Purification, molecular cloning, and expression of a novel growth-promoting factor for retinal pigment epithelial cells, REF-1/TFPI-2.
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.
Tanaka
Yasuhiko
Y
National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan. ytanaka@ntmc.hosp.go.jp
Utsumi
Jun
J
Matsui
Mizuo
M
Sudo
Tetsuo
T
Nakamura
Noriko
N
Mutoh
Masato
M
Kajita
Akemi
A
Sone
Saburo
S
Kigasawa
Kazuteru
K
Shibuya
Masahiko
M
Reddy
Venkat N
VN
Zhang
Qiang
Q
Iwata
Takeshi
T
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
D017077:Culture Media, Conditioned; D006023:Glycoproteins; D006133:Growth Substances; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C021240:tissue-factor-pathway inhibitor 2