Abstract
Purpose :
Inositol-requiring enzyme 1 (IRE1) regulates the most ancient branch of the unfolded protein response (UPR), and is present in the endoplasmic reticulum (ER) of all eukaryotes. IRE1 signaling is mediated by its splicing of X-box binding protein 1 (sXBP1), a transcription factor that is essential for expression of UPR genes that are required for ER stress relief. We previously reported that ER stress induced HtrA1 expression in retinal pigment epithelial (RPE) cells. This up-regulation was protective and essential for surviving proteotoxic cell death. We tested the hypothesis that IRE1 influences HtrA1 expression during ER stress in the RPE.
Methods :
Chronic protein misfolding was stimulated in cultured ARPE-19 cells using tunicamycin. HtrA1 expression was assayed by immunoblot and real time qRT-PCR. Parallel experiments were performed in the presence of 4μ8C (100 nM), an active site inhibitor of IRE1α’s endoribonuclease activity. To test the effect of HtrA1 suppression on IRE1α signaling, we stably expressed HtrA1-specific shRNA or control GFP shRNA. The total RNA pool was used in a UPR array (Qiagen) and gene expression was compared for IRE1α -related targets in vehicle- or tunicamycin-treated cells. For randomly selected hits, qRT-PCR results were validated by immunoblot. Data are representative of n>3 experiments, and *p<0.05 was considered statistically significant.
Results :
We mimicked conditions of proteotoxicity in RPE by preventing the glycosylation and ER-to-Golgi export of nascent peptides and found that intracellular HtrA1 protein accumulated in a tunicamycin dose (0.01–11 μM) and time (0-24h) – dependent manner. HtrA1 protein levels correlated with enhanced mRNA transcription. The IRE1α ribonuclease blocker 4μ8C potently inhibited the accumulation of sXBP1 and HtrA1 mRNA during chronic protein misfolding. We examined the effect of HtrA1 on IRE1α signaling in the face of ER stress. We found that expression of IRE1α protein, and consequently that of sXBP1, was suppressed after HtrA1 knockdown. Reduction in IRE1α and sXBP1 altered the mRNA and protein expression of members of the IRE1 signaling cascade. These include proteins which are critical to folding, glycosylation, and export of nascent peptides.
Conclusions :
Our results unravel a positive feedback loop that integrates HtrA1 into the IRE1α pathway, and which has direct consequences on RPE's toolbox for combating proteotoxic stress.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.