Abstract
Purpose: :
X-box binding protein 1 (XBP1) is a master coordinator of the unfolded protein response (UPR) that protects cells against endoplasmic reticulum (ER) stress. We recently reported that ER stress contributes to retinal inflammation and vascular leakage in diabetic retinopathy. The purpose of this study is to determine whether activation of the UPR by ER stress preconditioning can protect retinal endothelial cells from inflammatory damage and whether this is mediated by XBP1.
Methods: :
Primary human retinal endothelial cells (HREC) were used in in vitro studies. ER stress preconditioning was induced by transient exposure of HREC to a low dose of tunicamycin. Over-expression of active XBP1 was achieved by adenoviral infection, while down-regulation of XBP1 was manipulated by siRNA or quinotriaxin, a specific inhibitor of XBP1 activation. After pre-treatment, cells were treated with TNF-α (10 ng/ml) for 0-24 h and expression of active XBP1, VCAM-1 and ICAM-1 was determined by real-time RT-PCR and Western blot analysis. Activation of NF-ΚB was analyzed by NF-ΚB transcription activity assay, oligonucleotide pull-down assay and phosphorylation of the p65 subunit. The in vivo effects of ER stress preconditioning on retinal inflammation and vascular leakage were also determined.
Results: :
Exposure of HREC to tunicamycin (0.1 µg/ml) for 6 h (preconditioning) induced a robust increase in activation of XBP1, but did not affect expression of adhesion molecules. Treatment with TNF-α elicited a rapid and a delayed NF-ΚB activation, and significantly increased expression of VCAM-1 and ICAM-1. These changes were abolished or attenuated in preconditioned cells. Importantly, pharmaceutical inhibition of XBP1 activation or depletion of XBP1 by siRNA abrogated the inhibitory effects of preconditioning on inflammation. Moreover, knockdown of XBP1 alone increased ICAM-1 and VCAM-1 expression, suggesting that endogenous XBP1 is critical in regulation of the basal inflammatory state in retinal endothelial cells. In contrast, over-expression of active XBP1 in HREC inhibited TNF-α-induced phosphorylation of IKKβ (Ser180), NF-ΚB activation and adhesion molecule expression. Finally, preconditioning with periocular injection of tunicamycin significantly reduced TNF-α-induced retinal ICAM-1 and VCAM-1 expression and vascular leakage in non-diabetic mice.
Conclusions: :
Activation of XBP1 by ER stress preconditioning alleviates endothelial inflammatory response and attenuates TNF-α-induced retinal vascular leakage through inhibition of NF-ΚB activation.
Keywords: diabetic retinopathy • inflammation • stress response