Involvement of p27^KIP1 in the proliferation of the developing corneal endothelium

PURPOSE. To examine the involvement of p27^KIP1 in the regulation of the proliferation of the developing corneal endothelium. METHODS. Central and peripheral corneas in C57Bl6 mice at postnatal day (P)1, P11, and 12 weeks after birth were analyzed by immunocytochemistry with anti-p27^KIP1, -p57^KIP2, and -proliferating cell nuclear antigen (PCNA) antibodies. Nuclear staining was performed with 4′,6′-diamino-2-phenylindole (DAPI) in wholemounts of corneal endothelium of the center and peripheral cornea in wild-type and p27^KIP1 knockout (-/-) mice at 12 weeks of age. p27^KIP1-/- and control mice were injected with bromodeoxyuridine (BrdU) once on P7, twice per day on P8 and P9, and once on P10 and then were analyzed by a BrdU cell-proliferation assay on P11. RESULTS. On P1, p27 ^KIP1 immunoreactivity was detected in a small number of corneal endothelial cells, and many endothelial cells expressed PCNA. At P11 and 12 weeks after birth, p27^KIP1 immunoreactivity was detected in many corneal endothelial cells. PCNA-positive cells in the endothelium were rare on P11 and completely absent at 12 weeks after birth. p57^KIP2 was not detected in either corneal epithelium or endothelium at P1, P11, or 12 weeks after birth. In wholemounts of corneal endothelium at 12 weeks of age, the number of endothelial nuclei in the p27^KIP1-/- mice was significantly higher than that in wild-type mice in both the center and peripheral regions of the cornea. In the BrdU assay, positive cells were abundant in the corneal endothelium of p27^KIP1-/- mice, whereas there were few positive cells in control mice. PCNA immunoreactivity in the endothelium of the p27 ^KIP1-/- mice was completely absent at 12 weeks after birth. CONCLUSIONS. These results suggest that p27^KIP1 is involved in the regulation of proliferation in the endothelium of the developing cornea.