Abstract
Purpose :
Retinal neovascularization (RNV) is a crucial component of ischemic retinopathies such as retinopathy of prematurity and diabetic retinopathy. Inflammation has been shown to play a key role in RNV. Tumor necrosis factor alpha-converting enzyme (TACE) is a key regulator of inflammation through ectodomain shedding of a number of cytokines, cytokine receptors, growth factors, and adhesion molecules. TACE has been shown to critically contribute to pathological neovascularization. The goal of this study was to identify the molecular mechanism(s) involved in TACE activation during RNV. Specifically, we have tested the hypothesis that endoplasmic reticulum (ER) stress-mediated up-regulation of the ER proteins iRhoms leads to TACE activation in response to hypoxia.
Methods :
TACE mRNA and protein expression were measured in human retinal endothelial cells (HREC) in response to 2% O2 (1-12 hours) by quantitative PCR (qPCR) and Western blotting analysis, respectively. TACE activity was evaluated by fluorimetric assay. Induction of ER stress was assessed by evaluating expression of GRP78 by qPCR and Western blotting analysis. Blocking and activation of ER stress were performed by treating HREC with 4-phenyl butyric acid (PBA; 1 mM) and thapsigargin (1-10 nM), respectively. iRhom1/2 gene silencing was achieved by transfection of the cells with specific siRNAs.
Results :
Hypoxia promoted a significant elevation of TACE mRNA and proteins levels as well as an increase in TACE activity in HREC. Treatment of the cells with hypoxia was associated with induction of ER stress as evidenced by a significant increase in GRP78 expression, and correlated with up-regulation of ER residing iRhom1 on both mRNA and protein levels. Silencing of iRhom1 with iRhom1 specific siRNA reverted hypoxia-induced activation of TACE. Furthermore, inhibition of ER stress with PBA ameliorated hypoxia-mediated elevation of both iRhom1 and TACE expression. In addition, ER stress activator thapsigargin promoted an increase in iRhom1 as well as TACE expression.
Conclusions :
The obtained results indicate that hypoxia-mediated induction of ER stress contributes to TACE activation through up-regulation of iRhom1. The elucidated mechanism of TACE activation in ischemic retina can contribute to retinal neovascularization.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.