Abstract
PurposeThe retinal pigment epithelium (RPE) tight junctions play a pivotal role in maintaining the homeostatic environment of the neural retina. Herein, we investigated the role of X-box binding protein 1 (XBP1), an endoplasmic reticulum (ER) stress-responsive transcription factor, in regulation of RPE tight junctions.MethodsHuman RPE cell line (ARPE-19) and primary primate RPE cells were used for in vitro experiments and RPE-specific XBP1 knockout (KO) mice were used for in vivo study. Endoplasmic reticulum stress was induced by a sublethal dose of thapsigargin or tunicamycin. XBP1 activation was manipulated by IRE inhibitor 4μ8C, which suppresses XBP1 mRNA splicing. The integrity of tight junctions and the involvement of calcium-dependent RhoA/Rho kinase pathway were examined.ResultsInduction of ER stress by thapsigargin, but not tunicamycin, disrupted RPE tight junctions in ARPE-19 cells. Inhibition of XBP1 activation by 4μ8C resulted in a remarkable downregulation of tight junction proteins (ZO-1 and occludin) and defects in tight junction formation in the presence or absence of ER stress inducers. Overexpression of active XBP1 partially reversed 4μ8C-induced anomalies in tight junctions. Mechanistically, XBP1 inhibition resulted in increased intracellular Ca2+ concentration, upregulation of RhoA expression, redistribution of F-actin, and tight junction damage, which was attenuated by Rho kinase inhibitor Y27632. In vivo, deletion of XBP1 in the RPE resulted in defective RPE tight junctions accompanied by increased VEGF expression.ConclusionsTaken together, these results suggest a protective role of XBP1 in maintaining RPE tight junctions possibly through regulation of calcium-dependent RhoA/Rho kinase signaling and actin cytoskeletal reorganization.
Highlights
The retinal pigment epithelium (RPE) tight junctions play a pivotal role in maintaining the homeostatic environment of the neural retina
These results suggest a protective role of X-box binding protein 1 (XBP1) in maintaining RPE tight junctions possibly through regulation of calcium-dependent RhoA/Rho kinase signaling and actin cytoskeletal reorganization
The activation of the unfolded protein response (UPR) is mediated by three transmembrane proteins, namely inositolrequiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like endoplasmic reticulum (ER) kinase (PERK)
Summary
Human RPE cell line (ARPE-19) and primary primate RPE cells were used for in vitro experiments and RPE-specific XBP1 knockout (KO) mice were used for in vivo study. Endoplasmic reticulum stress was induced by a sublethal dose of thapsigargin or tunicamycin. XBP1 activation was manipulated by IRE inhibitor 4l8C, which suppresses XBP1 mRNA splicing. The integrity of tight junctions and the involvement of calcium-dependent RhoA/Rho kinase pathway were examined. Generation of RPE-specific XBP1 knockout mice was described elsewhere.[21] Cre negative, XBP1 floxed littermates were used as control. 8-weekold mice were given five consecutive intraperitoneal injections of streptozotocin (STZ, 50 mg/kg/d; Sigma-Aldrich Corp., St. Louis, MO, USA) or vehicle as control. Four weeks after STZ injection, mice were euthanized and eyes were harvested for analyses
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