title: Purification, molecular cloning, and expression of a novel growth-promoting factor for retinal pigment epithelial cells, REF-1/TFPI-2.
abstract: OBJECTIVE
  Retinal pigment epithelial (RPE) cells are known to play important roles in maintaining the homeostasis of the retina and in controlling choroidal neovascularization. The purpose of this study was to identify a factor or factors that would stimulate RPE cells to proliferate.
  
  
  METHODS
  To isolate such a factor, 100 L of human-fibroblast-conditioned medium underwent ion-exchange, hydrophobic, and reverse-phase chromatographies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The growth-promoting activity of the factor was examined in a human K-1034 RPE cell line and human primary RPE cells.
  
  
  RESULTS
  The different chromatographic processes isolated a 31-kDa factor that had RPE cell growth-promoting properties. This factor, which we have named RPE cell factor (REF)-1, promotes growth of RPE cells but not of human umbilical vein endothelial cells (HUVECs). The amino-terminal sequence and molecular cloned cDNA of REF-1 were identical with those of tissue-factor pathway inhibitor (TFPI)-2, a family of TFPIs, and placental protein (PP)-5, a serine protease inhibitor. The cDNA expression of REF-1/TFPI-2 with pcDL-pSRalpha vector in Chinese hamster ovary (CHO) cells confirmed the growth-promoting activity for RPE cells. The major component of the recombinant REF-1/TFPI-2 expressed in CHO cells had a molecular mass of 31 kDa and exerted growth-promoting activity in RPE cells but not in human endothelial cells and fibroblasts in vitro. REF-1/TFPI-2 also had protease inhibitory activity. The other family factor, TFPI-1, did not promote RPE cell growth.
  
  
  CONCLUSIONS
  REF-1/TFPI-2 is a novel growth-promoting factor for RPE cells but not for endothelial cells and fibroblasts. Its properties make it potentially beneficial for intraocular therapy for the repair and maintenance of RPE cells.
authors.affiliation: National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan. ytanaka@ntmc.hosp.go.jp
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Jun
authors.initials: J
authors.lastname: Utsumi
authors.forename: Mizuo
authors.initials: M
authors.lastname: Matsui
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Sudo
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakamura
authors.forename: Masato
authors.initials: M
authors.lastname: Mutoh
authors.forename: Akemi
authors.initials: A
authors.lastname: Kajita
authors.forename: Saburo
authors.initials: S
authors.lastname: Sone
authors.forename: Kazuteru
authors.initials: K
authors.lastname: Kigasawa
authors.forename: Masahiko
authors.initials: M
authors.lastname: Shibuya
authors.forename: Venkat N
authors.initials: VN
authors.lastname: Reddy
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Takeshi
authors.initials: T
authors.lastname: Iwata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017077:Culture Media, Conditioned; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D015870:Gene Expression; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010857:Pigment Epithelium of Eye / Q000166:cytology*; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D014162:Transfection
chemical_list: D017077:Culture Media, Conditioned; D006023:Glycoproteins; D006133:Growth Substances; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C021240:tissue-factor-pathway inhibitor 2