy activation in response to CMS in TM cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.