title: Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells.
abstract: OBJECTIVE
  Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells.
  
  
  METHODS
  Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins.
  
  
  RESULTS
  One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A.
  
  
  CONCLUSIONS
  Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.
authors.affiliation: Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Claudia S
authors.initials: CS
authors.lastname: Alge
authors.forename: Sabine
authors.initials: S
authors.lastname: Suppmann
authors.forename: Siegfried G
authors.initials: SG
authors.lastname: Priglinger
authors.forename: Aljoscha S
authors.initials: AS
authors.lastname: Neubauer
authors.forename: Christian A
authors.initials: CA
authors.lastname: May
authors.forename: Stefanie
authors.initials: S
authors.lastname: Hauck
authors.forename: Ulrich
authors.initials: U
authors.lastname: Welge-Lussen
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
authors.forename: Anselm
authors.initials: A
authors.lastname: Kampik
mesh_terms: D002454:Cell Differentiation; D002478:Cells, Cultured; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000032:analysis*; D015870:Gene Expression; D006801:Humans; D007525:Isoelectric Focusing; D008875:Middle Aged; D010449:Peptide Mapping; D010857:Pigment Epithelium of Eye / Q000737:chemistry* / Q000166:cytology*; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005136:Eye Proteins; D020543:Proteome