Phosphorylation of p27(KIP1) in the mitotic cells of the corneal epithelium.
OBJECTIVE
The mechanism in regulation of the cell cycle and proliferation of corneal epithelium in the homeostatic ocular surface remains unclear. The aim of this study is to examine the expression of p27(KIP1) and its phosphorylation in corneal epithelium.


METHODS
The eyes of C57BL/6 mice (7 weeks old) were enucleated. Formalin-fixed and paraffin-embedded tissue sections were examined using immunohistochemistry with anti-p27(KIP1), threonine 187 phosphorylated p27(KIP1) (T187-phospho-p27), and phosphorylated Histon H3 (pHiston H3) antibodies. Anti-T187-phospho-p27 and anti-pHiston H3 polyclonal antibodies were used for parallel immunofluorescent staining.


RESULTS
pHiston H3-immunopositive cells were noted in basal cells of the corneal epithelium. At high magnification of DAPI nuclear staining, mitotic and non-mitotic cells were observed in corneal basal layer. p27(KIP1)-positive nuclei were detected in corneal basal cells, where non-mitotic basal cells were located. In contrast, mitotic cells showed under detectable level on p27(KIP1) immunoreactivity. Immunoreactivity for T187-phospho-p27 was detected in basal cells of the corneal epithelium. At high magnification, it was confirmed that the immunopositive cells were mitotic cells. Immunoreactivity of T187-phospho-p27 as well as pHiston H3 was localized in the same corneal basal cells using double-staining immunohistochemistry.


CONCLUSIONS
These results suggested that degradation of p27(KIP1) regulates progression into mitosis in corneal basal cells.
Kase
Satoru
S
Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, Sapporo, Japan. kaseron@med.hokudai.ac.jp
Yoshida
Kazuhiko
K
Ohgami
Kazuhiro
K
Shiratori
Kenji
K
Ohno
Shigeaki
S
Nakayama
Keiichi I
KI
D000818:Animals; D002467:Cell Nucleus / Q000502:physiology; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D007211:Indoles; D051379:Mice; D008810:Mice, Inbred C57BL; D008938:Mitosis / Q000502:physiology*; D010766:Phosphorylation
C493961:Cdkn1b protein, mouse; D005456:Fluorescent Dyes; D007211:Indoles; D050760:Cyclin-Dependent Kinase Inhibitor p27; C007293:DAPI