Abstract
Purpose :
Dysfunctional autophagy mediates damage of the retinal pigment epithelium (RPE) and contributes to the pathogenesis of major blinding retinal diseases such as age-related macular degeneration (AMD). We showed that RPE homeostasis is under the control of Prominin-1 (Prom1/CD133)-dependent autophagy. Our recent studies demonstrate that STAT3 is an important regulator of RPE function. We examined the molecular cross-talk between Prom1 and STAT3 signaling pathways in RPE autophagy and health, and hypothesize that disruption of these pathways may trigger RPE pathology in retinal disease and other conditions in which Prom1 is a biomarker of the disease.
Methods :
A lentiviral construct was used to overexpress Prom1 in ARPE-19 cells and CRISPR/Cas9 lentivirus was used to knock out (KO) STAT3 and Prom1 in adult glioblastoma (GBM) and ARPE-19 cells. The STAT3-KO cells were transduced with either the wild-type STAT3 or the STAT3 phosphorylation-defective mutants (Y705F and S727A). Western blotting was used to quantify Beclin-1 and p62/SQSTM1 expression, and phosphorylation of AMPK, ULK. LC3-I/II processing in the presence and absence of Bafilomycin was used to assess induction of autophagy flux.
Results :
KO of STAT3 constitutively activates AMPK-alpha-Thr172 and ULK-Ser555/Ser638/Ser757 phosphorylation, increases Prom1 expression, and concomitantly increases autophagy flux in GBM cells. Rescue of KO cells with WT-STAT3 blocks AMPK/ULK signaling and represses autophagy, whereas rescue with phosphorylation defective mutants fails to suppress autophagy. Overexpression of Prom1 activates AMPK-ULK signaling and activates autophagy flux in ARPE-19 cells. On the other hand, KO of Prom1 decreases autophagy flux in GBM cells.
Conclusions :
We demonstrate that STAT3 activation represses autophagy through inhibition of AMPK-ULK and Prom1 signaling. STAT3’s canonical transcriptional activity is indispensable for autophagy inhibition in GBM cells and KO of STAT3 induces autophagy flux through activation of Prom1-dependent AMPK-ULK signaling. We predict that clarifying the crosstalk between Prom1 and STAT3-mediated autophagy regulation will provide novel strategies for the treatment of both RPE diseases and cancers like GBM.
This is a 2020 ARVO Annual Meeting abstract.