title: The Role of IRE-XBP1 Pathway in Regulation of Retinal Pigment Epithelium Tight Junctions.
abstract: OBJECTIVE
  The 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.
  
  
  METHODS
  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 4μ8C, which suppresses XBP1 mRNA splicing. The integrity of tight junctions and the involvement of calcium-dependent RhoA/Rho kinase pathway were examined.
  
  
  RESULTS
  Induction 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.
  
  
  CONCLUSIONS
  Taken 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.
authors.affiliation: Departments of Ophthalmology and Biochemistry, University at Buffalo, State University of New York, Buffalo, New York, United States 2SUNY Eye Institute, State University of New York, New York, United States 3Aier School of Ophthalmology, Central South University, Changsha, Hunan, China.
authors.forename: Jacey H
authors.initials: JH
authors.lastname: Ma
authors.affiliation: Departments of Ophthalmology and Biochemistry, University at Buffalo, State University of New York, Buffalo, New York, United States 2SUNY Eye Institute, State University of New York, New York, United States.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Wang
authors.affiliation: Departments of Ophthalmology and Biochemistry, University at Buffalo, State University of New York, Buffalo, New York, United States 2SUNY Eye Institute, State University of New York, New York, United States.
authors.forename: Junhua
authors.initials: J
authors.lastname: Li
authors.affiliation: Departments of Ophthalmology and Biochemistry, University at Buffalo, State University of New York, Buffalo, New York, United States 2SUNY Eye Institute, State University of New York, New York, United States 4Research Service, Veterans Administration Western New York Healthcare System, Buffalo, New York, United States.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Pfeffer
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: Departments of Ophthalmology and Biochemistry, University at Buffalo, State University of New York, Buffalo, New York, United States 2SUNY Eye Institute, State University of New York, New York, United States.
authors.forename: Sarah X
authors.initials: SX
authors.lastname: Zhang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D004195:Disease Models, Animal; D059865:Endoplasmic Reticulum Stress; D005786:Gene Expression Regulation; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000378:metabolism* / Q000473:pathology; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism; D000071717:X-Box Binding Protein 1 / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D000071717:X-Box Binding Protein 1; C000605544:Xbp1 protein, mouse