7-ketocholesterol stimulates differentiation of lens epithelial cells.
OBJECTIVE
To establish if oxysterols stimulate differentiation of lens epithelial cells (LEC).


METHODS
Primary cultures of lens epithelial cells were incubated with 7-ketocholesterol (7-keto), 25-hydroxycholesterol (25-OH) or cholesterol at 10 microg/ml for 10 days. Cells incubated with 100 ng/ml basic fibroblast growth factor (b-FGF) were used as positive controls for differentiation. The expression of the differentiation marker p57KIP2, proliferation marker PCNA (Proliferating Cell Nuclear Antigen) and fibers specific proteins gamma-crystallin, CP49, MIP26 following treatment with oxysterols was determined by western blot. Differentiation into fiber cells was further confirmed by counting the number of lentoid bodies formed following incubation with 7-keto.


RESULTS
LEC incubated with 7-keto presented higher levels of p57KIP2 and showed expression of fiber specific proteins such as MIP26 and CP49, compared to cells incubated with 25-OH or cholesterol. The differentiation marker p57KIP2 increased over time for cells incubated with 7-keto while there was a decline on the amount of the proliferation marker PCNA. The expression of the fiber specific proteins gamma-crystallin, MIP26 and CP49 was detected after 5 days of incubation with 7-keto. Differentiation was accompanied by a seven-fold increase in the number of lentoid bodies formed.


CONCLUSIONS
Results show for the first time that 7-keto inhibits proliferation and stimulates differentiation of lens epithelial cells into fiber cells. The presence of 7-keto in the lens may disrupt the highly regulated differentiation program of LEC, compromising normal lens growth and transparency.
Girão
Henrique
H
Centre of Ophthalmology, Biomedical Institute for Research in Light and Image (IBILI), Faculty of Medicine, University of Coimbra, Portugal.
Shang
Fu
F
Pereira
Paulo
P
D000818:Animals; D020346:Aquaporins; D015415:Biomarkers; D015153:Blotting, Western; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002784:Cholesterol / Q000494:pharmacology; D050761:Cyclin-Dependent Kinase Inhibitor p57; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006888:Hydroxycholesterols / Q000494:pharmacology; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007653:Ketocholesterols / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D038222:gamma-Crystallins / Q000378:metabolism
D020346:Aquaporins; D015415:Biomarkers; D050761:Cyclin-Dependent Kinase Inhibitor p57; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D006888:Hydroxycholesterols; D007381:Intermediate Filament Proteins; D007653:Ketocholesterols; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C030328:aquaporin 0; D038222:gamma-Crystallins; D016222:Fibroblast Growth Factor 2; C007997:25-hydroxycholesterol; D002784:Cholesterol; C003001:7-ketocholesterol