Phosphorylation of p27(KIP1) in lens epithelial cells after extraction of fiber cells.
Opacification of the posterior capsule depends on replication of the residual lens epithelial cells lining the capsule. However, the mechanisms in the regulation of lens cell proliferation have not been determined. The purpose of this study is to examine the expression of p27(KIP1), a cyclin-dependent kinase inhibitor, and its phosphorylation, and cyclin D1 in lens epithelial cells after extraction of fiber cells. C57Bl6 mice (12 weeks old) were anesthetized, and the lens fiber cells were surgically extracted. Eyeballs were collected and fixed at 15 min and 24 h after extraction with and without injection of a specific phosphorylated extracellular signal-regulated kinase (pERK) 1/2 inhibitor (PD98059) to the anterior chamber. Collected tissues were analyzed using immunohistochemistry with anti-p27(KIP1), anti-phosphorylated p27(KIP1) on serine 10 (s10-phospho-p27) and cyclin D1 antibodies. Human lens epithelial cells were cultured, and then were treated with and without 40 ng/ml human recombinant basic fibroblast growth factor (bFGF), which was analyzed by Western blot analysis. In the untreated lens, p27(KIP1) was not phosphorylated in the lens epithelial cells, although p27(KIP1)-positive nuclei were detected in the lens cells of the equatorial region. Immunoreactivity for cyclin D1 was hardly detected in the lens. Nuclear immunoreactivity for p27(KIP1) and s10-phospho-p27 was observed in several lens cells of the equatorial region 15 min after extraction of fiber cells. Western blotting demonstrated that the p27(KIP1) phosphorylation form was upregulated 15 min after bFGF treatment in cultured lens epithelial cells. Many cyclin D1-positive nuclei were noted 24 h after the surgery. p27(KIP1) phosphorylation and cyclin D1 induction were inhibited by PD98059. s10-phospho-p27 and p27(KIP1) immunoreactivity was undetected in the lens cells 24 h after the extraction of fiber cells. It is possible that the phosphorylation of p27(KIP1), and cyclin D1 expression are regulated by the ERK pathway in lens cells after the extraction of fiber cells.
Kase
Satoru
S
Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan. kaseron@med.hokudai.ac.jp
Yoshida
Kazuhiko
K
Jin
Xue-Hai
XH
Koyama
Yoshikazu
Y
Kitaichi
Nobuyoshi
N
Ohgami
Kazuhiro
K
Shiratori
Kenji
K
Ilieva
Iliyana
I
Ohno
Shigeaki
S
D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000166:cytology* / Q000378:metabolism* / Q000601:surgery*; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D015854:Up-Regulation
D004791:Enzyme Inhibitors; D005419:Flavonoids; D016222:Fibroblast Growth Factor 2; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D048049:Extracellular Signal-Regulated MAP Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one