7-ketocholesterol stimulates differentiation of lens epithelial cells

Abstract

PURPOSE: 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