Abstract
Purpose :
We recently showed that dynasore protect ocular surface epithelial cells and their mucosal glycocalyx against oxidative stress by shifting the unfolded protein response (UPR) in the endoplasmic reticulum (ER) towards homeostasis via calcium dynamics (PMID 32791188). Dynasore is a cell-permeable small molecule inhibitor of dynamin family GTPases responsible for membrane fusion/fission events, discovered after screening a small molecule library for inhibition of DNM2 GTPase activity and endocytosis (PMID:16740485). Our goal is to identify molecular target(s) of dynasore that determine cytoprotective effect.
Methods :
A cell culture model of human immortalized corneal limbal epithelial (HCLE) cells was used (PMID: 12766048). Cultures were stressed by application of t-butyl hydroperoxide (3mM) for 2 hrs while treating or not treating (control) with dynasore (Sigma). Cytoprotective effect was quantified using the WST-1 metabolic assay (Sigma). RNA was extracted with RNeasy kit (Qiagen). First strand cDNA was synthesized by reverse transcription. qPCR was performed with cDNA and specific primers using SYBR®Green. Western blot was performed with the total protein extracted using RIPA buffer.
Results :
We determined in dose-response experiments (0.5-80 µM) that dynasore protects cells with an EC50 of 6.0 ± 0.8 µM (n=16). qPCR was performed to determine which members of the dynamin family are expressed by HCLE cells (Fig. 1). Of the 3 classic dynamins, DNM2 is dominant while DNM1 has low expression. Of the 3 ER-localized atlastins, ATL3 is dominant while ATL1 has low expression. Of the 2 mitochondria-localized mitofusins, MNF2 is dominant. By western blot, we observed ATL3 dimerization increased in stressed cells, however, this was not inhibited by dynasore.
Conclusions :
We have identified, for the first time, dynamin family members expressed by ocular surface epithelia. Dimerization of ATL3, which brings two ER membranes into contact, increases in cells subjected to oxidative stress. This is consistent with UPR-mediate ER-phagy to eliminate misfolded proteins and restore metabolic homeostasis. ATL3 dimerization does not appear to be targeted by dynasore at the current condition.
This is a 2021 ARVO Annual Meeting abstract.