Hepatocyte growth factor promotes epithelial morphogenesis and occludin linkage to the cytoskeleton in cultured retinal pigment epithelial cells.
BACKGROUND
Although hepatocyte growth factor (HGF) is also known as scatter factor, it induces epithelial morphogenesis in cultured bovine retinal pigment epithelial (RPE) cells. To elucidate the mechanism of epithelial morphogenesis, we investigated the influence of HGF on occludin, a major component of tight junctions.


METHODS
RPE cells were plated on collagen type 1-coated chamber slides or dishes, 20 ng/ml HGF was added and the cells were incubated for 1 week. Cells were harvested at several time-points, and occludin expression was examined by immunohistochemistry. Detergent extraction protocols to identify the intensity of occludin linkage to the cytoskeleton were also used. Occludin expression was determined semiquantitatively by Western blotting.


RESULTS
Fluorescence microscopy revealed that HGF treatment increased the levels of insoluble occludin at the cell borders after detergent extraction. These level of insoluble occludin and the associated epithelial morphology were maintained for more than 3 weeks after withdrawal of HGF, whereas cells not treated with HGF had a fibroblastic appearance. Western blotting also showed that insoluble occludin was more abundant in HGF-treated cells. Furthermore, immunoreactive bands of insoluble occludin were somewhat larger than those of soluble occludin, suggesting that insoluble occludin may be modified in comparison to soluble occludin.


CONCLUSIONS
Our results suggest that HGF promotes linkage of occludin to the cytoskeleton. HGF may become a therapeutic candidate in physiological recovery of RPE cells and in preparation of RPE monolayers for transplantation.
Yanagihara
N
N
Department of Ophthalmology and Visual Sciences, Osaka City University Graduate School of Medicine, Osaka, Japan. nyanagi@med.osaka-cu.ac.jp
Miura
Y
Y
Moriwaki
M
M
Shiraki
K
K
Imamura
H
H
Kaida
M
M
Miki
T
T
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009024:Morphogenesis; D062793:Occludin; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
D008565:Membrane Proteins; D062793:Occludin; D017228:Hepatocyte Growth Factor