Molecular Vision 2001; 7:261-270 <http://www.molvis.org/molvis/v7/a37/> Received 11 October 2001 | Accepted 14 November 2001 | Published 20 November 2001

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Differential activity of TGF-b2 on the expression of p27^Kip1 and Cdk4 in actively cycling and contact inhibited rabbit corneal endothelial cells

Tae Yon Kim,^1,3 Won-Il Kim,¹ Ronald E. Smith,^1,2 EunDuck P. Kay^1,2
 
 

¹Doheny Eye Institute, ²Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA; ³Department of Ophthalmology, Kon Yang University, Seoul, Korea

Correspondence to: EunDuck P. Kay, Ph.D., Doheny Eye Institute, 1450 San Pablo Street, DVRC 203, Los Angeles, CA, 90033; Phone: (323) 442-6625; FAX: (323) 442-6688; email: ekay@hsc.usc.edu

Abstract

Purpose: To determine whether TGF-b2 exerts inhibitory action in a density dependent manner in primary, first passage, and second passage corneal endothelial cells (CEC).

Methods: Fifty percent confluent cultures were used for actively cycling cells and monolayers were used as contact inhibited cultures. Half of the experiments were performed in cells treated with TGF-b2 at 10 ng/ml for 24 h. Subcellular localization of cyclin dependent kinase 4 (Cdk4), p27^Kip1 (p27), and phosphorylated p27 (pp27) was determined by immunofluorescent staining followed by confocal laser microscopic analysis. Expression of proteins were analyzed by immunoblotting.

Results: Before colocalization between Cdk4 and p27 was studied, the two proteins were respectively stained, either in growing cells for the presence of Cdk4 or in contact inhibited cultures for the presence of p27. Nuclear Cdk4 was observed in FGF-2 treated cells while nuclear staining of Cdk4 was lost in mitogen deprived or TGF-b2 treated cells. On the other hand, a strong positive staining of nuclear p27 was observed in growth down regulated conditions, which was completely lost in growth up regulated conditions. When cells were double stained with Cdk4 and p27 antibodies, actively cycling cells contained nuclear Cdk4. Less than 10% of the primary cells were positive for Cdk4 staining, whereas all of the second passage CEC contained nuclear Cdk4. Conversely, p27 was not detected in actively cycling cells in either primary or passaged cells. Contact inhibited cells demonstrated nuclear p27 staining in all cells, but only a few cells were positive for nuclear Cdk4. Nuclear Cdk4 was absent when the actively cycling cells were treated with TGF-b2, whereas TGF-b2 did not induce the expression of nuclear p27 in the same cultures. In contact inhibited cells, TGF-b2 did not affect the staining profiles of p27. In the first passage CEC, TGF-b2 slightly increased the number of cells that were positive for nuclear Cdk4. When the effect of TGF-b2 at the level of protein synthesis was determined, TGF-b2 markedly downregulated Cdk4 synthesis and slightly upregulated p27 synthesis in actively cycling cells. On the other hand, TGF-b2 did not exert the same effect on Cdk4 synthesis in contact inhibited cells as it did on actively cycling cells. Contact inhibited cells contained a high level of p27, and TGF-b2 slightly upregulated p27 synthesis in these cells. When phosphorylated p27 was determined to be present, the nuclei of both actively cycling and contact inhibited cells contained phosphorylated p27 in the nuclei, regardless of the passage numbers. TGF-b2 inhibited phosphorylation of p27 in actively cycling cells, but it had no effect on phosphorylation of p27 in contact inhibited cells.

Conclusions: These data suggest that Cdk4 and p27 expression is density dependent, and TGF-b2 exerted its activity on actively cycling cells. In these cells, TGF-b2 downregulated Cdk4 expression and prevented the phosphorylation of p27, which is a prerequisite for nuclear export of the inhibitor molecule for degradation. Thus, TGF-b2 inhibits the G₁/S transition while it maintains p27 in an active form in the nuclei during the exponential growth cell stage.

Introduction

Corneal endothelium, a monolayer of differentiated cells located in the posterior portion of the cornea, is essential for maintaining corneal transparency. It has long been believed that the capacity for regeneration of corneal endothelium after injury is severely limited in humans; thus, corneal endothelium is considered a nonreplicating tissue [1,2]. However, recent work by Joyce et al. shows that corneal endothelial cells (CEC) in vivo are arrested in the G₁ phase of the cell cycle, suggesting that these cells possess proliferative potential [3-5]. Nonetheless, CEC mitosis is seldom observed in adult human eyes, even during the wound repair process, except in those clinical situations that produce retrocorneal fibrous membrane [6,7]. The underlying mechanisms that keep the endothelial cells from moving out of the G₁ phase are only partially understood; transforming growth factor b2 (TGF-b2), a resident in the aqueous humor of the anterior chamber [8,9], has been proposed to suppress mitotic activity of the cells [10]. Inhibition of cell proliferation is central to the TGF-b response in many cells, including endothelial cells [11,12]. Cell proliferation is ultimately governed at the level of the cell cycle, and progression through the cell cycle is regulated by the sequential activity of various cyclin dependent kinases (Cdks) [13-15]. The enzyme activity of Cdks is dependent on physical interactions with one of the cyclin proteins, which are the regulatory subunits of these complexes. In addition, Cdk activity can be negatively regulated by a group of proteins collectively termed Cdk inhibitors (CKIs); CKI levels, like cyclin levels, vary during the cell cycle, thus contributing to the timing of cyclin/Cdk activation. One family of CKIs includes p21^Cip1, p27^Kip1 (p27), and p57^Kip1; the N-termini of these CKIs share homology and can bind to and inhibit Cdks [16-18]. Overexpression of these inhibitors can attenuate the proliferative response, whereas a reduction of their expression increases proliferation.

The CKI p27 was initially found to be induced by an extracellular antimitogenic signal [19]. It accumulates in many situations in which cells are arrested in the G₀/G₁ phase. Its expression is elevated in contact inhibited or mitogen deprived cells, and it can negatively regulate G₁ phase progression in response to antimitogenic signals [20-22]. For example, TGF-b exerts antimitogenic effects through Cdks and p27. In TGF-b treated cells, Cdk4 synthesis is inhibited and p27 is mobilized from the cyclin D-Cdk4 complex into the cyclin E-Cdk2 complex, resulting in loss of activity of both kinases and concomitant G₁ arrest [11,20]. Thus, TGF-b inhibits cell cycle progression by increasing the interaction of p27 and Cdk2 containing complexes, rather than by altering the abundance of p27. TGF-b2 is the major TGF-b isoform in aqueous humor [8,9]. This growth factor in aqueous humor has been proposed to play a key role in maintaining CEC in a G₁ phase arrested state in vivo, although the mechanism of the G₁ arrest of CEC is not defined [10]. In our previous study, we attempted to elucidate the underlying mechanism of the inhibitory action of TGF-b2 on endothelial cell growth. We have shown that CEC, upon reaching confluency, contain a high level of nuclear p27, but that nuclear Cdks are undetectable in the same culture [23]. We have further shown that TGF-b2 has no effect on Cdk expression at the protein level, while the same growth factor blocks phosphorylation of p27 mediated by fibroblast growth factor 2 (FGF-2). Phosphorylation of Thr¹⁸⁷ of p27 is known to lead to nuclear export of p27, ubiquitination and subsequent degradation of p27 at the proteasome [24-27]. CEC treated with TGF-b2 are able to maintain p27 in an active form in the nuclei, thus continuously sustaining the cells in G₁ phase. Since these studies were performed in CEC reaching confluency, the contact inhibited cell state might have contributed to the high level expression of p27 and the undetectable amount of nuclear Cdks. These results, therefore, make it difficult to accurately assess the effect of TGF-b2 in actively cycling cells. One recent study demonstrated that the capacity of p27 to inhibit cell proliferation was a function of culture density, p27 effectively blocked the cell cycle progression of dense cultures, whereas actively cycling cultures were refractory to p27 mediated growth inhibition [28]. We, therefore, investigated the expression of p27 and Cdk4 in actively cycling cultures and contact inhibited monolayers in primary, first passage, and second passage CEC. Using the same cultures, we further determined the differential activity of TGF-b2 on the expression of p27 and Cdk4 and phosphorylation of p27.

Methods

Cell cultures

Isolation and establishment of rabbit CEC were performed as previously described [29,30]. Briefly, the Descemet's membrane and corneal endothelium complex was treated with 0.2% collagenase and 0.05% hyaluronidase (Worthington Biochemical, Lakewood, NJ) for 90 min at 37 °C. Cultured cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA) and 50 mg/ml of gentamicin (DMEM-10) in a 5% CO₂ incubator. This method has been shown to promote cell proliferation during the early phase of culture and to maintain the culture as a contact inhibited monolayer when the cells reach confluence. For subculture, contact inhibited monolayer cultures were treated with 0.2% trypsin and 5 mM EDTA for 3 to 5 min. The 50% confluent cultures were used as the actively cycling cultures, and the monolayer was used as contact inhibited cultures. In some experiments, both the actively cycling and contact inhibited cells were treated with 10 ng/ml TGF-b2 (R & D Systems, Minneapolis, MN) for 24 h to impair cell proliferation mediated by serum.

Immunofluorescent staining

Primary, first passage, and second passage cultures of rabbit CEC (4x10⁴ cells/chamber) were plated on 4 well chamber slides. The actively cycling cells and contact inhibited cultures were subjected to immunofluorescent staining. All washes were carried out in phosphate buffered saline (PBS) at room temperature. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min. After washing, cells were treated with 5% goat serum for 1 h at room temperature and then incubated with the primary antibodies (1:200 dilution) at 37 °C for 1 h. For single antibody staining, cells were rinsed and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:100 dilution) in the dark at room temperature for 30 min. For double staining experiments, after the first primary antibody incubation, cells were incubated with rhodamine-conjugated secondary antibody (1:200 dilution) for 30 min at room temperature in the dark. Cells were then extensively rinsed and incubated with the second primary antibody (1:200 dilution) for 1 h at 37 °C. After washing, cells were then incubated with FITC-conjugated secondary antibodies (1:100 dilution) in the dark at room temperature for 30 min. After extensive washing, the slides were mounted in a drop of Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA) to reduce photobleaching. For double staining experiments in which both primary antibodies were produced in the mouse, the experimental procedures were modified as follows [31]. Fixation, blocking, and rinsing procedures were conducted as described above. Cells were incubated with the first monoclonal antibody at 37 °C for 1 h, rinsed in PBS and incubated with the FITC-conjugated secondary antibody (anti-mouse IgG) at 37 °C for 30 min in the dark. After the first monoclonal reactions, the remaining mouse Fc sites were blocked by sequential incubation of cells with normal mouse serum and 20 mg/ml of goat anti-mouse IgG Fab fragment at 37 °C for 1 h. After extensive washing in PBS, the second monoclonal antibody was incubated at 37 °C for 1 h and cells were rinsed in PBS. The corresponding secondary rhodamine-conjugated antibody (anti-mouse IgG) was incubated for 30 min at room temperature in the dark. Antibody labeling was examined using a Zeiss LSM-510 laser scanning confocal microscope. Optical slices (1.8 mm) were taken perpendicular to the cell layer (apical to basal orientation). A 488 nm Argon laser was used in combination with a 499/505-530 excitation/emission filter set for fluorescein examination. For rhodamine, the 543 nm helium neon laser was used with a 543 excitation filter and 560 emission filter. Image analysis was performed using the standard system operating software provided with the Zeiss LSM-510 series microscope. Control experiments were performed in parallel with the omission of one of the primary antibodies.

Protein preparation and protein determination

Actively cycling and the contact inhibited cells maintained in DMEM-10 were treated with TGF-b2 for 2, 8, or 24 h. Cells were washed with ice cold PBS and then lysed with lysis buffer (20 mM HEPES, pH 7.2, 10% glycerol, 10 mM Na₃VO₄, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1% Triton X-100) on ice for 30 min with occasional rocking. The lysate was subjected to sonification and the cell homogenates were then centrifuged at 14,000x g for 10 min. The concentration of the resultant supernatant was assessed by Bradford assay, using bovine serum albumin as a standard, as previously described [32].

SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis

The conditions of electrophoresis were as described by Laemmli, using the discontinuous Tris-Glycine buffer system [33]. Protein (30 mg) was loaded on a 12% SDS-polyacrylamide gel and separated under reduced conditions. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane at 0.30 ampere for 16 h in a semidry transfer system (transfer buffer; 48 mM Tris-HCl, pH 8.3, 39 mM glycine, 0.037% SDS, 20% methanol). Immunoblot analysis was performed as described previously [23] using the ABC Vectastain kit (Vector Laboratories Inc., Burlingame, CA). All washes and incubations were carried out in TTBS (0.9% NaCl, 100 mM Tris-HCl, pH 7.5, 0.1% Tween 20) at room temperature. Briefly, the blotted PVDF membrane was immediately placed in blocking buffer (5% nonfat milk in TTBS) and kept for 2 h. The incubations were carried out with primary antibodies (1:5000 anti-p27 antibody and 1:5000 anti-Cdk4 antibody) for 2 h, with biotinylated secondary antibody (1:5000 dilution) for 1 h, and with ABC reagent for 30 min. The membrane was treated with the enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech, Buckinghamshire, England) for 1 min, and the ECL treated membrane was exposed to ECL film.

Antibodies

Mouse monoclonal antibodies against p27 and Cdk4 were purchased from Sigma (St. Louis, MO), and rabbit polyclonal anti-phosphorylated p27 antibody was purchased from Zymed Laboratories Inc. (San Francisco, CA). FITC-conjugated goat anti-mouse IgG, rhodamine-conjugated Fab goat anti-mouse IgG, rhodamine-conjugated Fab goat anti-rabbit IgG, and goat anti-mouse IgG Fab fragment were purchased from Jackson Immunoresearch Laboratories (West Grove, PA) and biotinylated goat anti-mouse IgG antibodies were purchased from Vector Laboratories Inc.

Results

p27 and Cdk4 in actively cycling and contact inhibited CEC

Rabbit CEC maintain the following three characteristics in culture; the primary cultures grow readily, the cells lose their proliferative potential as they are serially passaged (the second passage cells have a very limited growth potential), and the cells maintain type IV collagen expression and deposit a Descemet's membrane-like extracellular matrix [29,30]. We further demonstrated that, upon reaching confluency, the primary CEC contained high levels of nuclear p27 [23]. Since the expression of cell cycle regulatory proteins is highly dependent on the cell growth state, and since the capacity of p27 to inhibit cell proliferation has been reported to be a function of culture density [28], we determined the expression of p27 and Cdk4 in the present study as a function of cell growth stage in primary, first passage, and second passage CEC. However, before we studied the colocalization of p27 and Cdk4, the two proteins were separately stained to determine their subcellular localization using two respective growth conditions; growing first passage CEC were stained for Cdk4, and first passage CEC having reached confluence were stained for p27 (Figure 1). Most of the cells treated with FGF-2 contained nuclear Cdk4 (Figure 1E), however there was no Cdk4 staining in cells treated with TGF-b2 (Figure 1G) or in the absence of serum (D0, Figure 1C). On the other hand, nuclear p27 is absent under growth up regulated conditions (FGF-2, Figure 1F), but it shows positive nuclear staining in serum deprived (D0, Figure 1D) or TGF-b2 treated cells (Figure 1H). Control experiments performed in parallel in the absence of the primary antibodies showed negative staining profiles (Figure 1A,B). We then examined the colocalization of the two proteins (p27 and Cdk4, Figure 2). The actively cycling cultures demonstrated the absence of nuclear p27 (Figure 2B,D,F), but there was nuclear Cdk4 present in less than 10% of cell population in the primary CEC (Figure 2A). Interestingly, nuclear staining of Cdk4 was increased in the first passage CEC (Figure 2C), and a faint nuclear staining of p27 was observed in some of the first passage CEC (Figure 2D). All cells in the second passage CEC showed positive staining for nuclear Cdk4 (Figure 2E), whereas nuclear p27 staining was undetectable in the sparse second passage CEC (Figure 2F). When contact inhibited cultures were analyzed for expression of p27 and Cdk4 (Figure 3), the primary and first passage CEC showed negative staining of nuclear Cdk4 (Figure 3A,C), and a few cells in the second passage CEC showed positive staining for nuclear Cdk4 (Figure 3E). In contrast, all cells of the primary and first passage CEC showed strong nuclear p27 staining (Figure 3B,D), and most cells of the second passage CEC contained nuclear p27 (Figure 3F).

We have shown in our previous study that TGF-b2 exerts an antiproliferative effect on CEC through the mechanism by which the growth factor regulates p27 expression [23]. The effect of TGF-b2 on Cdk4 and p27 expression was examined in actively cycling and contact inhibited cultures. When actively cycling cultures were treated with TGF-b2 for 24 h, all cells in the primary (Figure 4A,B), first passage (Figure 4C,D), and second passage (Figure 4E,F) CEC showed negative staining for both Cdk4 and p27 in the nuclei. Interestingly, TGF-b2 does not alter p27 expression in actively cycling cultures, but it does downregulate the expression of nuclear Cdk4 in actively cycling first and second passage CEC. When contact inhibited cultures were treated with TGF-b2 for 24 h, the Cdk4 (Figure 5A,C,E) and p27 (Figure 5B,D,F) staining profiles and subcellular localization were not altered in primary CEC when compared to untreated cells. However, a few of the first passage CEC demonstrated nuclear Cdk4 (Figure 5C)staining in contrast to the untreated cells, but a strong nuclear p27 staining was observed in the first passage CEC (Figure 5D) as was in the untreated cells. The second passage CEC showed Cdk4 and p27 staining profiles identical to those observed in the untreated cells (Figure 5E,F). A few cells showed staining of the nuclear Cdk4, whereas most cells were stained for nuclear p27.

To investigate whether TGF-b2 affected protein synthesis, expression levels of Cdk4 and p27 were further determined using immunoblotting analysis as a function of exposure time. Actively cycling cells maintained in DMEM-10 contained a high level of Cdk4 and a low level of p27 in the absence of TGF-b2. When cells were treated with TGF-b2, Cdk4 expression markedly decreased and p27 expression slightly increased, both in a time dependent manner (Figure 6). On the other hand, contact inhibited cells demonstrate a detectable amount of Cdk4. Interestingly, TGF-b2 did not exert the same inhibitory effect on Cdk4 synthesis as it did on the actively cycling cells. Contact inhibited cells contained a high level of p27 in the absence of TGF-b2, in agreement with previous reports [20,23]. TGF-b2 slightly upregulated p27 synthesis in contact inhibited cells.

p27 and phosphorylated p27 in actively cycling and contact inhibited CEC

In our previous study, we reported that TGF-b2 prevented phosphorylation of p27 mediated by FGF-2 [23]. Since this study was performed under only one culture condition, we examined the effect of TGF-b2 on phosphorylation of p27 in actively cycling and contact inhibited cells. Figure 7 shows that actively cycling cells in primary (Figure 7A,B), first passage (Figure 7C,D), and second passage (Figure 7E,F) CEC contained nuclear phosphorylated p27 (pp27) in most of the cell populations. The antibody used to detect pp27 is specific to the Thr¹⁸⁷ phosphorylated form of p27 and does not react with unphosphorylated p27. Interestingly, cells containing pp27 did not show positive staining for nuclear p27. The negative staining of nuclear p27 confirmed our findings in Figure 2. The absence of positive staining of nuclear p27 may be attributed to an altered conformation of the molecule, perhaps due to phosphorylation, which is not subsequently recognized by the antibody used in the study. When contact inhibited cultures were examined, two populations were evident in the primary CEC; in approximately 20% of the primary CEC, nuclei contained pp27 (Figure 8A), but these cells were negative for p27 staining (Figure 8B). In the other major population, there was positive staining for nuclear p27, but there was no staining for pp27 (Figure 8A,B). On the other hand, most cells of the first (Figure 8C,D) and second passage (Figure 8E,F) CEC contained both p27 and pp27. These findings suggest that the cell cycle machinery may be differentially regulated in primary cultures and in passaged cells. We further determined the effect of TGF-b2 on phosphorylation of p27. Approximately 25% of the actively cycling primary CEC treated with TGF-b2 for 24 h demonstrated positive staining for pp27 (Figure 9A), and no positive staining for p27 (Figure 9B). Far fewer of the first (Figure 9C,D) and second passage (Figure 9E,F) CEC were positive for pp27 or p27 in the nuclei, compared to the untreated cells. These findings suggest that TGF-b2 inhibits phosphorylation of p27 mediated by the growth stimulatory factors present in fetal calf serum in actively cycling primary, first passage, and second passage CEC. All of the contact inhibited cells treated with TGF-b2 were positive for nuclear p27 (Figure 10B,D,F) and most contained pp27 in the nuclei, regardless of the passage (Figure 10A,C,E). The staining potentials of contact inhibited cells for p27 and pp27 were similar to those observed in untreated cells, suggesting that TGF-b2 may not hamper the phosphorylation of p27 in contact inhibited CEC.

Discussion

It has long been believed that the capacity for regeneration of corneal endothelium after injury is severely limited in humans, thus, the corneal endothelium is considered to be a nonreplicating tissue [1,2]. Recently, Joyce and her colleagues demonstrated that CEC in vivo are arrested in the G₁ phase of the cell cycle [3-5], as observed in most differentiated cells [34]. Although this information suggests that CEC possess proliferative potential under physiologic conditions, mitosis of CEC is seldom observed in adult humans, even during the wound repair process, except in those clinical conditions that produce retrocorneal fibrous membranes [6,7]. The underlying mechanisms that keep endothelial cells from moving out of the G₁ phase are partially explained by the following; adult CEC may markedly decrease in their response to growth stimulatory factors, TGF-b2, a resident in aqueous humor of the anterior chamber, may suppress mitogenic activity of the cells, causing CEC to remain in a G₁ phase arrested state in vivo, and, contact inhibition may block mitosis to avoid improper cell proliferation [5,10,35].

Inhibition of cell proliferation is central to the TGF-b response in many cell types, including endothelial cells [11,12]. TGF-b can induce antiproliferative responses at many points during the division cycle. However, these responses effectively inhibit cell cycle progression only during the G₁ phase. The known antiproliferative actions of TGF-b include inhibition of Cdk4 synthesis and Cdk enzyme activity through the action of p15^INK4b and mobilization of p27 from a cyclin D-Cdk4 complex to a cyclin E-Cdk2 complex. This mobilization, in turn, causes loss of activity of both kinases, and concomitant G₁ arrest [11,20,22]. Thus, TGF-b inhibits cell cycle progression by increasing the interaction of p27 and Cdk2 containing complexes, and p27 is thought to play a critical role in cell cycle progression. The concentration of p27 is regulated predominantly by a posttranslational mechanism [24,25], both the ubiquitin proteasome pathway and ubiquitin independent proteolytic cleavage degrade p27 [26]. The phosphorylation of Thr¹⁸⁷ of p27 is known to lead to ubiquitination and degradation of the molecule [27]. Recent data have also suggested that the F-box protein, destined to function as the receptor component of the ubiquitin ligase complex, binds to p27 only when Thr¹⁸⁷ is phosphorylated; such binding then results in the ubiquitination and degradation of p27 [27,36,37]. Therefore, phosphorylation of p27 plays a critical role in cell cycle progression by influencing the abundance of the active inhibitor to cell cycle progression.

A recent study suggested that the capacity of p27 to inhibit cell proliferation is a function of culture density [28]. Expression levels of cell cycle regulatory proteins as a function of cell density have not been studied; therefore, we examined the expression of Cdk4 and p27 and the phosphorylation of p27 in actively cycling and contact inhibited CEC in the present study. We further investigated the effect of TGF-b2 on the expression of Cdk4 and p27 and the phosphorylation of p27 in these cultures. Since CEC markedly lose their growth potential by the second passage, these studies were performed in primary, first passage, and second passage CEC, all of which have differential growth potentials. Our data demonstrate that actively cycling CEC contain nuclear Cdk4. Interestingly, the fewest number of cells that are positive for nuclear Cdk4 are seen in primary cultures, whereas all of the second passage CEC are positive for nuclear Cdk4. It is well established that cyclin D-Cdk4 complex supports cell cycle progression with a noncatalytic function, specifically, the sequestration of p27 [11,22]. Our data, therefore, suggest that the nuclear Cdk4 observed in the second passage CEC may be involved in sequestration of p27, an event that does not influence cell cycle progression [11]. Interestingly, actively cycling cells do not show positive staining of nuclear p27, but they do show phosphorylated p27 in nuclei. Phosphorylated p27 is subsequently exported out of the nucleus and rapidly degraded. As a consequence, p27 no longer inhibits cell cycle progression. These data confirm the previous report that activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas [38]. It is unclear why phosphorylated p27 in these actively cycling cells demonstrates negative staining for the anti-p27 antibody. The negative staining of nuclear p27 may be attributed to the phosphorylation of p27, which may alter the conformation and subsequently lead to loss of staining potential of p27 to the antibody. Since phosphorylation of p27 is known to reduce the stability of p27 [38], this scenario may well explain why phosphorylated p27 positive cells are negative for unphosphorylated p27 in actively cycling cells and in contact inhibited primary CEC. On the other hand, this possibility does not explain the coincidental staining of p27 and phosphorylated p27 in contact inhibited first and second passage CEC, a phenomenon that is not understood. One possible explanation for this phenomenon is the existence of an intermediate conformation of the p27 molecule between phosphorylated and unphosphorylated forms. This intermediate form observed in contact inhibited first and second passage CEC may be stained with anti-p27 antibody.

Our data further demonstrate that TGF-b2 shows differential activities in actively cycling and contact inhibited CEC. TGF-b2 treatment eliminates the nuclear staining of Cdk4 and significantly reduces the phosphorylated form of p27 in actively cycling CEC. In addition, TGF-b2 markedly inhibits Cdk4 synthesis while slightly upregulating p27 synthesis in the same culture. However, TGF-b2 does not affect the staining profiles of Cdk4, p27 and phosphorylated p27 in contact inhibited cells, and the growth factor exerts a low level of activity on Cdk4 and p27 synthesis in the same culture. These data indicate that TGF-b2 exerts dual control on the cell cycle progression of CEC by downregulating Cdk4 expression and phosphorylation of p27 only in the actively cycling cells. The mechanisms by which antimitogens prevent phosphorylation during the active growth stage are yet to be elucidated. On the other hand, contact inhibited cells that have achieved the already elevated p27 level in the absence of antiproliferative agents are able to maintain CEC in the p27 mediated G₁ arrest state. At this time, the degree of p27 phosphorylation may not influence the G₁ arrested state and TGF-b2 exerts no further activity on blocking the phosphorylation of p27 in contact inhibited cells. The mechanism by which contact inhibited cells are maintained in such a state is yet to be determined, however, growth suppressive spatial constraints imposed by cell-cell contact at high cell density have been proposed to be mediated by cadherins [28,39]. Recent studies demonstrated that EDTA releases human corneal endothelium from the confluent monolayer and further promotes proliferation in corneal endothelium from older donors [5]. These findings, taken together, suggest that cell cycle progression may be differentially regulated in a density dependent manner, perhaps using different mechanisms. p27 and its phosphorylation may be responsible for the onset and maintenance of the quiescent state in actively cycling cells by the action of TGF-b2 or other antimitogenic agents, whereas a calcium mediated cellular activity may be responsible for maintaining the quiescent state of contact inhibited cells. Characterization of these mechanisms should shed light on clinical applications that require CEC to move out of the G₁ arrested state, including the regenerative wound repair process or the provision of donor CEC with high growth potentials for corneal transplantation.

Acknowledgements

Support for this work was provided by NIH grants EY06431 and EY03040, and Research to Prevent Blindness, New York, NY.

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