title: Silencing of the CHM gene alters phagocytic and secretory pathways in the retinal pigment epithelium.
abstract: OBJECTIVE
  Choroideremia (CHM) is an X-linked progressive degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid caused by mutations in the CHM gene, which encodes Rab escort-protein-1 (REP-1). REP-1 enables posttranslational isoprenyl modification of Rab GTPases, proteins that control vesicle formation, movement, docking, and fusion. The aim of this study was to determine the effect of REP-1 depletion on vesicular trafficking in phagocytic and secretory pathways of human RPE.
  
  
  METHODS
  In vitro, REP-1 expression was inhibited in human fetal RPE (hfRPE) cells by siRNA knockdown and its effects measured on the uptake of bovine photoreceptor outer segments (POS), proteolysis of POS rhodopsin, phagosomal pH, phagosome fusion with early and late endosomes/lysosomes, and polarized secretion of cytokines.
  
  
  RESULTS
  Depletion of REP-1 in human RPE cells did not affect POS internalization but reduced phagosomal acidification and delayed POS protein clearance. REP-1 depletion also caused a decrease in the association of POS-containing phagosomes with late endosomal markers (Rab7, LAMP-1) and increases in the secretion of monocyte chemotactic protein (MCP-1) and interleukin (IL)-8 by hfRPE cells.
  
  
  CONCLUSIONS
  Lack of REP-1 protein expression in hfRPE cells leads to reduced degradation of POS most likely because of the inhibition of phagosome-lysosome fusion events and increased constitutive secretion of MCP-1 and IL-8. These observations may explain the accumulation of unprocessed outer segments within the phagolysosomes of RPE cells and the presence of inflammatory cells in the choroid of patients with CHM.
authors.affiliation: Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Nataliya V
authors.initials: NV
authors.lastname: Gordiyenko
authors.forename: Robert N
authors.initials: RN
authors.lastname: Fariss
authors.forename: Connie
authors.initials: C
authors.lastname: Zhi
authors.forename: Ian M
authors.initials: IM
authors.lastname: MacDonald
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D055214:Retinal Photoreceptor Cell Outer Segment / Q000502:physiology; D055213:Retinal Pigment Epithelium / Q000196:embryology / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D055571:Secretory Pathway / Q000502:physiology*; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000235:genetics*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C081787:CHM protein, human; D016207:Cytokines; D034741:RNA, Small Interfering; D012243:Rhodopsin; D020691:rab GTP-Binding Proteins