title: Aberrant accumulation of fibulin-3 in the endoplasmic reticulum leads to activation of the unfolded protein response and VEGF expression.
abstract: OBJECTIVE
  The inherited early-onset macular degenerative disease known as malattia leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) have been linked to a missense mutation leading to production of a mutant fibulin-3 protein (R345W). R345W is poorly secreted and accumulates in the RPE of ML/DHRD retinas. Accumulation of misfolded proteins within the endoplasmic reticulum (ER) causes activation of unfolded protein response (UPR) signaling and expression of ER stress-responsive genes, including vascular endothelial growth factor (VEGF). Therefore, we hypothesized that the expression of R345W activates the UPR, leading to VEGF expression.
  
  
  METHODS
  Adenoviral vectors were used to overexpress fibulin-3 wild-type (Wt) and R345W mutant proteins in ARPE-19 cells. Secretion and intracellular accumulation of Wt and R345W were compared by Western blot analysis and immunocytochemistry. Activation of the UPR was evaluated by measuring the expression of glucose-regulated protein 78 (GRP78 [BiP]) and editing of the X-box binding protein (XBP-1) mRNA. VEGF expression and transcriptional activation of the VEGF promoter were determined by Northern blot analysis, Western blot analysis, and use of a novel VEGF promoter-reporter construct containing 8.2 kb of the human VEGF gene.
  
  
  RESULTS
  R345W was poorly secreted by ARPE-19 cells and accumulated in the ER, leading to UPR activation and increased VEGF expression. Compared with Wt mutant proteins, the expression of R345W was more effective at causing UPR activation, increasing VEGF expression, and stimulating transcription from the VEGF promoter.
  
  
  CONCLUSIONS
  These findings demonstrated that the expression of mutated fibulin-3 caused UPR activation and increased VEGF expression. Expression of mutant fibulin proteins may contribute to macular degeneration and choroidal neovascularization by causing ER stress leading to RPE dysfunction and increased VEGF expression.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, USA.
authors.forename: C Nathaniel
authors.initials: CN
authors.lastname: Roybal
authors.forename: Lihua Y
authors.initials: LY
authors.lastname: Marmorstein
authors.forename: David L
authors.initials: DL
authors.lastname: Vander Jagt
authors.forename: Steve F
authors.initials: SF
authors.lastname: Abcouwer
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D016326:Extracellular Matrix Proteins / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics; D000071717:X-Box Binding Protein 1
chemical_list: D004268:DNA-Binding Proteins; C119630:EFEMP1 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; D016326:Extracellular Matrix Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D012333:RNA, Messenger; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human