Involvement of p27^KIP1 in the Proliferation of the Developing Corneal Endothelium

Abstract

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.

The corneal endothelium shows a monolayer of differentiated cells located in the posterior portion of the cornea. The capacity for regeneration of corneal endothelium after injury is severely limited in humans; thus, corneal endothelium is considered to be a nonreplicating tissue. The cellular mechanisms responsible for mitotic inhibition in the corneal endothelium are not clear but may be similar to the mechanisms that contribute to cessation of division in the newly formed endothelium of developing cornea. Early in corneal development, the putative endothelial cells actively divide to form the endothelium. ¹ As the endothelial monolayer matures, the cells establish gap and tight junctions, and proliferation essentially ceases. 

Cell cycle progression is controlled by a series of kinase complexes composed of cyclins and cyclin-dependent kinases (CDKs). ² Enzymatic activities of cyclin/CDK complexes are regulated by many mechanisms that reflect both the diversity of the signals that they integrate and the central importance of their roles in cell cycle control. These regulatory mechanisms include the actions of CDK inhibitors (CKIs) such as p27^KIP1 and p57^KIP2. ^{3 4 5} In various cell lines, elimination of p27^KIP1 during the late G₁ phase has been shown to be required for cell cycle progression to the S phase. ^{6 7 8 9} Consistent with this idea is the fact that forced expression of p27^KIP1 blocks cell cycle progression during the G₁ phase, whereas targeted p27^KIP1 mRNA antisense vectors increase the fraction of cells in S phase. 

A study has demonstrated that p27^KIP1 mRNA does fluctuate during the cell cycle, implying that control of p27^KIP1 expression is exerted posttranslationally. ¹⁰ The ubiquitin-mediated proteosome pathway has been suggested to be involved in p27^KIP1 degradation in mammals. ¹¹ This proteosome pathway is emerging as a major universal mechanism that regulates selective and time-controlled elimination of key short-lived regulatory proteins, such as CKIs ¹² and IκB. ¹³ In our recent paper, we suggested that this pathway is involved in the differentiation of corneal epithelial cells. ¹⁴ The ubiquitin-mediated pathway of protein degradation comprises two discrete steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate, and degradation of the polyubiquitylated protein by the 26S proteosome complex. The Skp1-Cullin-F-Box protein (SCF) complex is one of the major classes of ubiquitin ligase and determines the specificity of protein ubiquitylation. Skp2 is an F-box protein that is a component of the SCF complex. ¹⁵  

It has been reported that p27^KIP1 is specifically recognized by Skp2 and that this step was a rate-limiting component of the machinery that ubiquitinated and degraded p27^KIP1. ^{16 17} We have reported that p27^KIP1 is expressed in corneal epithelium. ¹⁸ This expression was not detected 24 hours after epithelial scraping, when there were many cells in S phase of the cell cycle in the corneal epithelium. Twenty-four hours after epithelial scraping in Skp2^−/− mice, the corneal epithelium was thinner than that in wild–type mice and had many p27^KIP1-positive cells and few cells in the S phase. These results suggest that degradation of p27^KIP1 by Skp2 is involved in the regulation of proliferation in response to wounding of the corneal epithelium. 

In the present study, we analyzed the expression of p27^KIP1 as well as proliferating cell nuclear antigen (PCNA), one of the essential replication factors, which was induced at late G₁ phase and reached a peak level in the S phase of the cell cycle ^{19 20} in the developing cornea. In addition, the corneal endothelium of p27^KIP1 knockout mice were also examined. 

Materials and Methods

Development of p27^KIP1 Knockout Mice

C57Bl6 mice were obtained from Hokudo Corp. (Sapporo, Japan). The p27^KIP1 knockout (−/−) and littermate control mice were obtained as previously reported. ^{21 22} Briefly, cloned genomic DNA corresponding to the p27^KIP1 loci was isolated from a 129/Sv mouse genomic library. To produce p27^KIP1−/− mice, a targeting vector was constructed by replacing a 2.5-kb SmaI-SmaI fragment containing the entire p27^KIP1 sequence with a PGK-neo-poly(A) cassette. Maintenance, transfection, and selection of ES cells were performed as described previously. ²¹ Mutant ES cells were microinjected into C57Bl6 mouse embryos, and the resultant male chimeras were mated with female C57Bl6 mice. 

Immunocytochemistry

Eyes were dissected from C57Bl6 mice at postnatal day (P)1, P11, and 12 weeks after birth. Eyes were also dissected from p27^KIP1 knockout mice at P11 and 12 weeks after birth. For the bromodeoxyuridine (BrdU) assay, p27^KIP1−/− mice and littermate controls were injected with BrdU (30 mg/kg) once on P7, twice per day on P8 and P9, and once on P10, and the eyes were dissected at P11. Dissected eyes were washed in saline and fixed in ice-cold 4% paraformaldehyde (PFA) in 0.1 M borate buffer (pH 9.5) for 2 hours, and processed for paraffin sectioning. The animal experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Sections were dewaxed and rehydrated and then rinsed twice in phosphate-buffered saline (PBS) and incubated with normal goat serum and then with a 1:1000 dilution of mouse monoclonal anti-p27^KIP1 antibody (BD-Pharmingen, San Diego, CA), or anti-PCNA (1:1000; Zymed, South San Francisco, CA), or anti-p57^KIP2 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). Anti-BrdU staining was performed as described by Yoshiki et al. ²³ Briefly, the sections were immersed in 0.4 mg/mL pepsin in 0.1 N HCl at 30°C for 1 minute and then in 2N HCl at 40°C for 1 hour. After the slides were washed with PBS, they were incubated with normal goat serum and then with anti-BrdU antibody (BD Biosciences, San Jose, CA) at a dilution of 1:1000. Binding of the primary antibody was localized by fluorescence microscopy using FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:200. 

Corneal Endothelium Wholemount

Eyes were dissected from p27^KIP1−/− and wild-type mice (12 weeks of age). The center and periphery of the cornea were removed and stained with 4′6-diamidino-2-phenylindole (DAPI; 10–4 μg/mL) in PBS for 5 minutes. The corneas then were laid flat on a glass slide with the endothelium facing up and examined with a microscope equipped for epifluorescence (Axiovert 35M; Carl Zeiss Meditec, Dublin, CA). 

Statistical Analysis

For the center of the cornea, a microscopic field of identical size (345.6 × 276.5 μm) was chosen at the center of each sample. ²⁴ For the peripheral region, the same size of microscopic field was chosen so that the peripheral margin was 310 μm from the iris route. Seven corneas each from p27^KIP1−/− mice and wild-type mice of each strain were used. The results were evaluated using the Aspin-Welch t-test. 

Results

In this study, the distribution of p27^KIP1 was examined in the corneas of mice at P1, P11, and 12 weeks after birth (Fig. 1) . On P1, p27^KIP1 immunoreactivity was detected in a small number of corneal endothelial cells (Figs. 1b 1e ; arrowheads), and PCNA was detected in many endothelial cells (Figs. 1c 1f) . At P11 and 12 weeks after birth, p27^KIP1 immunoreactivity was detected in many corneal endothelial cells (Figs. 1h 1k 1n 1q ; arrowheads). PCNA-positive cells in the endothelium were rare on P11 and completely absent at 12 weeks after birth (Figs. 1i 1l 1o 1r) . We examined six animals at each stage and four sections per animal to determine the positive rate of p27^KIP1 staining in corneal cells and had successive results. 

Figure 2 summarizes the number of cells positive for p27^KIP1 and PCNA among the endothelial cells of the central (left) and peripheral (right) cornea at P1, P11, and 12 weeks after birth. Expression of p27^KIP1 and PCNA appeared to be negatively correlated. p57^KIP2 immunoreactivity was detected in the equator of the P1 lens as previously described, ²⁵ but was not detected in either the corneal epithelium or endothelium at P1, P11, or 12 weeks after birth (data not shown). 

To examine the role of p27^KIP1 in the development of the corneal endothelium, nuclear staining with DAPI was performed with wholemounts of the endothelium of central and peripheral corneas. Figure 3 shows representative views of endothelium from 12-week-old wild-type and p27^KIP1−/− mice. More nuclei were seen in the endothelium of p27^KIP1−/− mice than in that of wild-type mice. 

The number of endothelial cells was then calculated within microscopic fields of identical size (345.6 × 276.5 μm) from the center and peripheral regions of the cornea. The number of endothelial nuclei in p27^KIP1−/− mice was significantly higher than that in wild-type mice (P < 0.0001; n = 7; Fig. 4 ) in both the center and peripheral regions of the cornea. The number of endothelial nuclei was not significantly different between the center and peripheral regions of the cornea in either wild-type or p27^KIP1−/− mice. 

To evaluate the proliferative activity of the corneal cells, we examined the incorporation of BrdU, which was incorporated only into cells that were in the S phase. ²³ When wild-type mice and littermate control animals were injected with BrdU between P7 and P10 and the eyes were analyzed at P11, BrdU-positive cells were detected among the epithelial cells (Fig. 5c , arrow) but they were not detected in corneal endothelial cells (Fig. 5c , arrowhead). In p27^KIP1−/− mice, BrdU-positive cells were detected in the corneal endothelium (Fig. 5d , arrowhead) and the epithelium (Fig. 5d , arrow). 

Finally, expression of PCNA was examined in the corneas of the p27^KIP1−/− mice at 12 weeks after birth (Fig. 6) . PCNA immunoreactivity was completely absent in the endothelium of the p27^KIP1−/− mice at 12 weeks after birth. 

Discussion

The p27^KIP1 protein prevents a cell in the G phase of the cell cycle from entering the S phase ^{6 7 8 9} PCNA is an essential replication factor ²⁶ that is induced in the late G₁ phase and reaches a peak level in the S phase of the cell cycle. ^{19 20} In this study, p27^KIP1 was expressed in a small number of corneal endothelial cells, whereas many endothelial cells expressed PCNA on P1. In contrast, many endothelial cells in the central and peripheral cornea showed nuclear immunoreactivity for p27^KIP1 at P11 and 12 weeks after birth. PCNA-positive cells in the endothelium were rare on P11 and were completely absent at 12 weeks after birth. These results suggest that expression of p27^KIP1 in the developing corneal endothelium is well correlated with the cessation of the proliferation in wild-type mice. 

To examine the role of p27^KIP1 in the proliferation of the corneal endothelium, DAPI was used for nuclear staining in wholemounts of corneal endothelium in the wild-type and the p27^KIP1−/− mice at 12 weeks of age. The number of endothelial nuclei of the p27^KIP1−/− mice was significantly higher than that of wild-type mice. When mice were injected with BrdU between P7 and P10, and analyzed at P11, BrdU-positive cells were detected among the endothelial cells of the corneas in p27^KIP1−/− mice, but they were not detected among those of wild-type mice. These results suggest that the period of proliferation of the corneal endothelium is prolonged in the p27^KIP1−/− mice. 

The number of endothelial nuclei was not significantly different between the center and peripheral region of the cornea in either wild-type or p27^KIP1−/− mice. These results are in contrast to those in reports about the human cornea showing that the peripheral density of nuclei is higher than the central density. ^{27 28}  

The specific mechanisms that inhibit proliferation on maturation of the corneal endothelium in vivo are unknown, but may be similar to the mechanisms that inhibit the proliferation of confluent cells. It has been reported that N-cadherin–mediated signaling is involved in contact inhibition of growth by inducing elevation of p27^KIP1 levels. ²⁹ Together with recent reports showing that N-cadherin is expressed in the developing corneal endothelium, ^{30 31} these findings suggest that N-cadherin–mediated signaling may be involved in the inhibition of growth of corneal endothelial cells through the elevation of p27^KIP1 expression. 

Recently, it was reported that p27^KIP1 is specifically recognized by Skp2 and that this step is a rate-limiting component of the machinery that ubiquitinates and degrades p27^KIP1. ¹⁶ An increase in p27^KIP1 expression has also been shown to be achieved by translational upregulation of p27^KIP1 synthesis ³³ or by an increase in the amount of p27 polyribosome-bound mRNA. ³⁴ Identification of the mechanism that regulates the protein level of the p27^KIP1 should be valuable in delineating the molecular pathways regulating the proliferation of corneal endothelial cells. 

PCNA immunoreactivity in the endothelia of p27^KIP1−/− mice was completely absent at 12 weeks after birth. This result suggested that proliferation of the endothelial cells in p27^KIP1−/− mice stops until 12 weeks after birth and also that there must be other mechanisms that eliminate the excess endothelial cells. 

 Submitted for publication November 13, 2003; revised January 26, 2004; accepted February 13, 2004.

 Disclosure: K. Yoshida, None; S. Kase, None; K. Nakayama, None; H. Nagahama, None; T. Harada, None; H. Ikeda, None; C. Harada, None; J. Imaki, None; K. Ohgami, None; K. Shiratori, None; I.B. Ilieva, None; S. Ohno, None; S. Nishi, None; K.I. Nakayama, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Nuclear staining with DAPI (a, d, g, j, m, p) and expression of p27^KIP1 (b, e, h, k, n, q) and PCNA (c, f, i, l, o, r) in the central (d–f, j–l, p–r) and peripheral (a–c, g–i, m–o) corneas of mice at P1 (a– f), P11 (g–l), or 12 weeks (m–r) after birth. p27^KIP1 was expressed in a small number of corneal endothelial cells (b, e, arrowhead), whereas many endothelial cells expressed PCNA on P1 (c, f). In contrast, many endothelial cells in the central and peripheral cornea showed nuclear immunoreactivity for p27^KIP1 at P11 and 12 weeks after birth (h, k, n, q; arrowhead). PCNA-positive cells in the endothelium were rare on P11 (i, l) and were completely absent at 12 weeks (o, r) after birth. Magnification: (a– l) ×630; (m– r) ×400 .

Figure 2.

 

The number of cells positive for p27^KIP1 and PCNA relative to the number of nuclei stained with DAPI among the endothelial cells of the central (left) and peripheral (right) cornea at P1, P11, or 12 weeks (12w) after birth.

Figure 3.

 

Nuclear staining with DAPI in wholemounts of endothelium from the center (a, b) and periphery (c, d) of the corneas of wild-type (a, c) and p27^KIP1−/− (b, d) mice at 12 weeks of age. In the peripheral region, the peripheral margin was 310 μm from the iris root. Magnification, ×400.

Figure 4.

 

The number of nuclei in the corneal endothelium in wild-type and p27^KIP1−/− mice at 12 weeks of age. Microscopic fields of identical size (345.6 × 276.5 μm) were chosen at the center and periphery of the cornea. For the peripheral region, the same-sized microscopic field was chosen at a position so that the peripheral margin was 310 μm from the iris root. Seven animals each were used for wild-type and p27^KIP1−/− mice. P < 0.0001 for both center and peripheral retina.

Figure 5.

 

Hematoxylin staining (a, b) and immunodetection with BrdU (c, d) in the central cornea of wild-type (a, c) and p27^KIP1−/− (b, d) mice. The mice were injected with BrdU between P7 and P10 and analyzed at P11. BrdU-positive cells were distributed among epithelial cells (c, arrow), but not among endothelial cells (c, arrowhead) of the central corneas in wild-type mice. In p27^KIP1−/− mice, corneal endothelial cells (d, arrowhead) and epithelial cells (d, arrow) showed nuclear immunoreactivity for BrdU. Seven eyes each from wild-type and p27^KIP1−/− mice were examined. Magnification, ×400.

Figure 6.

 

Nuclear staining with DAPI (a, c) and expression of PCNA (b, d) in the central (a, b) and peripheral (c, d) corneas of the p27^KIP1−/− mice on 12 weeks after birth. Immunoreactivity for p27^KIP1 in the endothelium was completely absent at 12 weeks after birth (b, d, arrowhead). Magnification, ×400.

The authors thank Takae Ohyama for skillful technical assistance.